WO2021187152A1 - Method for controlling soil infectious disease damage of soybean - Google Patents

Method for controlling soil infectious disease damage of soybean Download PDF

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Publication number
WO2021187152A1
WO2021187152A1 PCT/JP2021/008600 JP2021008600W WO2021187152A1 WO 2021187152 A1 WO2021187152 A1 WO 2021187152A1 JP 2021008600 W JP2021008600 W JP 2021008600W WO 2021187152 A1 WO2021187152 A1 WO 2021187152A1
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soybean
glutamic acid
pseudomonas
soil
present
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PCT/JP2021/008600
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French (fr)
Japanese (ja)
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香純 竹内
昌杰 姜
茂美 瀬尾
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国立研究開発法人農業・食品産業技術総合研究機構
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Publication of WO2021187152A1 publication Critical patent/WO2021187152A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/08—Immunising seed
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G13/00—Protecting plants
    • 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
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
    • A01N25/04—Dispersions, emulsions, suspoemulsions, suspension concentrates or gels
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • 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 method for controlling soybean from a soil-borne disease. More specifically, the present invention relates to a method for controlling soybean stalk epidemic using glutamic acid, and compositions and kits that can be used in the method.
  • Soybean is one of the important food crops produced in Japan.
  • soybean has become a particularly important item as a conversion crop in paddy fields, and it is said that about 80% of soybeans produced in Japan are cultivated in paddy field conversion fields.
  • the paddy field conversion field has poor drainage, and if rainfall continues, soybeans cultivated in the paddy field conversion field are vulnerable to moisture damage and soil diseases.
  • soybean stalk epidemics, which prefer stagnant water, are increasing in number in various parts of Japan, and have become a major problem as soil diseases of soybeans.
  • Soybean stalk epidemic is a disease caused by bacteria of the genus Phytophthora belonging to oomycetes, and occurs throughout the growth of soybeans. Soybeans affected by stalk plague develop water-soaked streaks or oval lesions on the main stems and branches above the stalks, and wither about one week after infection, and finally. Wither. Since the infectious bacteria of soybean stem epidemic can remain in the soil in the state of follicles for a long period of time, the damage of the stem epidemic is characteristically long-term. In addition, the infectious bacteria of stem epidemic that remain in the soil can be a further source of transmission.
  • soybean stalk epidemics Currently, the technology for controlling soybean stalk epidemics is extremely limited. Measures such as high ridge cultivation and drainage measures may be taken to control soybean stalk epidemics, but the effects are not always high and cannot be easily carried out from the viewpoint of labor.
  • a chemical pesticide for controlling soybean stalk epidemic a drug containing a siazophamide agent, an amisulbrom agent, or the like is registered.
  • existing chemical pesticides may not be very effective, and non-natural chemical pesticides have a high impact on the environment and adversely affect plants and human bodies due to residual pesticides and pesticide exposure. There is also concern that it may have an effect.
  • Patent Document 1 A technique for controlling soybean stalk epidemic has been reported.
  • Patent Document 2 A technique for controlling soybean stalk epidemic has been reported.
  • Patent Document 3 the use of bacteria as biological pesticides has attracted particular attention, and the use of bacteria of the genus Pseudomonas has been shown to be effective in controlling soil diseases caused by bacteria of the genus Pythium and bacteria of the genus Fusarium. The result has been reported so far (Patent Document 2).
  • an object of the present invention is to provide a method, composition and kit capable of effectively controlling stalk epidemics in soybean without using non-natural chemical pesticides.
  • the present inventor focused on the use of amino acids as natural compounds, and found that glutamic acid is particularly effective in controlling soybean stalk epidemics.
  • the present inventor has found that such a control effect can be further enhanced by using glutamic acid in combination with Pseudomonas bacteria. Based on these findings, the present inventor has completed the present invention.
  • a method for controlling stalk epidemics in soybean which comprises a step of applying glutamic acid to soybean or soil containing soybean.
  • Aspect 2 The method according to Aspect 1, further comprising a step of applying Pseudomonas bacteria to soybean or soil containing soybean.
  • Aspect 3 The method according to aspect 1 or 2, wherein the soybean is in the state of seeds or seedlings.
  • Aspect 4 The method according to any one of aspects 1 to 3, wherein glutamic acid is applied before sowing of soybean or within 3 days after sowing.
  • Glutamic acid used in the present invention is one of the natural amino acids, and is considered to have higher safety than general non-natural chemical pesticides.
  • the Pseudomonas bacterium used in the present invention has a track record of use as a biopesticide, and is considered to be as safe as glutamic acid.
  • the technique of the present invention can be easily used because the control effect of soybean stalk epidemic can be obtained only by one component of a chemical substance or a total of two components in which this is combined with one type of bacterium. ..
  • FIG. 1 is a graph showing the weight of soybean plants after cultivation for 2 weeks.
  • FIG. 2 is a graph showing the number of surviving soybeans during the cultivation period. The horizontal axis of the graph represents the cultivation period (days), and the vertical axis of the graph represents the number of surviving soybeans (pieces).
  • soybean is a type of plant belonging to the genus Soybean of the family Fabales, and is a plant referred to by the scientific name as Glycine max.
  • Glutamic acid is a kind of amino acid and is a compound represented by the chemical formula HOOC (CH 2 ) 2 CH (NH 2 ) COOH (or C 5 H 9 NO 4).
  • glutamate is classified into hydrophilic amino acids, polar amino acids, polar charged amino acids (polar negative charged amino acids), and acidic amino acids, and may be represented by the abbreviation Glu or E.
  • Glutamic acid is contained in plants and animals, and is specifically known to be contained in seaweed, wheat flour, soybeans, sugar cane and the like.
  • glutamic acid can be any of D-form (D-glutamic acid), L-form (L-glutamic acid), and DL-form (DL-glutamic acid), and is not particularly limited.
  • L-form glutamic acid (L-glutamic acid) is preferable.
  • the CAS number of D-form glutamic acid is 6893-26-1, and the CAS number of L-form glutamic acid is 56-86-0.
  • the method for obtaining glutamic acid is not particularly limited, and it may be either a natural glutamic acid isolated and purified from animals or plants, or a glutamic acid obtained by a chemical synthesis method, a fermentation method, or the like.
  • glutamic acid a self-purified product may be used, or a commercially available product may be used, and the use thereof is not particularly limited.
