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WO2023157928A1 - Resistance inducer for plants - Google Patents

Resistance inducer for plants Download PDF

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Publication number
WO2023157928A1
WO2023157928A1 PCT/JP2023/005501 JP2023005501W WO2023157928A1 WO 2023157928 A1 WO2023157928 A1 WO 2023157928A1 JP 2023005501 W JP2023005501 W JP 2023005501W WO 2023157928 A1 WO2023157928 A1 WO 2023157928A1
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WIPO (PCT)
Prior art keywords
humic acid
mass
less
plants
plant
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PCT/JP2023/005501
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French (fr)
Japanese (ja)
Inventor
一馬 本田
藤樹 飯野
義弘 鳴坂
真理 鳴坂
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デンカ株式会社
岡山県
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Publication of WO2023157928A1 publication Critical patent/WO2023157928A1/en

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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
    • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides

Definitions

  • the present invention relates to a plant resistance inducer.
  • Patent Document 1 discloses a plant pathogen control agent containing a metal chelate or salt as an active ingredient.
  • Patent Document 2 discloses a plant virus disease control agent containing at least one of zinc gluconate and copper gluconate as an active ingredient.
  • JP 2020-132552 A Japanese Patent No. 6634325
  • the purpose of the present invention is to provide a new plant resistance inducer.
  • the present invention relates to the following inventions.
  • a new plant resistance inducer can be provided.
  • FIG. 4 is a graph showing the results of PR1 gene expression analysis in Test Example 1.
  • FIG. 4 is a graph showing the expression analysis results of the PR1 gene in Test Example 2.
  • FIG. 10 is a graph showing the results of confirming the effect of suppressing the severity of disease by applying a humic acid extract in Test Example 3.
  • FIG. 10 is a graph showing the results of confirming the effect of suppressing the severity of disease by applying a humic acid extract in Test Example 4.
  • FIG. 10 is a graph showing the results of confirming the effect of suppressing the severity of disease by applying a humic acid extract in Test Example 5.
  • FIG. 10 is a graph showing the results of confirming the effect of suppressing the severity of disease by applying a humic acid extract in Test Example 6.
  • a numerical range indicated using “to” indicates a range including the numerical values before and after “to” as the minimum and maximum values, respectively. Unless otherwise specified, the units of numerical values before and after "-" are the same.
  • the upper limit value or lower limit value of the numerical range at one step may be replaced with the upper limit value or lower limit value of the numerical range at another step.
  • the upper or lower limits of the numerical ranges may be replaced with the values shown in the examples. The upper and lower limits described individually can be combined arbitrarily.
  • the plant resistance inducer according to this embodiment contains humic acid as an active ingredient.
  • the plant resistance-inducing agent according to the present embodiment can enhance disease resistance of plants compared to when it is not used. Since the plant resistance inducer according to the present embodiment has a mild effect, it can enhance disease resistance of plants without causing phytotoxicity.
  • the plant resistance inducer according to this embodiment can be suitably used for crop protection. Induction of resistance can be confirmed, for example, by the expression level of an infection-specific protein (PR protein) gene (eg, PR1 gene).
  • PR protein infection-specific protein
  • Plants that are targets of the resistance inducer are not particularly limited, but include, for example, Solanaceae plants, Cucurbitaceae plants, Rosaceae plants, Brassicaceae plants, Gramineae plants, Araceae plants, and leguminous plants.
  • Solanaceous plants include tomatoes, eggplants, green peppers, hot peppers, tobacco, potatoes and the like.
  • Cucurbitaceous plants include cucumbers, pumpkins, melons and the like.
  • Rosaceae plants include strawberries, apples, pears, plums, and peaches.
  • Cruciferous plants include, for example, Chinese cabbage, bok choy, cabbage, Japanese mustard spinach, Arabidopsis thaliana and the like.
  • the gramineous plants include rice, wheat and the like.
  • Aroaceae plants include taro and the like. Examples of leguminous plants include soybeans and peas.
  • the plant resistance inducer according to this embodiment can enhance resistance to various diseases depending on the plant species.
  • the relationship between diseases and plants (host plants) is listed, for example, in the Japanese plant disease name database (National Biological Resources Genebank).
  • Pathogenic bacteria include, for example, tomato leaf spot bacteria (eg, Pseudomonas syringae) and cruciferous vegetable black spot bacteria (eg, Pseudomonas cannabina pv. alisalensis).
  • “Humic acid” as used herein includes humic acid and fulvic acid.
  • Humic acid includes one or more selected from the group consisting of humic acid and humic acid salts.
  • Humic acid has agricultural advantages such as promoting the growth of crop bodies and making them less susceptible to environmental stress (for example, the effects of global warming). In addition to inducing plant disease resistance, it is possible to obtain effects such as promoting the growth of plants and making them less susceptible to environmental stress (for example, the effects of global warming).
  • humic acid examples include natural humic acid produced naturally in peat and weathered coal, artificial humic acid artificially produced by nitric acid oxidation of lignite, and natural humic acid or artificial humic acid containing sodium, potassium, Examples include humic acid salts neutralized with alkaline substances such as ammonia, calcium and magnesium.
  • Humic acids include fulvic acid, humic acid, nitrohumic acid, ammonium humate, calcium humate, magnesium humate, ammonium nitrohumate, calcium nitrohumate and magnesium nitrohumate, potassium humate, potassium nitrohumate and the like.
  • the active ingredient may be a humic acid extract.
  • the humic acid extract may be an extract obtained by extracting nitric oxide from young coal with an extraction solvent containing water and, if necessary, alkali.
  • Young coal is coal that has a lower carbon content than bituminous coal, etc., and is defined as having a carbon content of 83% by mass or less. Young coal includes, for example, peat, lignite, lignite, sub-bituminous coal, and the like. Young coal may be used singly or in combination of two or more. The humic acid may be derived from lignite for its resistance-inducing effect.
  • Nitric oxide of young coal is obtained by oxidative decomposition of young coal with nitric acid.
  • Concentrated nitric acid is preferred as the nitric acid. From the viewpoint of safety and reactivity, it is preferable to use nitric acid with a concentration of 40 to 60% by mass.
  • the amount of nitric acid (HNO 3 ) used in the oxidative decomposition may be 10 parts by mass or more, or 20 parts by mass or more with respect to 20 parts by mass of young coal, and may be 300 parts by mass or less, 250 parts by mass or less, 200 parts by mass or less. It may be no more than 150 parts by mass, no more than 100 parts by mass, no more than 50 parts by mass, no more than 36 parts by mass, or no more than 20 parts by mass.
  • the amount of nitric acid (HNO 3 ) used may be 10 to 20 parts by mass, and may be 20 to 36 parts by mass, with respect to 20 parts by mass of young coal.
  • the amount of nitric acid used is a value converted to 100% nitric acid (100% HNO 3 ).
  • the temperature during oxidative decomposition may be, for example, 70 to 95°C.
  • heating to 70 to 95° C. in a hot water bath or the like facilitates rapid progress of the oxidation reaction.
  • the reaction time may be, for example, 20 minutes or more, 0.5 hours or more, or 1 hour or more, and may be 6 hours or less, 4 hours or less, or 1 hour or less.
  • the humic acid extract is prepared as a liquid by, for example, stirring nitric oxide of young coal (hereinafter referred to as crude humic acid) and an extraction solvent containing water and alkali, and then performing a solid-liquid separation step. can get.
  • the alkali includes hydroxide, ammonia, and the like.
  • Hydroxides include alkali metal hydroxides, ammonium hydroxide and the like.
  • an alkali metal hydroxide is preferable.
  • alkali metal hydroxides include potassium hydroxide and sodium hydroxide.
  • the hydroxide one or more of potassium hydroxide, sodium hydroxide, and ammonium hydroxide (ammonia water) are preferable.
  • the pH of the extraction solvent may be 0.5-7.0, 0.5-4.0 or 1.0-3.0.
  • the temperature (extraction temperature) at which the humic acid crude product is extracted with an extraction solvent may be, for example, 40 to 90°C from the viewpoint of further suppressing freezing and quality deterioration of the extract.
  • the time for extracting the humic acid crude product with the extraction solvent may be, for example, 0.5 hours or longer, 24 hours or shorter, or 1 hour or shorter.
  • the solid-liquid ratio is defined as the amount of extraction solvent relative to the amount of raw young coal used to prepare the humic acid crude product. For example, when 100 g (100 mL) of extraction solvent (water) is added to a crude product prepared from 20 g of young coal, the solid-liquid ratio (extraction solvent/young coal) is 5.
  • the solid-liquid ratio may be 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, and 15 or less, 13 or less, 11 or less, 9 or less, 7 or less, or 6 or less.
  • the solid-liquid ratio can be adjusted by adding water.
  • the solid-liquid ratio may be adjusted so as to achieve the desired solid-liquid ratio after adjusting the pH.
  • the solid-liquid separation method may be centrifugation, filter press, or the like.
  • the total organic carbon concentration (TOC) of the humic acid extract is 15,000 mg/L or more, 15,300 mg/L or more, 15,500 mg/L or more, 16,000 mg/L or more, 16,500 mg/L or more, 17, 000 mg/L or more, 17,500 mg/L or more, 18,000 mg/L or more, 18,500 mg/L or more, 19,000 mg/L or more, 19,500 mg/L or more, 20,000 mg/L or more, or 20, It may be 500 mg/L or more.
  • the TOC of the humic acid extract is 75,000 mg/L or less, 70,000 mg/L or less, 65,000 mg/L or less, 60,000 mg/L or less, 55,000 mg/L or less, 50,000 mg/L or less, 45,000 mg/L or less, 40,000 mg/L or less, 35,000 mg/L or less, 30,000 mg/L or less, 25,000 mg/L or less, 24,000 mg/L or less, 23,000 mg/L or less, or It may be 22,000 mg/L or less.
  • the method of measuring the TOC of the humic acid extract is defined as follows.
  • the humic acid extract was centrifuged at 3,000 ⁇ g, and the supernatant was measured using a total organic carbon meter (TOC-L manufactured by Shimadzu Corporation) by combustion catalytic oxidation.
  • TOC-L total organic carbon meter
  • the above (humic acid fraction and fulvic acid fraction) are separated according to the International Humic Substances Society method (Fujitake, Humic Substances Research Vol3, P1-9). and measure the TOC of the humic acid extract.
  • the melanic index (MI) of humic acid may be, for example, 1.5 or higher, 2.0 or higher, 2.2 or higher, 2.5 or higher, 3.0 or higher, or 3.5 or higher.