  • commercially available glutamic acid is preferably used.
  • the method of the present invention may also further include a step of imparting Pseudomonas bacteria to soybean or soil containing soybean.
  • the bacterium of the genus Pseudomonas is a bacterium belonging to the family Pseudomonas of the phylum Proteobacteria, Gammaproteobacteria, and is characterized by being a gram-negative aerobic rod.
  • Pseudomonas bacteria have a wide habitat and are known to inhabit soil, freshwater, seawater, plants, or animals.
  • the Pseudomonas bacterium in the present invention is not particularly limited, and specific examples thereof include Pseudomonas protegens, Pseudomonas fluorescens, Pseudomonas syringe, Pseudomonas pseudomonas. Santa (Pseudomonas synxantha), Pseudomonas brushcasearum (Pseudomonas brassicareum), Pseudomonas putida, and Pseudomonas rodesia (Pseudomonas). Further, in the present invention, Pseudomonas sp.
  • Os17 strain and Pseudomonas sp. St29 strains can also be used, and these bacteria can be used by the National Institute of Technology and Evolution (NITE) Biotechnology Center Patent Microorganisms Depositary Center (NPMD) (Kazusaka, Kisarazu City, Chiba Prefecture, Japan). At Kazusakamatari 2-5-8), deposits were made under the accession number NITE P-02053 (consignment date: May 20, 2015) and the entrustment number NITE P-02054 (consignment date: May 20, 2015), respectively. There is.
  • Pseudomonas protegens Pseudomonas sp.
  • the St29 strain is used in the present invention.
  • the bacterial strains of Pseudomonas protegens the Pseudomonas protegens CHA0 strain and the Pseudomonas protegens Cab57 strain are preferably used, but are not particularly limited thereto.
  • Pseudomonas bacteria may be those isolated by themselves, those sold from the facility, or Pseudomonas bacteria (ie, commercial products) available on the market as biopesticide products. May be used, and the use thereof is not particularly limited.
  • both glutamic acid and Pseudomonas bacteria are applied to soybean or soil containing soybean.
  • soil as used herein means soil in which soybeans can grow.
  • the soil used in the present invention may be, for example, a soil represented by a name such as hilling soil, potting soil, seedling raising soil, or seedling raising soil.
  • the soil of the mountains that has not been treated in any way may be used as it is.
  • the particle size of the soil is not particularly limited, and any soil may be used as long as soybean can grow.
  • artificial soil for soil improvement such as vermiculite may be used, but it is particularly preferable to use seedling raising soil among various soils.
  • Seedling cultivation soil is soil containing fertilizer that is the basis of cultivation, and is classified as bed soil.
  • the seedling raising soil used in the present invention may contain nitrogen, phosphoric acid, and potassium, and preferably contains an organic acid. It is preferable that the seedling raising soil used in the present invention does not contain a large amount of minerals such as silicon oxide, magnesium oxide and aluminum oxide. Further, the seedling raising soil used in the present invention is preferably a seedling raising soil that has not been heat-treated at a temperature of 800 ° C. or higher.
  • the soil containing soybean means that the soil is in a state where soybean exists in or on the soil. If the soybean is present in the soil, the whole soybean may be buried in the soil, or a part of the soybean is buried in the soil and the rest is out of the soil. It may be.
  • the soil to be imparted in the present invention is preferably soil in the vicinity of soybean (for example, soil within a range of 10 cm from soybean).
  • the state of soybean to be imparted in the present invention is not particularly limited and may be a state in which seeds or seedlings or soybeans have already grown, but is preferably in the state of seeds or seedlings, and more preferably in the state of seeds. Is. By being in the early stage of growth such as seeds and seedlings, it is possible to suppress the onset of stem epidemic and more effectively control stem epidemic in soybean. In addition, since the tissue of soybean is young, glutamic acid and Pseudomonas bacteria easily permeate the inside of soybean, and in that respect, the control of stem epidemic can be performed more effectively.
  • the soybean to be imparted in the present invention may be in the state of rooted seeds or in the state of non-rooted seeds, but may be in the state of non-rooted seeds. preferable. When soybean is already in a grown state, glutamic acid and Pseudomonas bacteria can be applied to any part of the root, leaf, main stem, branch, etc. of soybean.
  • glutamic acid and Pseudomonas bacteria are applied to soybean or soil containing soybean, but the method and means for the application are not particularly limited as long as the effects of the present invention can be obtained.
  • glutamic acid and Pseudomonas bacteria are suspended or dissolved together or separately, and water (aqueous solution) is prepared, and the prepared water (aqueous solution) is brought into contact with soybean or soil containing soybean to carry out the donation operation. It can be carried out.
  • the morphology of glutamic acid and Pseudomonas bacteria may be liquid or solid.
  • glutamic acid and Pseudomonas bacteria can be applied to soybean or soil containing soybean by an operation such as spraying, dropping, or dipping.
  • a container may be prepared separately, and a perforated container containing soybean or soil containing soybean may be placed in the container containing the liquid to perform the work.
  • the morphology of glutamic acid and Pseudomonas is solid, it may be imparted by placing the solid on or in the soil, or by contacting the solid with the surface of soybean. May be good.
  • the amount of glutamic acid and Pseudomonas bacteria to be added to soybean or soil containing soybean is not particularly limited and can be appropriately set according to the form of application and the like.
  • glutamic acid when glutamic acid is given to soybean, it can be given in an amount of 0.01 mM to 1 M, preferably 0.1 mM to 100 mM per soybean.
  • Pseudomonas bacteria when Pseudomonas bacteria are given to soybean, the number of bacteria can be 10 4 to 10 20 CFU, preferably 10 6 to 10 10 CFU per soybean.
  • the amount of glutamic acid and Pseudomonas bacteria to be added can be adjusted according to the concentration in the preparation containing these and the amount of the preparation.
  • glutamic acid is a salt
  • the amount of glutamic acid can be calculated based on the molecular weight corresponding to its free form (glutamic acid ion).
  • the amount of glutamic acid and Pseudomonas bacteria to be added can be adjusted according to the concentration in the preparation containing these and the amount of the preparation.
  • soybeans or the soil containing the soybeans should be applied at any time before, at the time of sowing, and after sowing (including the seedling raising period, the planting period, etc.). It is also good, and is not particularly limited, but preferably within 3 days before or after sowing of soybean.