  • the MI of humic acid may be 6.5 or less, 6.0 or less, 5.5 or less, 5.0 or less, 4.5 or less, 4.0 or less, 3.5 or less, or 3.0 or less .
  • MI is an index used to classify humic acid, and is the ratio (A 450 / A 520 ). (Ky ⁇ ichi Kumada, Soil Organic Matter Chemistry, 2nd Edition, Gakkai Shuppan Center (1981), Japan Journal of Soil and Fertilizer Science, No. 71, No. 1, pp. 82-85 (2000)).
  • MI is calculated by the following method.
  • the sample is ground to a 250 ⁇ m sieve using a mortar and 250 ⁇ m sieve. About 10 g of it is placed in a weighing bottle with a known mass and accurately weighed. This weighing bottle is left in a dryer maintained at a temperature of 105° C. for about 12 hours, then returned to room temperature of 20° C. in a desiccator, and then accurately weighed again.
  • the moisture content of the sample is determined by considering the weight loss as moisture.
  • the absorbance at 450 nm shows 1.0 or more
  • add 0.1 mol/L sodium hydroxide aqueous solution to adjust the absorbance to 0.8 or more and less than 1.0, then measure the absorbance at 520 nm. do.
  • the ratio of absorbance at 450 nm to absorbance at 520 nm is calculated as MI.
  • the mass average molecular weight of humic acid may be 100-6,000.
  • the lower limit of the mass average molecular weight of humic acid may be, for example, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1,000 or more.
  • the upper limit of the mass average molecular weight of humic acid is, for example, 5,500 or less, 5,000 or less, 4,500 or less, 4,000 or less, 3,500 or less, 3,000 or less, 2,500 or less, 2,000 1,500 or less, 1,200 or less, or 1,000 or less.
  • the mass average molecular weight of humic acid is measured by the HPSEC method (GPC method) using Alliance HPLC System manufactured by Waters.
  • the column is SB-803HQ manufactured by Showa Denko KK, the standard sample is sodium polystyrene sulfonate, and the detection wavelength is 260 nm.
  • the mobile phase is 10 mmol/L sodium phosphate buffer containing 25% by mass of acetonitrile, the flow rate is 0.8 ml/min, and the column temperature is 40° C. (column oven setting).
  • the dosage form of the plant resistance inducer may be, for example, liquid or powder. Powders can be obtained as redissolvable powders by, for example, drying up a liquid plant resistance inducer by freeze-drying or the like.
  • the plant resistance inducer may consist of humic acid only, or may contain other ingredients than humic acid.
  • the content of humic acid in the plant resistance inducer is, for example, 5% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, based on the total mass of the plant resistance inducer. 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, or 95% by mass or more, 100% by mass or less, 99% by mass or less, Or it may be 95% by mass or less.
  • Other ingredients include, for example, spreading agents, fertilizers, and plant activators.
  • Fertilizers include, for example, ammonium sulfate, potassium nitrate, and ammonium phosphate.
  • Plant active agents include, for example, seaweed extracts and amino acids.
  • Amino acids include, for example, glycine, proline, and glutamic acid.
  • the total content of other components is, for example, 80% by mass or less, 60% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, based on the total mass of the plant resistance inducer. It may be no more than 5% by mass, no more than 1% by mass, or no more than 0% by mass, no less than 1% by mass, or no less than 5% by mass.
  • the method for enhancing disease resistance of plants according to this embodiment includes applying humic acid to plants.
  • Examples of the method of applying humic acid to plants include a method of spraying, coating, etc. on plants, and a method of soil irrigation, soil mixing, and the like.
  • the application amount and application period of humic acid are not particularly limited, and in the case of soil application, the total organic carbon concentration is 0.1 to 1,000 mg / L 1 to 30 times a month, or the total organic carbon concentration is 0.1 to 100 mg. /L about every day, in the case of hydroponics, the total organic carbon concentration can be 0.1 to 1,000 mg / L for the entire cultivation period, and in the case of foliar application, the total organic carbon The concentration can be 0.1 to 1,000 mg/L and can be 1 to 12 times a month.
  • the present invention described above can also be regarded as the use of humic acid to induce plant resistance.
  • the invention described above can also be viewed as humic acid for use as a plant resistance inducer.
  • the present invention described above can also be regarded as the use (application) of humic acid for the production of a plant resistance inducer.
  • the MI of humic acid in humic acid extract A was 2.2.
  • the total organic carbon concentration (TOC) of humic acid extract A was 34,000 mg/L.
  • the mass average molecular weight of humic acid in humic acid extract A was 4,300.
  • the MI of humic acid in humic acid extract B was 4.8.
  • the total organic carbon concentration (TOC) of humic acid extract B was 22,000 mg/L.
  • the mass average molecular weight of humic acid in humic acid extract B was 530.
  • the mass average molecular weight of humic acid was measured by HPSEC method (GPC method) using Alliance HPLC System manufactured by Waters.
  • the column was SB-803HQ manufactured by Showa Denko KK, the standard sample was sodium polystyrene sulfonate, and the detection wavelength was 260 nm.
  • the mobile phase was 10 mmol/L sodium phosphate buffer containing 25 mass % acetonitrile, the flow rate was 0.8 ml/min, and the column temperature was 40° C. (column oven setting).
  • TOC Total organic carbon concentration
  • MI Melt Index
  • the sample was ground to a 250 ⁇ m sieve product using a mortar and 250 ⁇ m sieve. About 10 g of it was placed in a weighing bottle with a known mass and accurately weighed. This weighing bottle was allowed to stand for about 12 hours in a dryer maintained at a temperature of 105° C., then returned to room temperature of 20° C. in a desiccator, and then accurately weighed again. The moisture content of the sample was determined by considering the mass decrease as moisture.
  • the PR1 gene in plants is a marker that serves as an indicator of plant disease resistance induction, and increased PR1 expression indicates that plant disease resistance is induced.
  • it is AtPR1 (Arabidopsis thaliana PR1) in Arabidopsis thaliana and SlPR1 (Solanum lycopersicum PR1) in tomato.
  • Figure 1 is a graph showing the results of expression analysis of the PR1 gene. As shown in FIG. 1, the application of humic acid increased the expression of the PR1 gene.
  • FIG. 2 is a graph showing the results of expression analysis of the SlPR1 gene. As shown in FIG. 2, even when tomatoes were used, the application of humic acid increased the expression of the PR1 gene.
  • Test Example 3 Pathogen inoculation test to tomato when using humic acid extract A
  • Control plots, test plots (humic acid extract A (150 ppm)) and positive control agents were prepared by the following method.
  • a spray solution was prepared by adding 0.1% spreading agent (approach BI) to water.
  • the humic acid extract A total organic carbon concentration: 34,000 mg/L
  • 0.1% spreading agent approximately BI was added to the spray solution.
  • Tomatoes (cultivar Regina) (13 days old after seeding, 2.3 true leaves) were sprayed with each agent and allowed to stand in an incubator (24°C, 16h/8h cycle under light and dark, 50% humidity). . The plants were sprayed three times at intervals of one week, and Pseudomonas syringae (1 ⁇ 10 7 cfu/ml) was spray-inoculated two days after the third spraying. Five days after the inoculation, the severity of disease was investigated.
  • the disease severity is represented by the following formula.
  • the disease incidence was investigated by classifying the severity of disease into the following five categories. 0: no symptoms, 1: fine spots, 2: lesions are observed in less than 25% area of the leaves, 3: lesions are observed in 25% or more and less than 50% area of the leaves, 4: leaf Lesions are observed in 50% or more of the area. 5: defoliation or death n1 to n5 indicate the number of individuals.
  • the phenomenon of suppressing the severity of disease by the above test method has been confirmed by a test using a positive control.
  • Tomatoes (cultivar Regina) (10 days old after seeding, 2 undeveloped true leaves) were sprayed with each agent and left in an incubator (24 ° C., 16h/8h cycle under light and dark, 50% humidity). ). The plants were sprayed three times at intervals of one week, and Pseudomonas syringae (1 ⁇ 10 7 cfu/ml) was spray-inoculated two days after the third spraying. Five days after the inoculation, the severity of disease was investigated. The severity results are shown in FIG.
  • Test Example 5 pathogen inoculation test to tomato when combined with glycine (Gly)
  • Control plot, test plot 1 (humic acid extract B40ppm), test plot 2 (humic acid extract B40ppm+Gly50ppm), test plot 3 (Gly50ppm) and positive control agents were prepared by the following method.
  • a spray solution was prepared by adding 0.1% spreading agent (approach BI) to water.
  • humic acid extract B total organic carbon concentration: 22,000 mg/L
  • glycine manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.
  • a spray solution of 40 mg/L of humic acid extract B + 50 mg/L of Gly was prepared. 0.1% spreading agent (approach BI) was added to the spray solution.
  • a spray solution containing 50 mg/L of Gly was prepared by diluting glycine with water. 0.1% spreading agent (approach BI) was added to the spray solution.
  • Tomatoes (cultivar Regina) (17 days old after seeding, 2.3 true leaves) were sprayed with each agent and allowed to stand in an incubator (24°C, 16h/8h cycle under light and dark, 50% humidity). .
  • Pseudomonas syringae (1 ⁇ 10 7 cfu/ml) was inoculated by spraying.
  • the above plants were cultured in a moist room (24°C, 16h/8h cycle of light and dark), and symptoms were examined 5 days after inoculation. The severity results are shown in FIG.
  • Test Example 6 Pathogen inoculation test to tomato Verification of root irrigation effect
  • test plot 1 humic acid extract A 150 ppm
  • test plot 2 humic acid extract B 100 ppm
  • test plot 3 humic acid extract B 40 ppm
  • test plot 4 BTH 100 ppm
  • BTH is acibenzolar S-methyl.
  • BTH is a material that can suppress the severity of disease by irrigation. Only water was applied in the control plot.
  • test group 1 a chemical having a total organic carbon concentration of 150 mg/L was prepared by diluting humic acid extract A (total organic carbon concentration of 34,000 mg/L) with water.
  • humic acid extract B total organic carbon concentration: 22,000 mg/L
  • BTH total organic carbon concentration: 22,000 mg/L
  • BTH manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.
  • Tomatoes (cultivar Regina) (11 days old after seeding, 2 undeveloped true leaves) were irrigated with each agent (15 ml/pot) and left to stand in an incubator (24° C., 16 h/8 h under light and dark conditions). cycle, 50% humidity). Irrigation was performed twice weekly (3 times in total). Two days after the final treatment, Pseudomonas syringae (5 ⁇ 10 6 cfu/ml) was spray-inoculated (4-5 sprays per plant). The above plants were cultured in a moist room (24°C, 16h/8h cycle of light and dark), and 5 days after inoculation, disease symptoms were examined. The results are shown in FIG.

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Abstract

One aspect of the present invention relates to a plant resistance inducer comprising humic acid as an active ingredient.

Description

植物用抵抗性誘導剤Plant resistance inducer
　本発明は、植物用抵抗性誘導剤に関する。 The present invention relates to a plant resistance inducer.
　植物を病害から保護するために、病原菌、ウイルスの防除剤が用いられる場合がある。例えば、特許文献１には、金属のキレート又は塩を有効成分とする、植物病原菌の防除剤が開示されている。例えば、特許文献２には、グルコン酸亜鉛及びグルコン酸銅の少なくとも１つを有効成分とする、植物ウイルス病の防除剤が開示されている。 In order to protect plants from disease, pathogenic bacteria and virus control agents are sometimes used. For example, Patent Document 1 discloses a plant pathogen control agent containing a metal chelate or salt as an active ingredient. For example, Patent Document 2 discloses a plant virus disease control agent containing at least one of zinc gluconate and copper gluconate as an active ingredient.
特開２０２０－１３２５５２号公報JP 2020-132552 A 特許第６６３４３２５号公報Japanese Patent No. 6634325
　本発明は、新たな植物用抵抗性誘導剤を提供することを目的とする。 The purpose of the present invention is to provide a new plant resistance inducer.
　本発明は、以下の各発明に関する。
［１］腐植酸を有効成分として含有する、植物用抵抗性誘導剤。
［２］メラニックインデックスが２．０以上である、［１］に記載の植物用抵抗性誘導剤。
［３］腐植酸の質量平均分子量が１００～６，０００である、［１］又は［２］に記載の植物用抵抗性誘導剤。
［４］有効成分が腐植酸抽出液であり、腐植酸抽出液の全有機炭素濃度が１５，０００ｍｇ／Ｌ以上である、［１］～［３］のいずれかに記載の植物用抵抗性誘導剤。
［５］褐炭由来である、［１］～［４］のいずれかに記載の植物用抵抗性誘導剤。
［６］植物の病害抵抗性を高める方法であって、植物に腐植酸を施用することを含む、方法。 The present invention relates to the following inventions.
[1] A plant resistance inducer containing humic acid as an active ingredient.
[2] The plant resistance inducer of [1], which has a melanic index of 2.0 or more.
[3] The plant resistance inducer according to [1] or [2], wherein the humic acid has a mass average molecular weight of 100 to 6,000.
[4] The resistance inducer for plants according to any one of [1] to [3], wherein the active ingredient is a humic acid extract, and the total organic carbon concentration of the humic acid extract is 15,000 mg/L or more. agent.
[5] The plant resistance inducer according to any one of [1] to [4], which is derived from lignite.
[6] A method of increasing disease resistance of a plant, comprising applying humic acid to the plant.
　本発明によれば、新たな植物用抵抗性誘導剤を提供することができる。 According to the present invention, a new plant resistance inducer can be provided.
試験例１におけるＰＲ１遺伝子の発現解析結果を示すグラフである。4 is a graph showing the results of PR1 gene expression analysis in Test Example 1. FIG. 試験例２におけるＰＲ１遺伝子の発現解析結果を示すグラフである。4 is a graph showing the expression analysis results of the PR1 gene in Test Example 2. FIG. 試験例３において腐植酸抽出液の施用による発病度の抑制効果を確認した結果を示すグラフである。10 is a graph showing the results of confirming the effect of suppressing the severity of disease by applying a humic acid extract in Test Example 3. FIG. 試験例４において腐植酸抽出液の施用による発病度の抑制効果を確認した結果を示すグラフである。10 is a graph showing the results of confirming the effect of suppressing the severity of disease by applying a humic acid extract in Test Example 4. FIG. 試験例５において腐植酸抽出液の施用による発病度の抑制効果を確認した結果を示すグラフである。10 is a graph showing the results of confirming the effect of suppressing the severity of disease by applying a humic acid extract in Test Example 5. FIG. 試験例６において腐植酸抽出液の施用による発病度の抑制効果を確認した結果を示すグラフである。10 is a graph showing the results of confirming the effect of suppressing the severity of disease by applying a humic acid extract in Test Example 6. FIG.
　以下、本発明を実施するための形態について詳細に説明する。ただし、本発明は以下の実施形態に限定されるものではない。本明細書中、「～」を用いて示された数値範囲は、「～」の前後に記載される数値をそれぞれ最小値及び最大値として含む範囲を示す。具体的に明示する場合を除き、「～」の前後に記載される数値の単位は同じである。本明細書中に段階的に記載されている数値範囲において、ある段階の数値範囲の上限値又は下限値は、他の段階の数値範囲の上限値又は下限値に置き換えてもよい。本明細書中に記載されている数値範囲において、その数値範囲の上限値又は下限値は、実施例に示されている値に置き換えてもよい。個別に記載した上限値及び下限値は任意に組み合わせ可能である。 Hereinafter, the embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments. In this specification, a numerical range indicated using "to" indicates a range including the numerical values before and after "to" as the minimum and maximum values, respectively. Unless otherwise specified, the units of numerical values before and after "-" are the same. In the numerical ranges described stepwise in this specification, the upper limit value or lower limit value of the numerical range at one step may be replaced with the upper limit value or lower limit value of the numerical range at another step. In the numerical ranges described herein, the upper or lower limits of the numerical ranges may be replaced with the values shown in the examples. The upper and lower limits described individually can be combined arbitrarily.
〔植物用抵抗性誘導剤〕
　本実施形態に係る植物用抵抗性誘導剤は、腐植酸を有効成分として含有する。本実施形態に係る植物用抵抗性誘導剤は、これを使用しない場合と比べて、植物の病害抵抗性を高めることができる。本実施形態に係る植物用抵抗性誘導剤は、その効果がマイルドであるため、薬害を発生させることなく、植物の病害抵抗性を高めることができる。本実施形態に係る植物用抵抗性誘導剤は、作物保護に好適に用いることができる。抵抗性が誘導されていることは、例えば、感染特異的タンパク質（ＰＲタンパク質）遺伝子（例えば、ＰＲ１遺伝子）の発現量によって確認することができる。 [Plant resistance inducer]
The plant resistance inducer according to this embodiment contains humic acid as an active ingredient. The plant resistance-inducing agent according to the present embodiment can enhance disease resistance of plants compared to when it is not used. Since the plant resistance inducer according to the present embodiment has a mild effect, it can enhance disease resistance of plants without causing phytotoxicity. The plant resistance inducer according to this embodiment can be suitably used for crop protection. Induction of resistance can be confirmed, for example, by the expression level of an infection-specific protein (PR protein) gene (eg, PR1 gene).
　抵抗性誘導剤の対象となる植物は、特に制限されないが、例えば、ナス科植物、ウリ科植物、バラ科植物、アブラナ科植物、イネ科植物、サトイモ科植物、マメ科植物が挙げられる。ナス科植物としては、トマト、ナス、ピーマン、トウガラシ、タバコ、ジャガイモ等が挙げられる。ウリ科植物としては、キュウリ、カボチャ、メロン等が挙げられる。バラ科植物としては、イチゴ、リンゴ、ナシ、ウメ、モモ等が挙げられる。アブラナ科植物としては、例えば、ハクサイ、チンゲンサイ、キャベツ、コマツナ、シロイヌナズナ等が挙げられる。イネ科植物としては、イネ、コムギ等が挙げられる。サトイモ科植物としては、サトイモ等が挙げられる。マメ科植物としては、ダイズ、エンドウ等が挙げられる。 Plants that are targets of the resistance inducer are not particularly limited, but include, for example, Solanaceae plants, Cucurbitaceae plants, Rosaceae plants, Brassicaceae plants, Gramineae plants, Araceae plants, and leguminous plants. Solanaceous plants include tomatoes, eggplants, green peppers, hot peppers, tobacco, potatoes and the like. Cucurbitaceous plants include cucumbers, pumpkins, melons and the like. Rosaceae plants include strawberries, apples, pears, plums, and peaches. Cruciferous plants include, for example, Chinese cabbage, bok choy, cabbage, Japanese mustard spinach, Arabidopsis thaliana and the like. The gramineous plants include rice, wheat and the like. Aroaceae plants include taro and the like. Examples of leguminous plants include soybeans and peas.
　本実施形態に係る植物用抵抗性誘導剤は、植物種に応じた種々の病害に対する抵抗性を高めることができる。病害と、植物（宿主植物）との関係は、例えば、日本植物病名データベース（農業生物資源ジーンバンク）に収載されている。病原菌としては、例えば、トマト斑葉細菌病菌（例えば、Ｐｓｅｕｄｏｍｏｎａｓ　ｓｙｒｉｎｇａｅ）、アブラナ科野菜黒斑細菌病菌（例えば、Ｐｓｅｕｄｏｍｏｎａｓ　ｃａｎｎａｂｉｎａ　ｐｖ．　ａｌｉｓａｌｅｎｓｉｓ）が挙げられる。 The plant resistance inducer according to this embodiment can enhance resistance to various diseases depending on the plant species. The relationship between diseases and plants (host plants) is listed, for example, in the Japanese plant disease name database (National Biological Resources Genebank). Pathogenic bacteria include, for example, tomato leaf spot bacteria (eg, Pseudomonas syringae) and cruciferous vegetable black spot bacteria (eg, Pseudomonas cannabina pv. alisalensis).
＜腐植酸＞
　本明細書における「腐植酸」には、フミン酸及びフルボ酸が含まれる。腐植酸は、腐植酸及び腐植酸塩からなる群より選択される１種以上を含む。腐植酸は、作物体の生育を促進する、環境ストレス（例えば、温暖化の影響）を受けにくくなる等の農業上の利点を有するため、本実施形態に係る植物用抵抗性誘導剤によれば、植物病害の抵抗性を誘導するとともに、植物の生育を促進する、及び、環境ストレス（例えば、温暖化の影響）を受けにくくなる等の効果を得ることができる。 <humic acid>
"Humic acid" as used herein includes humic acid and fulvic acid. Humic acid includes one or more selected from the group consisting of humic acid and humic acid salts. Humic acid has agricultural advantages such as promoting the growth of crop bodies and making them less susceptible to environmental stress (for example, the effects of global warming). In addition to inducing plant disease resistance, it is possible to obtain effects such as promoting the growth of plants and making them less susceptible to environmental stress (for example, the effects of global warming).