  • “sowing” in the present specification means planting soybeans in soil as a starting point for growth, and includes, for example, sowing seeds in soil and planting seedlings in soil.
  • the specific timing is not particularly limited, but for example, within 7 days before sowing, within 6 days before sowing, within 5 days before sowing, and 4 before sowing. Within days, within 3 days before sowing, within 2 days before sowing, within 1 day before sowing, within 12 hours before sowing, within 6 hours before sowing, within 3 hours before sowing, within 1 hour before sowing, within 30 minutes before sowing , Within 10 minutes before sowing, within 5 minutes before sowing, within 1 minute before sowing, or within 30 seconds before sowing.
  • the soybean When the soybean is given after sowing, it may be given within 7 days after sowing, within 6 days after sowing, within 5 days after sowing, or within 4 days after sowing, or within 2 days after sowing. , Within 1 day after sowing, within 12 hours after sowing, within 6 hours after sowing, within 3 hours after sowing, within 1 hour after sowing, within 30 minutes after sowing, within 10 minutes after sowing, within 5 minutes after sowing, within 5 minutes after sowing It may be within 1 minute after sowing or within 30 seconds after sowing.
  • the above-mentioned granting time is typically within 7 days before and after sowing.
  • the period between the above-mentioned feeding time and soybean sowing time is short.
  • glutamic acid and Pseudomonas bacteria may be added simultaneously or separately.
  • glutamic acid and Pseudomonas bacteria are simultaneously applied. Even if both components are applied at the same time, some time interval is allowed. In that case, the time interval is, for example, 10 minutes or less, 5 minutes or less, 3 minutes or less, 1 minute or less, or 30 seconds or less.
  • the interval between the two grant periods may be within 2 days, within 1 day, within 12 hours, within 6 hours, within 3 hours, or within 1 hour.
  • the interval between the application periods of both can be, for example, 30 minutes or more, 1 hour or more, 6 hours or more, or 12 hours or more.
  • the Pseudomonas bacterium is considered to have a function of enhancing the soybean stalk epidemic control effect of glutamic acid, and therefore it is preferable that the interval between the two application periods is not long.
  • the interval between the application times of both glutamic acid and Pseudomonas bacteria is at least one day or less.
  • the above-mentioned soybean seeding can be applied with glutamic acid and Pseudomonas bacteria as the time when either one is given, and the other can be given within 3 days after the soybean seeding.
  • Glutamic acid and Pseudomonas bacteria may be given first. That is, glutamic acid may be given first, and then Pseudomonas bacteria may be given, or Pseudomonas bacteria may be given first, and then glutamic acid may be given.
  • the method of the present invention can also include the step of adding glutamic acid and Pseudomonas bacteria and then further adding glutamic acid. Further addition of glutamic acid is considered to be able to sustain the enhancing effect of glutamic acid and Pseudomonas bacteria for controlling soybean stalk epidemic, and is expected to be able to more effectively control soybean stalk epidemic. Further application of glutamic acid can be performed once or more, and may be performed twice or more, three times or more, four times or more, or five times or more. Further addition of glutamic acid is typically 1 to 20 times, preferably 2 to 10 times, more preferably 3 to 8 times.
  • the interval at which glutamic acid is further applied is not particularly limited, but may be, for example, 6 hours or more, 12 hours or more, 1 day or more, 2 days or more, 3 days or more, or 4 days or more, and 10 It may be less than a day, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, or less than 12 hours.
  • the interval at which glutamic acid is further applied is typically 6 hours to 10 days, preferably 12 hours to 7 days, and more preferably 1 to 3 days.
  • the form and means for further adding glutamic acid are not particularly limited, and the same contents as described above can be used. Further, the amount of glutamic acid to be further added is not particularly limited, and the same can be applied as described above.
  • Stem plague in soybean is usually caused by fungi of the genus Phytophthora.
  • the genus Oomycete is a fungus belonging to the class Oomycete among fungi, and is known to be a soil fungus that grows in plant tissues after infection.
  • a typical example of Phytophthora spp. That causes stalk epidemics in soybean is Phytophthora sojae (more specifically, Phytophthora sojae N1).
  • composition for Controlling Stem Epidemic in Soybean is a composition for controlling stem epidemic in soybean containing glutamic acid.
  • the composition of the present invention may further include Pseudomonas bacteria.
  • the glutamic acid and Pseudomonas bacteria used in the composition of the present invention are as described above.
  • the amount of glutamic acid contained in the composition of the present invention can be appropriately set according to the type of dosage form and the like.
  • the content of glutamic acid in the composition of the present invention is, for example, 0.01 mM to 1 M, preferably 0.1 mM to 100 mM per 100 g of the composition, but is not particularly limited.
  • the amount of Pseudomonas bacteria contained in the composition of the present invention can be appropriately set according to the bacterial species, the properties of the bacteria (drought resistance, etc.), the type of dosage form, and the like.
  • the content of Pseudomonas bacteria in the composition of the present invention is, for example, 10 4 to 10 20 CFU, preferably 10 6 to 10 12 CFU per 100 g of the composition, but is not particularly limited.
  • the method of adding Pseudomonas bacteria to the composition of the present invention is not particularly limited, and may be added as it is.
  • bacteria of the genus Pseudomonas are cultivated by a method known to those skilled in the art, and if it is a liquid medium, it is recovered by centrifugation or the like, and if it is a solid medium, the formed colonies are recovered by a loop loop or the like to obtain the composition of the present invention.
  • the bacterium of the genus Pseudomonas may be added to the composition of the present invention as a solid by freeze-drying the product while it is stored in a liquid by a method known per se.
  • the composition of the present invention may contain additives such as excipients, thickeners, binders, stabilizers, preservatives, pH adjusters, colorants, and flavoring agents.
  • additives such as excipients, thickeners, binders, stabilizers, preservatives, pH adjusters, colorants, and flavoring agents.
  • the various additives are not particularly limited, but materials known in the technical field of biopesticide can be used, and the blending amount thereof can be appropriately adjusted based on the known techniques of those skilled in the art.
  • the form of the composition of the present invention may be any form such as liquid, solid, gel, paste, etc., and can be appropriately set according to the usage situation and the like.
  • the composition of the present invention may be in the form of a concentrate.