　腐植酸としては、泥炭及び風化炭等の天然に産出される天然腐植酸、褐炭の硝酸酸化等により人工的に製造される人工腐植酸、及び、天然腐植酸又は人工腐植酸をナトリウム、カリウム、アンモニア、カルシウム及びマグネシウム等のアルカリ物質で中和した腐植酸塩等が挙げられる。腐植酸としては、フルボ酸、フミン酸、ニトロフミン酸、フミン酸アンモニウム、フミン酸カルシウム、フミン酸マグネシウム、ニトロフミン酸アンモニウム、ニトロフミン酸カルシウム及びニトロフミン酸マグネシウム、フミン酸カリウム、ニトロフミン酸カリウム等が挙げられる。 Examples of humic acid include natural humic acid produced naturally in peat and weathered coal, artificial humic acid artificially produced by nitric acid oxidation of lignite, and natural humic acid or artificial humic acid containing sodium, potassium, Examples include humic acid salts neutralized with alkaline substances such as ammonia, calcium and magnesium. Humic acids include fulvic acid, humic acid, nitrohumic acid, ammonium humate, calcium humate, magnesium humate, ammonium nitrohumate, calcium nitrohumate and magnesium nitrohumate, potassium humate, potassium nitrohumate and the like.
　有効成分は、腐植酸抽出液であってよい。腐植酸抽出液は、若年炭の硝酸酸化物を、水と必要によりアルカリを含む抽出溶媒により抽出した抽出物であってよい。 The active ingredient may be a humic acid extract. The humic acid extract may be an extract obtained by extracting nitric oxide from young coal with an extraction solvent containing water and, if necessary, alkali.
　若年炭とは、瀝青炭等に比べ炭素含有量の少ない石炭であり、炭素含有率が８３質量％以下であるものと定義される。若年炭としては、例えば、泥炭、亜炭、褐炭、亜瀝青炭等が挙げられる。若年炭は、１種を単独で、又は２種以上を組み合わせて使用してよい。腐植酸は、抵抗性誘導効果の点から、褐炭由来であってよい。 Young coal is coal that has a lower carbon content than bituminous coal, etc., and is defined as having a carbon content of 83% by mass or less. Young coal includes, for example, peat, lignite, lignite, sub-bituminous coal, and the like. Young coal may be used singly or in combination of two or more. The humic acid may be derived from lignite for its resistance-inducing effect.
　若年炭の硝酸酸化物は、若年炭を硝酸で酸化分解させて得られる。硝酸としては濃硝酸が好ましい。安全性と反応性の点で、濃度４０～６０質量％の硝酸を用いることが好ましい。酸化分解の際の硝酸（ＨＮＯ３）の使用量は、若年炭２０質量部に対して、１０質量部以上、又は２０質量部以上であってよく、３００質量部以下、２５０質量部以下、２００質量部以下、１５０質量部以下、１００質量部以下、５０質量部以下、３６質量部以下、又は２０質量部以下であってよい。硝酸（ＨＮＯ３）の使用量は、若年炭２０質量部に対して、１０～２０質量部であってよく、２０～３６質量部であってよい。ここで、硝酸の使用量は１００％硝酸（１００％ＨＮＯ３）に換算した値である。 Nitric oxide of young coal is obtained by oxidative decomposition of young coal with nitric acid. Concentrated nitric acid is preferred as the nitric acid. From the viewpoint of safety and reactivity, it is preferable to use nitric acid with a concentration of 40 to 60% by mass. The amount of nitric acid (HNO 3 ) used in the oxidative decomposition may be 10 parts by mass or more, or 20 parts by mass or more with respect to 20 parts by mass of young coal, and may be 300 parts by mass or less, 250 parts by mass or less, 200 parts by mass or less. It may be no more than 150 parts by mass, no more than 100 parts by mass, no more than 50 parts by mass, no more than 36 parts by mass, or no more than 20 parts by mass. The amount of nitric acid (HNO 3 ) used may be 10 to 20 parts by mass, and may be 20 to 36 parts by mass, with respect to 20 parts by mass of young coal. Here, the amount of nitric acid used is a value converted to 100% nitric acid (100% HNO 3 ).
　酸化分解の際の温度は、例えば、７０～９５℃であってよい。酸化反応のスターターとして、湯浴等で７０～９５℃に加温すると酸化反応が速やかに進行しやすい。反応時間は、例えば、２０分間以上、０．５時間以上、又は１時間以上であってよく、６時間以下、４時間以下、又は１時間以下であってよい。 The temperature during oxidative decomposition may be, for example, 70 to 95°C. As a starter for the oxidation reaction, heating to 70 to 95° C. in a hot water bath or the like facilitates rapid progress of the oxidation reaction. The reaction time may be, for example, 20 minutes or more, 0.5 hours or more, or 1 hour or more, and may be 6 hours or less, 4 hours or less, or 1 hour or less.
　腐植酸抽出液は、例えば、若年炭の硝酸酸化物（以下、腐植酸粗製物という）と、水及びアルカリを含む抽出溶媒とを攪拌した後、固液分離工程を行うことにより、液状物として得られる。 The humic acid extract is prepared as a liquid by, for example, stirring nitric oxide of young coal (hereinafter referred to as crude humic acid) and an extraction solvent containing water and alkali, and then performing a solid-liquid separation step. can get.
　アルカリとしては、水酸化物、アンモニア等が挙げられる。水酸化物としては、アルカリ金属の水酸化物、水酸化アンモニウム等が挙げられる。水酸化物としては、アルカリ金属の水酸化物が好ましい。アルカリ金属の水酸化物としては、水酸化カリウム、水酸化ナトリウム等が挙げられる。水酸化物としては、水酸化カリウム、水酸化ナトリウム、及び水酸化アンモニウム（アンモニア水）のうちの１種以上が好ましい。抽出溶媒のｐＨは、０．５～７．０、０．５～４．０又は１．０～３．０であってよい。 　The alkali includes hydroxide, ammonia, and the like. Hydroxides include alkali metal hydroxides, ammonium hydroxide and the like. As the hydroxide, an alkali metal hydroxide is preferable. Examples of alkali metal hydroxides include potassium hydroxide and sodium hydroxide. As the hydroxide, one or more of potassium hydroxide, sodium hydroxide, and ammonium hydroxide (ammonia water) are preferable. The pH of the extraction solvent may be 0.5-7.0, 0.5-4.0 or 1.0-3.0.
　腐植酸粗製物を抽出溶媒で抽出する際の温度（抽出温度）は、抽出液の凍結及び品質低下を更に抑制する観点から、例えば、４０～９０℃であってよい。腐植酸粗製物を抽出溶媒で抽出する時間（抽出時間）は、例えば、０．５時間以上であってよく、２４時間以下であってよく、１時間以下であってもよい。 The temperature (extraction temperature) at which the humic acid crude product is extracted with an extraction solvent may be, for example, 40 to 90°C from the viewpoint of further suppressing freezing and quality deterioration of the extract. The time for extracting the humic acid crude product with the extraction solvent (extraction time) may be, for example, 0.5 hours or longer, 24 hours or shorter, or 1 hour or shorter.
　腐植酸粗製物を調製するために用いた原料の若年炭の量に対する抽出溶媒の量を、固液比と定義する。例えば、若年炭２０ｇから調製された粗製物に抽出溶媒（水）１００ｇ（１００ｍＬ）を添加した場合、固液比（抽出溶媒／若年炭）は５となる。固液比は３以上、４以上、５以上、６以上、７以上、８以上、９以上又は１０以上であってよく、１５以下、１３以下、１１以下、９以下、７以下、又は６以下であってよい。固液比は、水の添加によって調整することができる。固液比は、ｐＨを調整後に目的の固液比となるように調整されてよい。固液分離する方法は、遠心分離、フィルタープレス等であってよい。 The solid-liquid ratio is defined as the amount of extraction solvent relative to the amount of raw young coal used to prepare the humic acid crude product. For example, when 100 g (100 mL) of extraction solvent (water) is added to a crude product prepared from 20 g of young coal, the solid-liquid ratio (extraction solvent/young coal) is 5. The solid-liquid ratio may be 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, and 15 or less, 13 or less, 11 or less, 9 or less, 7 or less, or 6 or less. can be The solid-liquid ratio can be adjusted by adding water. The solid-liquid ratio may be adjusted so as to achieve the desired solid-liquid ratio after adjusting the pH. The solid-liquid separation method may be centrifugation, filter press, or the like.
　腐植酸抽出液の全有機炭素濃度（ＴＯＣ）は１５，０００ｍｇ／Ｌ以上、１５，３００ｍｇ／Ｌ以上、１５，５００ｍｇ／Ｌ以上、１６，０００ｍｇ／Ｌ以上、１６，５００ｍｇ／Ｌ以上、１７，０００ｍｇ／Ｌ以上、１７，５００ｍｇ／Ｌ以上、１８，０００ｍｇ／Ｌ以上、１８，５００ｍｇ／Ｌ以上、１９，０００ｍｇ／Ｌ以上、１９，５００ｍｇ／Ｌ以上、２０，０００ｍｇ／Ｌ以上、又は２０，５００ｍｇ／Ｌ以上であってよい。腐植酸抽出液のＴＯＣは、７５，０００ｍｇ／Ｌ以下、７０，０００ｍｇ／Ｌ以下、６５，０００ｍｇ／Ｌ以下、６０，０００ｍｇ／Ｌ以下、５５，０００ｍｇ／Ｌ以下、５０，０００ｍｇ／Ｌ以下、４５，０００ｍｇ／Ｌ以下、４０，０００ｍｇ／Ｌ以下、３５，０００ｍｇ／Ｌ以下、３０，０００ｍｇ／Ｌ以下、２５，０００ｍｇ／Ｌ以下、２４，０００ｍｇ／Ｌ以下、２３，０００ｍｇ／Ｌ以下、又は２２，０００ｍｇ／Ｌ以下であってよい。 The total organic carbon concentration (TOC) of the humic acid extract is 15,000 mg/L or more, 15,300 mg/L or more, 15,500 mg/L or more, 16,000 mg/L or more, 16,500 mg/L or more, 17, 000 mg/L or more, 17,500 mg/L or more, 18,000 mg/L or more, 18,500 mg/L or more, 19,000 mg/L or more, 19,500 mg/L or more, 20,000 mg/L or more, or 20, It may be 500 mg/L or more. The TOC of the humic acid extract is 75,000 mg/L or less, 70,000 mg/L or less, 65,000 mg/L or less, 60,000 mg/L or less, 55,000 mg/L or less, 50,000 mg/L or less, 45,000 mg/L or less, 40,000 mg/L or less, 35,000 mg/L or less, 30,000 mg/L or less, 25,000 mg/L or less, 24,000 mg/L or less, 23,000 mg/L or less, or It may be 22,000 mg/L or less.
　腐植酸抽出液のＴＯＣの測定方法は、次のように定義される。腐植酸抽出液を、３，０００×ｇで遠心分離した上澄み液を、全有機体炭素計（島津製作所製ＴＯＣ－Ｌ）を用いて燃焼触媒酸化方式で測定した値である。肥料成分である尿素等の非腐植物質を含む場合は、国際腐植物質学会法（藤嶽、ＨｕｍｉｃＳｕｂｓｔａｎｃｅｓ　Ｒｅｓｅａｒｃｈ　Ｖｏｌ３、Ｐ１－９）に準じて分別したもの（フミン酸画分及びフルボ酸画分）を上記の手法にて定量し、腐植酸抽出液のＴＯＣを測定する。 The method of measuring the TOC of the humic acid extract is defined as follows. The humic acid extract was centrifuged at 3,000×g, and the supernatant was measured using a total organic carbon meter (TOC-L manufactured by Shimadzu Corporation) by combustion catalytic oxidation. When non-humic substances such as urea, which is a fertilizer component, are included, the above (humic acid fraction and fulvic acid fraction) are separated according to the International Humic Substances Society method (Fujitake, Humic Substances Research Vol3, P1-9). and measure the TOC of the humic acid extract.
　腐植酸のメラニックインデックス（ＭＩ）は、例えば、１．５以上、２．０以上、２．２以上、２．５以上、３．０以上、又は３．５以上であってよい。腐植酸のＭＩは、６．５以下、６．０以下、５．５以下、５．０以下、４．５以下、４．０以下、３．５以下、又は３．０以下であってよい。 The melanic index (MI) of humic acid may be, for example, 1.5 or higher, 2.0 or higher, 2.2 or higher, 2.5 or higher, 3.0 or higher, or 3.5 or higher. The MI of humic acid may be 6.5 or less, 6.0 or less, 5.5 or less, 5.0 or less, 4.5 or less, 4.0 or less, 3.5 or less, or 3.0 or less .
　ＭＩとは、腐植酸の分類に用いられている指標であり、水酸化ナトリウム抽出液の吸収スペクトルの波長４５０ｎｍにおける吸光度（Ａ４５０）と５２０ｎｍにおける吸光度（Ａ５２０）との比（Ａ４５０／Ａ５２０）である。（熊田恭一著、土壌有機物の化学第２版　学会出版センター（１９８１）、日本土壌肥料学雑誌　第７１号　第１号　ｐ．８２～８５（２０００））。 MI is an index used to classify humic acid, and is the ratio (A 450 / A 520 ). (Kyōichi Kumada, Soil Organic Matter Chemistry, 2nd Edition, Gakkai Shuppan Center (1981), Japan Journal of Soil and Fertilizer Science, No. 71, No. 1, pp. 82-85 (2000)).
　より具体的には、ＭＩとは、次の方法によって算出されるものである。試料を乳鉢と２５０μｍ篩を用い２５０μｍ篩下品に粉砕する。その約１０ｇを、質量が既知の秤量ビンに取り精秤する。この秤量ビンを温度１０５℃に保持した乾燥機で約１２時間放置し、その後、デシケーター中で室温２０℃に戻してから再度精秤する。その質量減少分を水分とみなして試料の含水率を求める。次に、５０ｍｌ遠沈管に、上記２５０μｍ篩下品を乾燥質量相当量で０．１０ｇと、０．５ｍｏｌ／Ｌ水酸化ナトリウム水溶液４５ｍｌとを入れ、室温２０℃で約１時間、２５０ｒｐｍの速度で振とうした後、３，０００×ｇ、約１０分間の遠心分離を実施し、その上澄み液をアドバンテック社製Ｎｏ．５Ｃの濾紙で濾過する。濾液の４５０ｎｍの吸光度と５２０ｎｍの吸光度を、蒸留水をブランクとして測定する。この場合、４５０ｎｍの吸光度が１．０以上を示したならば、０．１ｍｏｌ／Ｌ水酸化ナトリウム水溶液を添加し吸光度が０．８以上１．０未満に調整してから、５２０ｎｍの吸光度を測定する。５２０ｎｍでの吸光度に対する４５０ｎｍでの吸光度の比（４５０ｎｍでの吸光度／５２０ｎｍでの吸光度）を算出し、ＭＩとする。 More specifically, MI is calculated by the following method. The sample is ground to a 250 μm sieve using a mortar and 250 μm sieve. About 10 g of it is placed in a weighing bottle with a known mass and accurately weighed. This weighing bottle is left in a dryer maintained at a temperature of 105° C. for about 12 hours, then returned to room temperature of 20° C. in a desiccator, and then accurately weighed again. The moisture content of the sample is determined by considering the weight loss as moisture. Next, in a 50 ml centrifuge tube, 0.10 g of the above 250 μm sieve product (dry mass equivalent) and 45 ml of 0.5 mol/L sodium hydroxide aqueous solution were placed, and the mixture was shaken at room temperature of 20° C. for about 1 hour at a speed of 250 rpm. After centrifugation, centrifugation was carried out at 3,000×g for about 10 minutes, and the supernatant was centrifuged in Advantech No. Filter through 5C filter paper. The absorbance at 450 nm and 520 nm of the filtrate is measured using distilled water as a blank. In this case, if the absorbance at 450 nm shows 1.0 or more, add 0.1 mol/L sodium hydroxide aqueous solution to adjust the absorbance to 0.8 or more and less than 1.0, then measure the absorbance at 520 nm. do. The ratio of absorbance at 450 nm to absorbance at 520 nm (absorbance at 450 nm/absorbance at 520 nm) is calculated as MI.
　腐植酸の質量平均分子量は、１００～６，０００であってよい。腐植酸の質量平均分子量の下限は、例えば、２００以上、３００以上、４００以上、５００以上、６００以上、７００以上、８００以上、９００以上、又は１，０００以上であってよい。腐植酸の質量平均分子量の上限は、例えば、５，５００以下、５，０００以下、４，５００以下、４，０００以下、３，５００以下、３，０００以下、２，５００以下、２，０００以下、１，５００以下、１，２００以下、又は１，０００以下であってよい。 The mass average molecular weight of humic acid may be 100-6,000. The lower limit of the mass average molecular weight of humic acid may be, for example, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1,000 or more. The upper limit of the mass average molecular weight of humic acid is, for example, 5,500 or less, 5,000 or less, 4,500 or less, 4,000 or less, 3,500 or less, 3,000 or less, 2,500 or less, 2,000 1,500 or less, 1,200 or less, or 1,000 or less.
　腐植酸の質量平均分子量は、Ｗａｔｅｒｓ社製Ａｌｌｉａｎｃｅ　ＨＰＬＣ　Ｓｙｓｔｅｍを用い、ＨＰＳＥＣ法（ＧＰＣ法）により測定される。カラムは昭和電工株式会社製ＳＢ－８０３ＨＱ、標準試料はポリスチレンスルホン酸ナトリウムを用い、検出波長は２６０ｎｍとする。移動相は２５質量％アセトニトリル含有の１０ｍｍｏｌ／Ｌリン酸ナトリウム緩衝液とし、流速は０．８ｍｌ／分とし、カラムの温度は４０℃（カラムオーブンの設定値）とする。 The mass average molecular weight of humic acid is measured by the HPSEC method (GPC method) using Alliance HPLC System manufactured by Waters. The column is SB-803HQ manufactured by Showa Denko KK, the standard sample is sodium polystyrene sulfonate, and the detection wavelength is 260 nm. The mobile phase is 10 mmol/L sodium phosphate buffer containing 25% by mass of acetonitrile, the flow rate is 0.8 ml/min, and the column temperature is 40° C. (column oven setting).
　植物用抵抗性誘導剤の剤型は、例えば、液剤又は粉剤であってよい。粉剤は、例えば、液剤である植物用抵抗性誘導剤を凍結乾燥等によってドライアップすることにより、再溶解可能な粉剤として得ることができる。 The dosage form of the plant resistance inducer may be, for example, liquid or powder. Powders can be obtained as redissolvable powders by, for example, drying up a liquid plant resistance inducer by freeze-drying or the like.
　植物用抵抗性誘導剤は、腐植酸のみからなっていてよく、腐植酸以外の他の成分を含んでいてよい。植物用抵抗性誘導剤中の腐植酸の含有量は、植物用抵抗性誘導剤の全質量を基準として、例えば、５質量％以上、１０質量％以上、２０質量％以上、３０質量％以上、４０質量％以上、５０質量％以上、６０質量％以上、７０質量％以上、８０質量％以上、９０質量％以上、又は９５質量％以上であってよく、１００質量％以下、９９質量％以下、又は９５質量％以下であってよい。
　他の成分としては、例えば、展着剤、肥料、植物活性剤が挙げられる。肥料としては、例えば、硫酸アンモニウム、硝酸カリウム、リン酸アンモニウムが挙げられる。植物活性剤としては例えば海藻抽出エキス、アミノ酸が挙げられる。アミノ酸としては、例えば、グリシン、プロリン、グルタミン酸が挙げられる。植物用抵抗性誘導剤がグリシンを含む場合、病害の抑制効果を更に高めることができる。他の成分の総含有量は、植物用抵抗性誘導剤の全質量を基準として、例えば、８０質量％以下、６０質量％以下、４０質量％以下、３０質量％以下、２０質量％以下、１０質量％以下、５質量％以下、又は１質量％以下であってよく、０質量％超、１質量％以上、又は５質量％以上であってよい。 The plant resistance inducer may consist of humic acid only, or may contain other ingredients than humic acid. The content of humic acid in the plant resistance inducer is, for example, 5% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, based on the total mass of the plant resistance inducer. 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, or 95% by mass or more, 100% by mass or less, 99% by mass or less, Or it may be 95% by mass or less.
Other ingredients include, for example, spreading agents, fertilizers, and plant activators. Fertilizers include, for example, ammonium sulfate, potassium nitrate, and ammonium phosphate. Plant active agents include, for example, seaweed extracts and amino acids. Amino acids include, for example, glycine, proline, and glutamic acid. When the plant resistance inducer contains glycine, the effect of suppressing disease can be further enhanced. The total content of other components is, for example, 80% by mass or less, 60% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, based on the total mass of the plant resistance inducer. It may be no more than 5% by mass, no more than 1% by mass, or no more than 0% by mass, no less than 1% by mass, or no less than 5% by mass.