  • the concentration ratio is not particularly limited, and can be, for example, 2 to 1000 times, 5 to 100 times, or 10 to 50 times.
  • the composition of the present invention can be appropriately diluted with a solvent such as water, and the diluted product can be applied to soybean or soil containing soybean.
  • glutamic acid and Pseudomonas bacteria their content in the composition of the present invention can be set according to the concentration ratio.
  • the composition of the present invention may be configured as a single preparation containing glutamic acid and Pseudomonas bacteria, or two preparations containing glutamic acid and Pseudomonas bacteria separately. It may be configured as.
  • the composition of the present invention is composed of two preparations, it is preferable that the two preparations are used in combination (that is, they are concomitant agents).
  • the composition of the present invention is composed of two formulations, the two formulations may be in different types of dosage forms or may be in the same type of dosage form.
  • composition of the present invention is used for controlling stalk epidemics in soybean.
  • the composition of the present invention can also be used as a biopesticide or a microbial pesticide. Therefore, when Pseudomonas bacteria are used, the composition of the present invention can also be referred to as a pesticide composition.
  • Kit for controlling stem epidemic in soybean One aspect of the present invention is a kit for controlling stem epidemic in soybean containing glutamate.
  • the kit of the present invention may further include Pseudomonas bacteria.
  • the glutamic acid and Pseudomonas bacteria used in the kit of the present invention are as described above.
  • glutamic acid and Pseudomonas bacteria their forms may be reagents or preparations, and are not particularly limited.
  • glutamic acid and Pseudomonas bacteria may be contained in the same preparation, or may be separately contained in separate preparations.
  • glutamic acid and Pseudomonas bacteria are separated into separate formulations, the formulations may be in different dosage forms or in the same dosage form.
  • the form of the pharmaceutical product used in the kit of the present invention is not particularly limited, and may be any form such as liquid, solid, gel, and paste.
  • the contents of glutamic acid and Pseudomonas bacteria in the preparation are not particularly limited, and can be arbitrarily set according to the composition of the present invention described above.
  • the formulation used in the kit of the present invention may be individually packaged in a single-use amount for single use, or may be used a plurality of times (for example, 2 times, 3 times, 4 times, 5 times, 10 times, etc.). Or more) may be packaged in a form containing the usage amount.
  • the container to be used is not particularly limited, and can be appropriately set according to the amount of the pharmaceutical product used and the like.
  • kits of the present invention When glutamic acid and Pseudomonas bacteria are separated into separate reagents or formulations in the kit of the present invention, they do not have to be packaged in the same package, but are packaged separately and used together at the time of use. It may be.
  • the kit of the present invention may also include an instruction manual for the use of glutamic acid and Pseudomonas bacteria.
  • the kit of the present invention is used for controlling stalk epidemics in soybean.
  • the kit of the present invention can also be used as a biopesticide or a microbial pesticide.
  • Test plant Soybean seed (variety: Enrei) Infectious bacteria of soybean stem epidemic (Phytophthora genus): Phytophthora sojae N1 Pseudomonas bacteria: Pseudomonas protegens Cab57 Soil: Raising seedling cultivation soil (plantation Iwamoto)
  • V8 medium (Medium composition) V8 Vegetable Juice Original (Campbell) 40mL CaCO 3 0.6g 315mL respect supernatant 35mL of H 2 O V8 vegetable juice and mixtures CaCO 3 Agar, Powder (Wako) 4.36g (Preparation method) 1) 40 mL of V8 vegetable juice Original and 0.6 g of CaCO 3 were put into a 100 mL beaker, and the mixture was stirred with a stirrer for 5 to 10 minutes. 2) The solution obtained in 1) was transferred to a 50 mL Falcon tube and centrifuged (3,000 rpm, 3 minutes).
  • Nutrient Agar (NA) medium Blood agar base (Oxoid) 40g Yeast extract (Oxoid) 5g H 2 O 1000mL (After preparing the medium, autoclave was performed and dispensed into a petri dish.)
  • Nutrient yeast bouillon (NYB) medium Nutrient bouillon (Oxoid) 25g Yeast extract (Oxoid) 5g H 2 O 1000mL (After preparing the medium, autoclaving was performed.)
  • the seedling raising soil to be used was sterilized by an autoclave, and the genus Fightoftra was cultivated in V8 medium.
  • the genus Fightoftra was cultivated in V8 medium.
  • the liquid was prepared.
  • the soybean seeds to be tested were placed in a moisturizing box and stored at 4 ° C. for 24 hours in the dark.
  • the V8 medium in which the genus Fightoftra was cultured was passed through a 10 mL syringe (with an injection needle (18 G)) and crushed into small pieces, and the culture medium was added to the seedling culture soil and mixed thoroughly.
  • the amount of the Fightoftra genus culture V8 medium added was 4.63 g per 1 L of the nursery soil.
  • 180 mL of the seedling raising soil was evenly poured into a seedling raising tray (6 cm square, 5.5 cm deep) having a hole at the bottom and allowing water to permeate. Then, five soybean seeds were sown on the nursery soil with the hypocotyl part facing down.
  • test plots were set: (a) water only, (b) Pseudomonas bacteria only, (c) glutamic acid only, and (d) glutamic acid and Pseudomonas bacteria.
  • the specific procedure is as follows.
  • the seedling raising tray was covered with plastic wrap and left in the plant incubator. Soybeans were cultivated at 25 ° C. for 2 weeks at 16 hours in the light period and 8 hours in the dark period. Water in the vat or a 10 mM glutamic acid aqueous solution was replenished every 5 days during the cultivation period. The amount of water to be replenished or the 10 mM glutamic acid aqueous solution was set to 50 mL per section of the seedling raising tray in the same manner as described above. If water or a 10 mM glutamic acid aqueous solution remained in the vat at the time of replenishment, it was discarded and a new water or 10 mM glutamic acid aqueous solution was replenished.
  • the weight of the soybean plant after cultivation is as shown in FIG. 1, and it was found that the control effect of soybean stem epidemic can be obtained by adding glutamic acid, and the effect by using the Pseudomonas bacterium in combination. It became clear that
  • the number of surviving soybeans during the cultivation period is as shown in Fig. 2. As of 13 days after the start of cultivation, the number of surviving soybeans was 7 in the test plots to which only water was given and the test plots to which only Pseudomonas bacteria were given, whereas the number of soybeans alive in the test plots to which only glutamic acid was given. The number was 9, and the number of surviving soybeans was 10 in the test plots fed with glutamate and Pseudomonas bacteria. From this result, it was shown that the control effect of soybean stem epidemic was obtained by the addition of glutamic acid, and the effect was enhanced by the combined use of Pseudomonas bacteria.