　本実施形態に係る植物の病害抵抗性を高める方法は、植物に腐植酸を施用することを含む。 The method for enhancing disease resistance of plants according to this embodiment includes applying humic acid to plants.
　植物に腐植酸を施用する方法として、植物に対して散布、塗付等を行う方法、土壌潅注又は土壌混和等を行う方法が挙げられる。
　腐植酸の施用量及び施用期間は特に限られず、土壌施用の場合は全有機炭素濃度として０．１～１，０００ｍｇ／Ｌを月に１～３０回若しくは全有機炭素濃度として０．１～１００ｍｇ／Ｌ程度を毎日とすることができ、水耕栽培の場合は全有機炭素濃度として０．１～１，０００ｍｇ／Ｌを全栽培期間とすることができ、葉面施用の場合は全有機炭素濃度として０．１～１，０００ｍｇ／Ｌで月に１～１２回とすることができる。 Examples of the method of applying humic acid to plants include a method of spraying, coating, etc. on plants, and a method of soil irrigation, soil mixing, and the like.
The application amount and application period of humic acid are not particularly limited, and in the case of soil application, the total organic carbon concentration is 0.1 to 1,000 mg / L 1 to 30 times a month, or the total organic carbon concentration is 0.1 to 100 mg. /L about every day, in the case of hydroponics, the total organic carbon concentration can be 0.1 to 1,000 mg / L for the entire cultivation period, and in the case of foliar application, the total organic carbon The concentration can be 0.1 to 1,000 mg/L and can be 1 to 12 times a month.
　上述した本発明は、植物の抵抗性を誘導するための腐植酸の使用と捉えることもできる。上述した本発明は、植物の抵抗性誘導剤に使用するための腐植酸と捉えることもできる。上述した本発明は、植物用抵抗性誘導剤の製造のための腐植酸の使用（応用）と捉えることもできる。 The present invention described above can also be regarded as the use of humic acid to induce plant resistance. The invention described above can also be viewed as humic acid for use as a plant resistance inducer. The present invention described above can also be regarded as the use (application) of humic acid for the production of a plant resistance inducer.
　以下、実施例に基づいて本発明をより具体的に説明する。但し、本発明は、以下の実施例により限定されるものではない。 The present invention will be described more specifically below based on examples. However, the present invention is not limited by the following examples.
［腐植酸抽出液Ａの準備］
　ドラフト中で、炭素含有率が７７質量％の褐炭５００ｇを１,０００ｍｌのビーカーに入れて、濃度４８質量％の硝酸６２５ｇ（若年炭１００質量部に対して１００％硝酸６０質量部）を添加した。８０℃の水浴中で３時間酸化反応を行った。この操作で得た腐植酸を含む粗製物を以下の抽出操作に供した。
　この粗製物１００ｇに０．５ｍｏｌ／Ｌの水酸化カリウム水溶液を約９００ｍＬ加え、ｐＨ計でモニタしながら１.０ｍｏｌ／Ｌの水酸化カリウム水溶液を適宜加えｐＨ６．５とした。固液比（抽出溶媒／若年炭）１０：１となるように水を加え、８０℃で１時間抽出した。この抽出液を、３，０００×ｇで遠心分離し、得られた上澄み液は適宜希釈し、質量平均分子量、全有機炭素濃度（ＴＯＣ）及びメラニックインデックス（ＭＩ）を測定した。 [Preparation of humic acid extract A]
In a fume hood, 500 g of brown coal with a carbon content of 77% by mass was placed in a 1,000 ml beaker, and 625 g of nitric acid with a concentration of 48% by mass (60 parts by mass of 100% nitric acid per 100 parts by mass of young coal) was added. . An oxidation reaction was carried out in a water bath at 80°C for 3 hours. The crude product containing humic acid obtained by this operation was subjected to the following extraction operation.
About 900 mL of a 0.5 mol/L potassium hydroxide aqueous solution was added to 100 g of this crude product, and the pH was adjusted to 6.5 by adding a 1.0 mol/L potassium hydroxide aqueous solution appropriately while monitoring with a pH meter. Water was added so that the solid-liquid ratio (extraction solvent/juvenile charcoal) was 10:1, and the mixture was extracted at 80° C. for 1 hour. This extract was centrifuged at 3,000×g, the obtained supernatant was diluted appropriately, and the weight average molecular weight, total organic carbon concentration (TOC) and melanic index (MI) were measured.
　腐植酸抽出液Ａ中の腐植酸のＭＩは２．２であった。腐植酸抽出液Ａの全有機炭素濃度（ＴＯＣ）は、３４，０００ｍｇ／Ｌであった。腐植酸抽出液Ａ中の腐植酸の質量平均分子量は４，３００であった。 The MI of humic acid in humic acid extract A was 2.2. The total organic carbon concentration (TOC) of humic acid extract A was 34,000 mg/L. The mass average molecular weight of humic acid in humic acid extract A was 4,300.
［腐植酸抽出液Ｂの準備］
　ドラフト中で、炭素含有率が７７質量％の褐炭５００ｇを１,０００ｍｌのビーカーに入れて、濃度４８質量％の硝酸１５６２．５ｇ（若年炭１００質量部に対して１００％硝酸１５０質量部）を添加した。８０℃の水浴中で３時間酸化反応を行った。この操作で得た腐植酸を含む粗製物を以下の抽出操作に供した。
　この粗製物１００ｇに０．５ｍｏｌ／Ｌの水酸化カリウム水溶液を約４５０ｍＬ加え、ｐＨ計でモニタしながら１.０ｍｏｌ／Ｌの水酸化カリウム水溶液を適宜加えｐＨ２．０とした。固液比（抽出溶媒／若年炭）５：１となるように水を加え、８０℃で１時間抽出した。この抽出液を、３，０００×ｇで遠心分離し、得られた上澄み液は適宜希釈し、質量平均分子量、全有機炭素濃度（ＴＯＣ）及びメラニックインデックス（ＭＩ）を測定した。 [Preparation of humic acid extract B]
In a fume hood, 500 g of brown coal with a carbon content of 77% by mass is placed in a 1,000 ml beaker, and 1562.5 g of nitric acid with a concentration of 48% by mass (150 parts by mass of 100% nitric acid for 100 parts by mass of young coal) is added. added. An oxidation reaction was carried out in a water bath at 80°C for 3 hours. The crude product containing humic acid obtained by this operation was subjected to the following extraction operation.
About 450 mL of a 0.5 mol/L potassium hydroxide aqueous solution was added to 100 g of this crude product, and the pH was adjusted to 2.0 by adding a 1.0 mol/L potassium hydroxide aqueous solution appropriately while monitoring with a pH meter. Water was added so that the solid-liquid ratio (extraction solvent/juvenile charcoal) was 5:1, and extraction was carried out at 80° C. for 1 hour. This extract was centrifuged at 3,000×g, the obtained supernatant was diluted appropriately, and the weight average molecular weight, total organic carbon concentration (TOC) and melanic index (MI) were measured.
　腐植酸抽出液Ｂ中の腐植酸のＭＩは４．８であった。腐植酸抽出液Ｂの全有機炭素濃度（ＴＯＣ）は、２２,０００ｍｇ／Ｌであった。腐植酸抽出液Ｂ中の腐植酸の質量平均分子量は５３０であった。 The MI of humic acid in humic acid extract B was 4.8. The total organic carbon concentration (TOC) of humic acid extract B was 22,000 mg/L. The mass average molecular weight of humic acid in humic acid extract B was 530.
［質量平均分子量］
　腐植酸の質量平均分子量は、Ｗａｔｅｒｓ社製Ａｌｌｉａｎｃｅ ＨＰＬＣ Ｓｙｓｔｅｍを用い、ＨＰＳＥＣ法（ＧＰＣ法）により測定した。カラムは昭和電工株式会社製ＳＢ－８０３ＨＱ、標準試料はポリスチレンスルホン酸ナトリウムを用い、検出波長は２６０ｎｍとした。移動相は２５質量％アセトニトリル含有の１０ｍｍｏｌ／Ｌリン酸ナトリウム緩衝液とし、流速は０．８ｍｌ／分とし、カラムの温度は４０℃（カラムオーブンの設定値）とした。 [Mass average molecular weight]
The mass average molecular weight of humic acid was measured by HPSEC method (GPC method) using Alliance HPLC System manufactured by Waters. The column was SB-803HQ manufactured by Showa Denko KK, the standard sample was sodium polystyrene sulfonate, and the detection wavelength was 260 nm. The mobile phase was 10 mmol/L sodium phosphate buffer containing 25 mass % acetonitrile, the flow rate was 0.8 ml/min, and the column temperature was 40° C. (column oven setting).
［全有機炭素濃度（ＴＯＣ）］
　腐植酸抽出液のＴＯＣは、全有機体炭素計（株式会社島津製作所製ＴＯＣ－Ｌ）を用い、燃焼触媒酸化方式で測定した。 [Total organic carbon concentration (TOC)]
The TOC of the humic acid extract was measured by a combustion catalytic oxidation method using a total organic carbon meter (TOC-L manufactured by Shimadzu Corporation).