  • the technique provided by the present invention is useful in the agricultural field related to soybean production.
  • the Pseudomonas bacterium and glutamic acid provided by the present invention can be used in the field of pesticides, particularly in the field of biopesticides.

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Abstract

The purpose of the present invention is to provide a method that enables effective control of soybean phytophthora rot without using non-natural chemical pesticides, and a composition and a kit.　A method for controlling soybean phytophthora rot, wherein a step for applying glutamic acid to soybeans or soil containing soybeans is carried out. Also provided are a glutamic acid-containing composition and a glutamic acid-containing kit for controlling soybean phytophthora rot.

Description

ダイズの土壌伝染性病害防除方法Soybean soil infectious disease control method
　本発明は、ダイズを土壌伝染性病害から防除する方法に関する。より具体的には、本発明は、グルタミン酸を利用したダイズ茎疫病の防除方法、並びに当該方法に利用可能な組成物及びキットに関する。 The present invention relates to a method for controlling soybean from a soil-borne disease. More specifically, the present invention relates to a method for controlling soybean stalk epidemic using glutamic acid, and compositions and kits that can be used in the method.
　ダイズは、日本でも生産されている重要な食用作物の一つである。近年では、ダイズは水田の転換作物として特に重要な品目となっており、日本国内で生産されるダイズの約８０％は水田転換畑で栽培されていると言われている。しかし、水田転換畑は排水性が悪く、降雨が続く場合には、水田転換畑で栽培されるダイズは湿害や土壌病害による被害を受けやすい。特に、停滞水を好むダイズ茎疫病は日本各地で発生が増加傾向にあり、ダイズの土壌病害として大きな問題となっている。 Soybean is one of the important food crops produced in Japan. In recent years, soybean has become a particularly important item as a conversion crop in paddy fields, and it is said that about 80% of soybeans produced in Japan are cultivated in paddy field conversion fields. However, the paddy field conversion field has poor drainage, and if rainfall continues, soybeans cultivated in the paddy field conversion field are vulnerable to moisture damage and soil diseases. In particular, soybean stalk epidemics, which prefer stagnant water, are increasing in number in various parts of Japan, and have become a major problem as soil diseases of soybeans.
　ダイズ茎疫病は、卵菌類に属するＰｈｙｔｏｐｈｔｈｏｒａ属の細菌によって引き起こされる病害であり、ダイズの生育全般を通じて発生する。茎疫病に罹ったダイズは、茎の地際部或いはそれよりも上位の主茎や分枝に水浸状の条斑又は楕円形の病変部を生じ、感染後１週間程度で萎凋し、最終的に枯死する。ダイズ茎疫病の感染菌は卵胞子の状態で長期間土壌に残存することができるため、茎疫病の被害は長期にわたることが特徴的である。また、土壌に残存する茎疫病の感染菌は、さらなる伝染源ともなり得る。 Soybean stalk epidemic is a disease caused by bacteria of the genus Phytophthora belonging to oomycetes, and occurs throughout the growth of soybeans. Soybeans affected by stalk plague develop water-soaked streaks or oval lesions on the main stems and branches above the stalks, and wither about one week after infection, and finally. Wither. Since the infectious bacteria of soybean stem epidemic can remain in the soil in the state of follicles for a long period of time, the damage of the stem epidemic is characteristically long-term. In addition, the infectious bacteria of stem epidemic that remain in the soil can be a further source of transmission.
　現状では、ダイズ茎疫病の防除方法に関する技術は極めて限定的である。ダイズ茎疫病を防除するために高畦栽培や排水対策などの処置がとられることがあるが、必ずしもその効果は高いとはいえず、労力の面からも容易に行うことはできない。また、ダイズ茎疫病の防除用の化学農薬として、シアゾファミド剤やアミスルブロム剤などを成分とする薬剤が登録されている。しかしながらダイズ茎疫病に関しては、既存の化学農薬ではあまり効果が見られないこともあり、さらに非天然の化学農薬は、環境への負荷が高かったり、残留農薬や農薬曝露によって植物や人体に悪影響を及ぼしたりする可能性があることも懸念されている。 Currently, the technology for controlling soybean stalk epidemics is extremely limited. Measures such as high ridge cultivation and drainage measures may be taken to control soybean stalk epidemics, but the effects are not always high and cannot be easily carried out from the viewpoint of labor. In addition, as a chemical pesticide for controlling soybean stalk epidemic, a drug containing a siazophamide agent, an amisulbrom agent, or the like is registered. However, with regard to soybean stalk epidemics, existing chemical pesticides may not be very effective, and non-natural chemical pesticides have a high impact on the environment and adversely affect plants and human bodies due to residual pesticides and pesticide exposure. There is also concern that it may have an effect.
　このような観点から近年では、有効性が高いだけではなく、安全性も高く、且つ低環境負荷型のダイズ茎疫病の防除技術が強く求められており、その一例として、Ｔｒｉｃｈｏｄｅｒｍａ属の細菌を利用したダイズ茎疫病の防除技術が報告されている（特許文献１）。このように生物農薬として細菌を利用することは最近では特に注目されており、Ｐｓｅｕｄｏｍｏｎａｓ属の細菌を利用して、Ｐｙｔｈｉｕｍ属細菌やＦｕｓａｒｉｕｍ属細菌を病原菌とする土壌病害に対して防除効果が見られたという結果もこれまでに報告されている（特許文献２）。 From this point of view, in recent years, there has been a strong demand for technology for controlling soybean stalk epidemics that is not only highly effective but also highly safe and has a low environmental load, and as an example, Trichoderma bacteria are used. A technique for controlling soybean stalk epidemic has been reported (Patent Document 1). In recent years, the use of bacteria as biological pesticides has attracted particular attention, and the use of bacteria of the genus Pseudomonas has been shown to be effective in controlling soil diseases caused by bacteria of the genus Pythium and bacteria of the genus Fusarium. The result has been reported so far (Patent Document 2).
特開２００８－１３７９４６号公報Japanese Unexamined Patent Publication No. 2008-137946 国際公開第２０１９／１９８７３５号International Publication No. 2019/198735
　上述したように、ダイズにおける茎疫病の防除技術は限定的であり、高い有効性と安全性とを兼ね備え、且つ低環境負荷型のダイズ茎疫病の防除技術が強く求められている。そこで、本発明は、非天然の化学農薬を用いることなく効果的にダイズにおける茎疫病を防除することのできる方法、組成物及びキットを提供することを目的とする。 As described above, the technology for controlling soybean stem epidemic is limited, and there is a strong demand for a technology for controlling soybean stem epidemic that has both high effectiveness and safety and has a low environmental load. Therefore, an object of the present invention is to provide a method, composition and kit capable of effectively controlling stalk epidemics in soybean without using non-natural chemical pesticides.
課題を解決しようとする手段Means to solve the problem
　本発明者は、上記の課題に鑑みて鋭意検討した結果、天然化合物としてアミノ酸の利用に着目し、その中から特にグルタミン酸がダイズ茎疫病の防除に有効であることを見出した。また、かかる防除効果は、グルタミン酸にシュードモナス属細菌を併用することによりさらに高められることを本発明者は見出した。これらの知見に基づき、本発明者は、本発明を完成するに至った。 As a result of diligent studies in view of the above problems, the present inventor focused on the use of amino acids as natural compounds, and found that glutamic acid is particularly effective in controlling soybean stalk epidemics. In addition, the present inventor has found that such a control effect can be further enhanced by using glutamic acid in combination with Pseudomonas bacteria. Based on these findings, the present inventor has completed the present invention.
　本発明は、好ましくは以下に記載するような態様により行われるが、これに限定されるものではない。
［態様１］グルタミン酸をダイズ又はダイズを含む土に付与する工程を含む、ダイズにおける茎疫病の防除方法。
［態様２］シュードモナス属細菌をダイズ又はダイズを含む土に付与する工程をさらに含む、態様１に記載の方法。
［態様３］ダイズが種子又は幼苗の状態である、態様１又は２に記載の方法。
［態様４］ダイズの播種前又は播種後３日以内にグルタミン酸を付与する、態様１～３のいずれか１に記載の方法。
［態様５］ダイズの播種前又は播種後３日以内にシュードモナス属細菌を付与する、態様２～４のいずれか１に記載の方法。
［態様６］グルタミン酸及びシュードモナス属細菌を同時に付与する、態様２～５のいずれか１に記載の方法。
［態様７］グルタミン酸及びシュードモナス属細菌を付与した後でさらにグルタミン酸を付与する工程を含む、態様２～６のいずれか１に記載の方法。
［態様８］グルタミン酸を含む、ダイズにおける茎疫病の防除用組成物。
［態様９］シュードモナス属細菌をさらに含む、態様８に記載の組成物。
［態様１０］グルタミン酸を含む、ダイズにおける茎疫病の防除用キット。
［態様１１］シュードモナス属細菌をさらに含む、態様１０に記載のキット。 The present invention is preferably performed in the manner described below, but is not limited thereto.
[Aspect 1] A method for controlling stalk epidemics in soybean, which comprises a step of applying glutamic acid to soybean or soil containing soybean.
[Aspect 2] The method according to Aspect 1, further comprising a step of applying Pseudomonas bacteria to soybean or soil containing soybean.
[Aspect 3] The method according to aspect 1 or 2, wherein the soybean is in the state of seeds or seedlings.
[Aspect 4] The method according to any one of aspects 1 to 3, wherein glutamic acid is applied before sowing of soybean or within 3 days after sowing.
[Aspect 5] The method according to any one of aspects 2 to 4, wherein the Pseudomonas bacterium is applied before sowing of soybean or within 3 days after sowing.
[Aspect 6] The method according to any one of aspects 2 to 5, wherein glutamic acid and Pseudomonas bacteria are simultaneously applied.
[Aspect 7] The method according to any one of aspects 2 to 6, comprising a step of adding glutamic acid and a bacterium of the genus Pseudomonas and then further adding glutamic acid.
[Aspect 8] A composition for controlling stem epidemic in soybean, which comprises glutamic acid.
[Aspect 9] The composition according to Aspect 8, further comprising a Pseudomonas bacterium.
[Aspect 10] A kit for controlling stem epidemic in soybean, which comprises glutamic acid.
[Aspect 11] The kit according to aspect 10, further comprising a Pseudomonas bacterium.
　本発明を利用することによって、非天然の化学農薬を使用することなく効果的にダイズにおける茎疫病を防除することができる。本発明において用いられるグルタミン酸は、天然アミノ酸のうちの一種であり、一般的な非天然の化学農薬よりも高い安全性を有するものと考えられる。また、本発明において使用されるシュードモナス属細菌も、生物農薬としての使用実績があり、グルタミン酸と同様に安全性は高いことが考えられる。また、本発明では化学物質１成分、或いはこれと１種類の細菌とを組み合わせた合計２成分のみでダイズ茎疫病の防除効果が得られることから、本発明の技術は簡便に利用することができる。 By using the present invention, it is possible to effectively control stalk epidemics in soybean without using unnatural chemical pesticides. Glutamic acid used in the present invention is one of the natural amino acids, and is considered to have higher safety than general non-natural chemical pesticides. In addition, the Pseudomonas bacterium used in the present invention has a track record of use as a biopesticide, and is considered to be as safe as glutamic acid. Further, in the present invention, the technique of the present invention can be easily used because the control effect of soybean stalk epidemic can be obtained only by one component of a chemical substance or a total of two components in which this is combined with one type of bacterium. ..
図１は、２週間の栽培後におけるダイズ植物体の重量を示すグラフである。FIG. 1 is a graph showing the weight of soybean plants after cultivation for 2 weeks. 図２は、栽培期間中のダイズの生存数を示すグラフである。グラフの横軸は栽培期間（日）を表し、グラフの縦軸はダイズの生存数（個）を表す。FIG. 2 is a graph showing the number of surviving soybeans during the cultivation period. The horizontal axis of the graph represents the cultivation period (days), and the vertical axis of the graph represents the number of surviving soybeans (pieces).