［メラニックインデックス（ＭＩ）］
　試料を乳鉢と２５０μｍ篩を用い２５０μｍ篩下品に粉砕した。その約１０ｇを、質量が既知の秤量ビンに取り精秤した。この秤量ビンを温度１０５℃に保持した乾燥機で約１２時間放置し、その後、デシケーター中で室温２０℃に戻してから再度精秤した。その質量減少分を水分とみなして試料の含水率を求めた。次に、５０ｍｌ遠沈管に、上記２５０μｍ篩下品を乾燥質量相当量で０．１０ｇと、０．５ｍｏｌ／Ｌ水酸化ナトリウム水溶液４５ｍｌとを入れ、室温２０℃で約１時間、２５０ｒｐｍの速度で振とうした後、３，０００×ｇ、約１０分間の遠心分離を実施し、その上澄み液をアドバンテック社製Ｎｏ．５Ｃの濾紙で濾過した。濾液の４５０ｎｍの吸光度と５２０ｎｍの吸光度を、蒸留水をブランクとして測定した。この場合、４５０ｎｍの吸光度が１．０以上を示したならば、０．１ｍｏｌ／Ｌ水酸化ナトリウム水溶液を添加し吸光度が０．８以上１．０未満に調整してから、５２０ｎｍの吸光度を測定した。５２０ｎｍでの吸光度に対する４５０ｎｍでの吸光度の比（４５０ｎｍでの吸光度／５２０ｎｍでの吸光度）を算出し、ＭＩとした。 [Melanic Index (MI)]
The sample was ground to a 250 μm sieve product using a mortar and 250 μm sieve. About 10 g of it was placed in a weighing bottle with a known mass and accurately weighed. This weighing bottle was allowed to stand for about 12 hours in a dryer maintained at a temperature of 105° C., then returned to room temperature of 20° C. in a desiccator, and then accurately weighed again. The moisture content of the sample was determined by considering the mass decrease as moisture. Next, in a 50 ml centrifuge tube, 0.10 g of the above 250 μm sieve product (dry mass equivalent) and 45 ml of 0.5 mol/L sodium hydroxide aqueous solution were placed, and the mixture was shaken at room temperature of 20° C. for about 1 hour at a speed of 250 rpm. After centrifugation, centrifugation was carried out at 3,000×g for about 10 minutes, and the supernatant was centrifuged in Advantech No. It was filtered through a 5C filter paper. Absorbance at 450 nm and 520 nm of the filtrate was measured using distilled water as a blank. In this case, if the absorbance at 450 nm shows 1.0 or more, add 0.1 mol/L sodium hydroxide aqueous solution to adjust the absorbance to 0.8 or more and less than 1.0, then measure the absorbance at 520 nm. did. The ratio of absorbance at 450 nm to absorbance at 520 nm (absorbance at 450 nm/absorbance at 520 nm) was calculated and designated as MI.
［試験例１：シロイヌナズナの葉面に腐植酸を噴霧した際の遺伝子発現］
　シロイヌナズナＣｏｌ－０（土植、播種から３週齢）に、０．１％アプローチＢＩ（農薬用展着剤、丸和バイオケミカル株式会社）を添加した２００倍（図１の×２００）希釈腐植酸抽出液Ａを噴霧処理し、１０、２４時間後にサンプリングを個体ごと（３サンプル）に行った。対照区（図１のＣｏｎｔ　Ａｐ）として０．１％アプローチＢＩのみを処理した植物のサンプリングを行った。リアルタイムＰＣＲにより、ＰＲ１；Ａｔ２ｇ１４６１０の遺伝子発現を解析した。サンプル間のノーマライズにはＡｔＣＢＰ２０遺伝子を用いた。 [Test Example 1: Gene expression when humic acid was sprayed on the leaves of Arabidopsis thaliana]
Arabidopsis thaliana Col-0 (soil planted, 3 weeks old from sowing), 0.1% approach BI (pesticide spreading agent, Maruwa Biochemical Co., Ltd.) added 200 times (× 200 in Fig. 1) diluted humic acid Extract A was sprayed, and 10 and 24 hours later, sampling was performed for each individual (3 samples). As a control plot (Cont Ap in FIG. 1), plants treated with 0.1% Approach BI alone were sampled. Gene expression of PR1;At2g14610 was analyzed by real-time PCR. AtCBP20 gene was used for normalization between samples.
　一般的に植物のＰＲ１遺伝子は、植物病害に対する抵抗性誘導の指標となるマーカーであり、ＰＲ１の発現上昇は、植物病害に対する抵抗性が誘導されていることを示す。例えば、シロイヌナズナではＡｔＰＲ１(Ａｒａｂｉｄｏｐｓｉｓ　ｔｈａｌｉａｎａ　ＰＲ１)、トマトではＳｌＰＲ１(Ｓｏｌａｎｕｍ　ｌｙｃｏｐｅｒｓｉｃｕｍ　ＰＲ１)である。 In general, the PR1 gene in plants is a marker that serves as an indicator of plant disease resistance induction, and increased PR1 expression indicates that plant disease resistance is induced. For example, it is AtPR1 (Arabidopsis thaliana PR1) in Arabidopsis thaliana and SlPR1 (Solanum lycopersicum PR1) in tomato.
　図１は、ＰＲ１遺伝子の発現解析結果を示すグラフである。図１のとおり、腐植酸の施用によりＰＲ１遺伝子の発現が上昇した。 Figure 1 is a graph showing the results of expression analysis of the PR1 gene. As shown in FIG. 1, the application of humic acid increased the expression of the PR1 gene.
［試験例２：トマトの葉面に腐植酸を噴霧した際の遺伝子発現］
　トマト（品種レジナ）（土植、播種から１７日齢、本葉２．５枚）に０．１％アプローチＢＩ（農薬用展着剤、丸和バイオケミカル株式会社）を添加した２００倍希釈腐植抽出液Ａ（図２の×２００）を噴霧処理し、５，１０，２４時間後にサンプリング（２個体から３サンプル）を行った。また、対照区（図２のＣｏｎｔ　Ａｐ）として０．１％アプローチＢＩのみを処理した植物のサンプリングを行った。次いで、ｔｏｔａｌ　ＲＮＡを調製し、ｃＤＮＡ合成を行った後に、リアルタイムＰＣＲにより、ＳｌＰＲ１遺伝子の発現を解析した。サンプル間のノーマライズにはＳｌＴＩＰ４１遺伝子を用いた。 [Test Example 2: Gene expression when tomato leaves are sprayed with humic acid]
200-fold diluted humus extraction by adding 0.1% approach BI (pesticide spreading agent, Maruwa Biochemical Co., Ltd.) to tomato (cultivar Regina) (planted in soil, 17 days old from sowing, 2.5 true leaves) Liquid A (×200 in FIG. 2) was sprayed, and sampling (3 samples from 2 individuals) was performed 5, 10 and 24 hours later. Also, as a control (Cont Ap in FIG. 2), plants treated with 0.1% Approach BI alone were sampled. After preparing total RNA and synthesizing cDNA, the expression of the SlPR1 gene was analyzed by real-time PCR. The SlTIP41 gene was used for normalization between samples.
　図２は、ＳｌＰＲ１遺伝子の発現解析の結果を示すグラフである。図２のとおり、トマトを用いた場合でも、腐植酸の施用によりＰＲ１遺伝子の発現が上昇した。 FIG. 2 is a graph showing the results of expression analysis of the SlPR1 gene. As shown in FIG. 2, even when tomatoes were used, the application of humic acid increased the expression of the PR1 gene.
［試験例３：腐植酸抽出液Ａを用いた場合のトマトへの病原菌接種試験］
　以下の方法で対照区、試験区（腐植酸抽出液Ａ（１５０ｐｐｍ））及び陽性対照（ポジティブコントロール）の薬剤を準備した。
　対照区では水に０．１％の展着剤（アプローチＢＩ）を添加して散布液を調製した。試験区では腐植酸抽出液Ａ（全有機炭素濃度３４，０００ｍｇ／Ｌ）を水で希釈することで全有機炭素濃度１５０ｍｇ／Ｌの散布液を調整した。散布液には０．１％の展着剤（アプローチＢＩ）を添加した。 [Test Example 3: Pathogen inoculation test to tomato when using humic acid extract A]
Control plots, test plots (humic acid extract A (150 ppm)) and positive control agents were prepared by the following method.
In the control plot, a spray solution was prepared by adding 0.1% spreading agent (approach BI) to water. In the test group, the humic acid extract A (total organic carbon concentration: 34,000 mg/L) was diluted with water to prepare a spray solution having a total organic carbon concentration of 150 mg/L. 0.1% spreading agent (approach BI) was added to the spray solution.
　トマト（品種レジナ）（播種後、１３日齢、本葉２．３枚）に、各剤を噴霧処理し、培養庫に静置した（２４℃、明暗下１６ｈ／８ｈサイクル、湿度５０％）。１週間おきに３回噴霧処理を行い、３回目噴霧から処理２日後にトマト斑葉細菌病菌Ｐｓｅｕｄｏｍｏｎａｓ　ｓｙｒｉｎｇａｅ（１×１０７ｃｆｕ／ｍｌ）を噴霧接種した。接種５日後に発病度を調査した。 Tomatoes (cultivar Regina) (13 days old after seeding, 2.3 true leaves) were sprayed with each agent and allowed to stand in an incubator (24°C, 16h/8h cycle under light and dark, 50% humidity). . The plants were sprayed three times at intervals of one week, and Pseudomonas syringae (1×10 7 cfu/ml) was spray-inoculated two days after the third spraying. Five days after the inoculation, the severity of disease was investigated.
　発病度は、以下の式で表される。発病度は結果を図３に示す。
発病度＝｛（１ｎ１＋２ｎ２＋３ｎ３＋４ｎ４＋５ｎ５）／（５×調査数）｝×１００
　発病調査は発病度を以下の５つに区分して調査した。
０：病徴なし、１：微小斑点、２：葉の２５％未満の面積に病斑が認められる、３：葉の２５％以上５０％未満の面積に病斑が認められる、４：葉の５０％以上の面積に病斑が認められる、５：落葉又は枯死
ｎ１からｎ５は個体数を示す。
　上記試験方法による発病度の抑制現象は、ポジティブコントロールを用いた試験により確認している。 The disease severity is represented by the following formula. The results of disease severity are shown in FIG.
Disease severity = {(1n1 + 2n2 + 3n3 + 4n4 + 5n5) / (5 x number of investigations)} x 100
The disease incidence was investigated by classifying the severity of disease into the following five categories.
0: no symptoms, 1: fine spots, 2: lesions are observed in less than 25% area of the leaves, 3: lesions are observed in 25% or more and less than 50% area of the leaves, 4: leaf Lesions are observed in 50% or more of the area. 5: defoliation or death n1 to n5 indicate the number of individuals.
The phenomenon of suppressing the severity of disease by the above test method has been confirmed by a test using a positive control.
　図３のとおり、腐植酸抽出液Ａの施用により、発病度が抑制されることが確認された。 As shown in Figure 3, it was confirmed that the application of humic acid extract A suppressed the severity of the disease.
［試験例４：腐植酸抽出液Ｂを用いた場合のトマトへの病原菌接種試験］
　以下の方法で対照区、及び試験区（腐植酸抽出液Ｂ（１００ｐｐｍ又は４０ｐｐｍ））の薬剤を準備した。
　対照区では水の０．１％の展着剤（アプローチＢＩ）を添加して散布液を調製した。
　試験区では腐植酸抽出液Ｂ（全有機炭素濃度２２，０００ｍｇ／Ｌ）を水で希釈することで全有機炭素濃度１００ｍｇ／Ｌ又は４０ｍｇ／Ｌの散布液を調製した。散布液には０．１％の展着剤（アプローチＢＩ）を添加した。 [Test Example 4: Pathogen inoculation test to tomato when using humic acid extract B]
Chemical agents for the control plot and the test plot (humic acid extract B (100 ppm or 40 ppm)) were prepared by the following method.