　以下に、本発明について詳述するが、本発明はこれらに限定されるものではない。本明細書で特段に定義されない限り、本発明に関連して用いられる科学用語及び技術用語は、当業者によって一般に理解される意味を有するものである。 The present invention will be described in detail below, but the present invention is not limited thereto. Unless otherwise defined herein, scientific and technical terms used in the context of the present invention have meanings commonly understood by those skilled in the art.
　（１）ダイズにおける茎疫病の防除方法
　本発明の一態様は、グルタミン酸をダイズ又はダイズを含む土に付与する工程を含む、ダイズにおける茎疫病の防除方法である。本明細書においてダイズとは、マメ目マメ科ダイズ属の植物の一種であり、学名でＧｌｙｃｉｎｅ　ｍａｘと称される植物である。 (1) Method for controlling stem epidemic in soybean One aspect of the present invention is a method for controlling stem epidemic in soybean, which comprises a step of applying glutamic acid to soybean or soil containing soybean. As used herein, soybean is a type of plant belonging to the genus Soybean of the family Fabales, and is a plant referred to by the scientific name as Glycine max.
　グルタミン酸は、アミノ酸の一種であり、化学式ＨＯＯＣ（ＣＨ２）２ＣＨ（ＮＨ２）ＣＯＯＨ（又はＣ５Ｈ９ＮＯ４）で表される化合物である。また、グルタミン酸は、親水性アミノ酸、極性アミノ酸、極性電荷アミノ酸（極性負電荷アミノ酸）、又は酸性アミノ酸に分類され、Ｇｌｕ又はＥの略号で表されることもある。グルタミン酸は、植物及び動物に含まれており、具体的には海藻、小麦粉、大豆、サトウキビ等に含まれることが知られている。 Glutamic acid is a kind of amino acid and is a compound represented by the chemical formula HOOC (CH 2 ) 2 CH (NH 2 ) COOH (or C 5 H 9 NO 4). In addition, glutamate is classified into hydrophilic amino acids, polar amino acids, polar charged amino acids (polar negative charged amino acids), and acidic amino acids, and may be represented by the abbreviation Glu or E. Glutamic acid is contained in plants and animals, and is specifically known to be contained in seaweed, wheat flour, soybeans, sugar cane and the like.
　本発明においてグルタミン酸は、Ｄ体（Ｄ－グルタミン酸）、Ｌ体（Ｌ－グルタミン酸）、及びＤＬ体（ＤＬ－グルタミン酸）のいずれもが使用可能であり、特に限定されない。本発明では、Ｌ体のグルタミン酸（Ｌ－グルタミン酸）が好ましい。なお、Ｄ体のグルタミン酸のＣＡＳ番号は６８９３－２６－１であり、Ｌ体のグルタミン酸のＣＡＳ番号は５６－８６－０である。 In the present invention, glutamic acid can be any of D-form (D-glutamic acid), L-form (L-glutamic acid), and DL-form (DL-glutamic acid), and is not particularly limited. In the present invention, L-form glutamic acid (L-glutamic acid) is preferable. The CAS number of D-form glutamic acid is 6893-26-1, and the CAS number of L-form glutamic acid is 56-86-0.
　グルタミン酸について、その入手方法は特に限定されず、動物や植物から単離精製された天然のもの、或いは化学合成法や発酵法等により得られたもののいずれであってもよい。グルタミン酸は、自ら精製したものを使用してもよいし、或いは市販品を使用してもよく、その使用に関しては特に限定されない。本発明では、好ましくは市販されているグルタミン酸が用いられる。 The method for obtaining glutamic acid is not particularly limited, and it may be either a natural glutamic acid isolated and purified from animals or plants, or a glutamic acid obtained by a chemical synthesis method, a fermentation method, or the like. As the glutamic acid, a self-purified product may be used, or a commercially available product may be used, and the use thereof is not particularly limited. In the present invention, commercially available glutamic acid is preferably used.
　本発明の方法ではまた、シュードモナス属細菌をダイズ又はダイズを含む土に付与する工程がさらに含まれていてもよい。シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属細菌は、プロテオバクテリア門ガンマプロテオバクテリア綱シュードモナス科に属する細菌であり、グラム陰性好気性桿菌であることを特徴とする。シュードモナス属細菌の生育域は幅広く、土壌、淡水、海水、植物、又は動物に生息することが知られている。 The method of the present invention may also further include a step of imparting Pseudomonas bacteria to soybean or soil containing soybean. The bacterium of the genus Pseudomonas is a bacterium belonging to the family Pseudomonas of the phylum Proteobacteria, Gammaproteobacteria, and is characterized by being a gram-negative aerobic rod. Pseudomonas bacteria have a wide habitat and are known to inhabit soil, freshwater, seawater, plants, or animals.