In the control plots, 0.1% water spreading agent (approach BI) was added to prepare a spray solution.
In the test group, the humic acid extract B (total organic carbon concentration: 22,000 mg/L) was diluted with water to prepare a spray solution with a total organic carbon concentration of 100 mg/L or 40 mg/L. 0.1% spreading agent (approach BI) was added to the spray solution.
　トマト（品種レジナ）（播種後、１０日齢、未展開の本葉２枚）に、各剤を噴霧処理し、培養庫に静置した（２４℃、明暗下１６ｈ／８ｈサイクル、湿度５０％）。１週間おきに３回噴霧処理を行い、３回目噴霧から処理２日後にトマト斑葉細菌病菌Ｐｓｅｕｄｏｍｏｎａｓ　ｓｙｒｉｎｇａｅ（１×１０７ｃｆｕ／ｍｌ）を噴霧接種した。接種５日後に発病度を調査した。発病度の結果を図４に示す。 Tomatoes (cultivar Regina) (10 days old after seeding, 2 undeveloped true leaves) were sprayed with each agent and left in an incubator (24 ° C., 16h/8h cycle under light and dark, 50% humidity). ). The plants were sprayed three times at intervals of one week, and Pseudomonas syringae (1×10 7 cfu/ml) was spray-inoculated two days after the third spraying. Five days after the inoculation, the severity of disease was investigated. The severity results are shown in FIG.
　図４のとおり、腐植酸抽出液Ｂの施用によっても、発病度が抑制されることが示された。 As shown in Figure 4, it was shown that the application of humic acid extract B also suppressed the severity of the disease.
［試験例５：グリシン（Ｇｌｙ）併用時のトマトへの病原菌接種試験］
　以下の方法で対照区、試験区１（腐植酸抽出液Ｂ４０ｐｐｍ）、試験区２（腐植酸抽出液Ｂ４０ｐｐｍ＋Ｇｌｙ５０ｐｐｍ）、試験区３（Ｇｌｙ５０ｐｐｍ）及びポジティブコントロールの薬剤を準備した。
　対照区では水に０．１％の展着剤（アプローチＢＩ）を添加して散布液を調製した。
　試験区１又は試験区２では腐植酸抽出液Ｂ（全有機炭素濃度２２，０００ｍｇ／Ｌ）及びグリシン（富士フイルム和光純薬株式会社製）を水で希釈することで腐植酸抽出液Ｂ４０ｍｇ／Ｌ又は腐植酸抽出液Ｂ４０ｍｇ／Ｌ＋Ｇｌｙ５０ｍｇ／Ｌの散布液を調製した。散布液には０．１％の展着剤（アプローチＢＩ）を添加した。
　試験区３ではグリシンを水で希釈することでＧｌｙ５０ｍｇ／Ｌの散布液を調製した。散布液には０．１％の展着剤（アプローチＢＩ）を添加した。 [Test Example 5: pathogen inoculation test to tomato when combined with glycine (Gly)]
Control plot, test plot 1 (humic acid extract B40ppm), test plot 2 (humic acid extract B40ppm+Gly50ppm), test plot 3 (Gly50ppm) and positive control agents were prepared by the following method.
In the control plot, a spray solution was prepared by adding 0.1% spreading agent (approach BI) to water.
In test group 1 or test group 2, humic acid extract B (total organic carbon concentration: 22,000 mg/L) and glycine (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) were diluted with water to give 40 mg/L of humic acid extract B. Alternatively, a spray solution of 40 mg/L of humic acid extract B + 50 mg/L of Gly was prepared. 0.1% spreading agent (approach BI) was added to the spray solution.
In Test Group 3, a spray solution containing 50 mg/L of Gly was prepared by diluting glycine with water. 0.1% spreading agent (approach BI) was added to the spray solution.
　トマト（品種レジナ）（播種後、１７日齢、本葉２．３枚）に、各剤を噴霧処理し、培養庫に静置した（２４℃、明暗下１６ｈ／８ｈサイクル、湿度５０％）。処理２日後にトマト斑葉細菌病菌Ｐｓｅｕｄｏｍｏｎａｓ　ｓｙｒｉｎｇａｅ（１×１０７ｃｆｕ／ｍｌ）を噴霧接種した。以上について湿室下（２４℃、明暗下１６ｈ／８ｈサイクル）にて培養し、接種５日後に病徴を検定した。発病度の結果を図５に示す。 Tomatoes (cultivar Regina) (17 days old after seeding, 2.3 true leaves) were sprayed with each agent and allowed to stand in an incubator (24°C, 16h/8h cycle under light and dark, 50% humidity). . Two days after the treatment, Pseudomonas syringae (1×10 7 cfu/ml) was inoculated by spraying. The above plants were cultured in a moist room (24°C, 16h/8h cycle of light and dark), and symptoms were examined 5 days after inoculation. The severity results are shown in FIG.
　図５のとおり、グリシン併用時に発病度の抑制効果が向上した。 As shown in Figure 5, the effect of suppressing the severity of disease was improved when glycine was used in combination.
［試験例６：トマトへの病原菌接種試験　株元潅注効果の検証］
　以下の方法で対照区、試験区１（腐植酸抽出液Ａ１５０ｐｐｍ）、試験区２（腐植酸抽出液Ｂ１００ｐｐｍ）、試験区３（腐植酸抽出液Ｂ４０ｐｐｍ）及び試験区４（ＢＴＨ１００ｐｐｍ）の薬剤を準備した。ＢＴＨは、アシベンゾラルＳメチルである。ＢＴＨは、潅注処理により、発病度を抑制可能な資材である。
　対照区では水のみを施用した。
　試験区１では腐植酸抽出液Ａ（全有機炭素濃度３４，０００ｍｇ／Ｌ）を水で希釈することで全有機炭素濃度１５０ｍｇ／Ｌの薬剤を準備した。
　試験区２又は試験区３では腐植酸抽出液Ｂ（全有機炭素濃度２２，０００ｍｇ／Ｌ）を水で希釈することで全有機炭素濃度１００ｍｇ／Ｌ又は４０ｍｇ／Ｌの薬剤を準備した。
　試験区４ではＢＴＨ（富士フイルム和光純薬株式会社製）を水で希釈することでＢＴＨ１００ｍｇ／Ｌの薬剤を準備した。 [Test Example 6: Pathogen inoculation test to tomato Verification of root irrigation effect]
Prepare chemicals for control plot, test plot 1 (humic acid extract A 150 ppm), test plot 2 (humic acid extract B 100 ppm), test plot 3 (humic acid extract B 40 ppm) and test plot 4 (BTH 100 ppm) by the following method. did. BTH is acibenzolar S-methyl. BTH is a material that can suppress the severity of disease by irrigation.
Only water was applied in the control plot.
In test group 1, a chemical having a total organic carbon concentration of 150 mg/L was prepared by diluting humic acid extract A (total organic carbon concentration of 34,000 mg/L) with water.
In test group 2 or test group 3, humic acid extract B (total organic carbon concentration: 22,000 mg/L) was diluted with water to prepare drugs with a total organic carbon concentration of 100 mg/L or 40 mg/L.
In test group 4, BTH (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was diluted with water to prepare a BTH 100 mg/L drug.
　トマト（品種レジナ）（播種後、１１日齢、未展開の本葉２枚）に、各剤を潅注処理（１５ｍｌ/ポット）し、培養庫に静置した（２４℃、明暗下１６ｈ／８ｈサイクル、湿度５０％）。１週間おきに２回（合計３回）潅注処理した。最終の処理２日後にトマト斑葉細菌病菌Ｐｓｅｕｄｏｍｏｎａｓ　ｓｙｒｉｎｇａｅ（５×１０６ｃｆｕ／ｍｌ）を噴霧接種した（個体ごとに４－５回噴霧）。以上について、湿室下（２４℃、明暗下１６ｈ／８ｈサイクル）にて培養し、接種５日後に病徴を検定した。結果を図６に示す。 Tomatoes (cultivar Regina) (11 days old after seeding, 2 undeveloped true leaves) were irrigated with each agent (15 ml/pot) and left to stand in an incubator (24° C., 16 h/8 h under light and dark conditions). cycle, 50% humidity). Irrigation was performed twice weekly (3 times in total). Two days after the final treatment, Pseudomonas syringae (5×10 6 cfu/ml) was spray-inoculated (4-5 sprays per plant). The above plants were cultured in a moist room (24°C, 16h/8h cycle of light and dark), and 5 days after inoculation, disease symptoms were examined. The results are shown in FIG.
　図６に示すとおり、潅注処理の場合であっても、腐植酸抽出液の施用により発病度が抑えられることが確認された。

  As shown in FIG. 6, it was confirmed that even in the case of irrigation treatment, the application of the humic acid extract suppresses the severity of the disease.

Claims (6)

  1. 　腐植酸を有効成分として含有する、植物用抵抗性誘導剤。 A plant resistance inducer containing humic acid as an active ingredient.
  2. 　メラニックインデックスが２．０以上である、請求項１に記載の植物用抵抗性誘導剤。 The resistance inducer for plants according to claim 1, which has a melanic index of 2.0 or more.
  3. 　前記腐植酸の質量平均分子量が１００～６，０００である、請求項１又は２に記載の植物用抵抗性誘導剤。 The plant resistance inducer according to claim 1 or 2, wherein the humic acid has a mass average molecular weight of 100 to 6,000.
  4. 　前記有効成分が腐植酸抽出液であり、
    　前記腐植酸抽出液の全有機炭素濃度が１５，０００ｍｇ／Ｌ以上である、請求項１又は２に記載の植物用抵抗性誘導剤。     The active ingredient is a humic acid extract,
    3. The plant resistance inducer according to claim 1, wherein the humic acid extract has a total organic carbon concentration of 15,000 mg/L or more.
  5. 　褐炭由来である、請求項１又は２に記載の植物用抵抗性誘導剤。 The plant resistance inducer according to claim 1 or 2, which is derived from lignite.
  6. 　植物の病害抵抗性を高める方法であって、
    　前記植物に腐植酸を施用することを含む、方法。

          A method for enhancing disease resistance of a plant, comprising:
    A method comprising applying humic acid to said plant.
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