　本発明におけるシュードモナス属細菌は特に限定されないが、その具体例としては、シュードモナス　プロテゲンス（Ｐｓｅｕｄｏｍｏｎａｓ　ｐｒｏｔｅｇｅｎｓ）、シュードモナス　フルオレッセンス（Ｐｓｅｕｄｏｍｏｎａｓ　ｆｌｕｏｒｅｓｃｅｎｓ）、シュードモナス　シリンゲ（Ｐｓｅｕｄｏｍｏｎａｓ　ｓｙｒｉｎｇａｅ）、シュードモナス　クロロラフィス（Ｐｓｅｕｄｏｍｏｎａｓ　ｃｈｌｏｒｏｒａｐｈｉｓ）、シュードモナス　シンキサンタ（Ｐｓｅｕｄｏｍｏｎａｓ　ｓｙｎｘａｎｔｈａ）、シュードモナス　ブラシカセアルム（Ｐｓｅｕｄｏｍｏｎａｓ　ｂｒａｓｓｉｃａｃｅａｒｕｍ）、シュードモナス　プチダ（Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ）、及びシュードモナス　ロデシア（Ｐｓｅｕｄｏｍｏｎａｓｒｈｏｄｅｓｉａｅ）等が挙げられる。また、本発明では、シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）ｓｐ．　Ｏｓ１７株、及びシュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）ｓｐ．　Ｓｔ２９株も利用することができ、これらの細菌は独立行政法人製品評価技術基盤機構（Ｎａｔｉｏｎａｌ　Ｉｎｓｔｉｔｕｔｅ　ｏｆ　Ｔｅｃｈｎｏｌｏｇｙ　ａｎｄ　Ｅｖａｌｕａｔｉｏｎ（ＮＩＴＥ））　バイオテクノロジーセンター　特許微生物寄託センター（ＮＰＭＤ）（日本国千葉県木更津市かずさ鎌足２－５－８）において、それぞれ受託番号ＮＩＴＥ　Ｐ－０２０５３（受託日：２０１５年５月２０日）及び受託番号ＮＩＴＥ　Ｐ－０２０５４（受託日：２０１５年５月２０日）として寄託されている。好ましくは、シュードモナス　プロテゲンス、シュードモナスｓｐ．　Ｏｓ１７株、及びシュードモナスｓｐ．　Ｓｔ２９株が本発明において使用される。シュードモナス　プロテゲンスの細菌株のうち、好ましくはシュードモナス　プロテゲンスＣＨＡ０株、及びシュードモナス　プロテゲンスＣａｂ５７株が用いられるが、特にこれらに限定されない。 The Pseudomonas bacterium in the present invention is not particularly limited, and specific examples thereof include Pseudomonas protegens, Pseudomonas fluorescens, Pseudomonas syringe, Pseudomonas pseudomonas. Santa (Pseudomonas synxantha), Pseudomonas brushcasearum (Pseudomonas brassicareum), Pseudomonas putida, and Pseudomonas rodesia (Pseudomonas). Further, in the present invention, Pseudomonas sp. Os17 strain and Pseudomonas sp. St29 strains can also be used, and these bacteria can be used by the National Institute of Technology and Evolution (NITE) Biotechnology Center Patent Microorganisms Depositary Center (NPMD) (Kazusaka, Kisarazu City, Chiba Prefecture, Japan). At Kazusakamatari 2-5-8), deposits were made under the accession number NITE P-02053 (consignment date: May 20, 2015) and the entrustment number NITE P-02054 (consignment date: May 20, 2015), respectively. There is. Preferably, Pseudomonas protegens, Pseudomonas sp. Os17 strain and Pseudomonas sp. The St29 strain is used in the present invention. Among the bacterial strains of Pseudomonas protegens, the Pseudomonas protegens CHA0 strain and the Pseudomonas protegens Cab57 strain are preferably used, but are not particularly limited thereto.
　シュードモナス属細菌は、自ら単離してきたものを使用してもよく、施設から分譲されたものを使用してもよく、或いは生物農薬製品として市場で利用可能なシュードモナス属細菌（即ち、市販品）を使用してもよく、その使用に関しては特に限定されない。 Pseudomonas bacteria may be those isolated by themselves, those sold from the facility, or Pseudomonas bacteria (ie, commercial products) available on the market as biopesticide products. May be used, and the use thereof is not particularly limited.
　本発明の方法において、グルタミン酸及びシュードモナス属細菌はいずれも、ダイズ又はダイズを含む土に付与される。ここで、本明細書において「土」とは、ダイズが生育可能な土壌を意味する。本発明で用いられる土は、例えば、培土、培養土、育苗土、又は育苗培土等の名称で示されるものであってよい。また、何らの処理も行われていない山野の土をそのまま用いてもよい。土の粒度も特に限定されず、ダイズが生育可能である限りいかなる土であってもよい。本発明では、バーミキュライト等の人工の土壌改良用の土を用いてもよいが、種々の土の中でも育苗培土を用いることが特に好ましい。育苗培土は、栽培のベースとなる肥料を含む土であり、床土に分類される。本発明で使用される育苗培土には、窒素、リン酸、及びカリウムが含まれていてよく、さらに有機酸が含まれていることが好ましい。本発明で使用される育苗培土には、酸化ケイ素、酸化マグネシウム、酸化アルミニウムなどの鉱物が多量に含まれていないことが好ましい。また、本発明で使用される育苗培土は、８００℃以上の温度で熱処理されていない育苗培土であることが好ましい。 In the method of the present invention, both glutamic acid and Pseudomonas bacteria are applied to soybean or soil containing soybean. Here, the term "soil" as used herein means soil in which soybeans can grow. The soil used in the present invention may be, for example, a soil represented by a name such as hilling soil, potting soil, seedling raising soil, or seedling raising soil. In addition, the soil of the mountains that has not been treated in any way may be used as it is. The particle size of the soil is not particularly limited, and any soil may be used as long as soybean can grow. In the present invention, artificial soil for soil improvement such as vermiculite may be used, but it is particularly preferable to use seedling raising soil among various soils. Seedling cultivation soil is soil containing fertilizer that is the basis of cultivation, and is classified as bed soil. The seedling raising soil used in the present invention may contain nitrogen, phosphoric acid, and potassium, and preferably contains an organic acid. It is preferable that the seedling raising soil used in the present invention does not contain a large amount of minerals such as silicon oxide, magnesium oxide and aluminum oxide. Further, the seedling raising soil used in the present invention is preferably a seedling raising soil that has not been heat-treated at a temperature of 800 ° C. or higher.
　本発明において、ダイズを含む土とは、ダイズが土の中、或いは土の上に存在する状態の土であることを意味する。ダイズが土の中に存在する場合、当該ダイズはその全体が土に埋入されていてもよく、或いはダイズの一部が土に埋入され、残りの部分が土の外に出ている状態であってもよい。本発明において付与対象とされる土は、ダイズの近辺の土（例えば、ダイズから１０ｃｍ以内の範囲にある土）であることが好ましい。 In the present invention, the soil containing soybean means that the soil is in a state where soybean exists in or on the soil. If the soybean is present in the soil, the whole soybean may be buried in the soil, or a part of the soybean is buried in the soil and the rest is out of the soil. It may be. The soil to be imparted in the present invention is preferably soil in the vicinity of soybean (for example, soil within a range of 10 cm from soybean).
　本発明において付与対象とされるダイズの状態は特に限定されず、種子若しくは幼苗、又は既にダイズが生育した状態であってよいが、好ましくは種子又は幼苗の状態であり、より好ましくは種子の状態である。種子や幼苗といった生育初期の段階であることにより、茎疫病の罹りはじめを抑制することができ、より効果的にダイズにおける茎疫病の防除を行うことができる。また、ダイズの組織が幼若であることにより、グルタミン酸及びシュードモナス属細菌はダイズの内部に浸透しやすくなり、その点でも茎疫病の防除をより効果的に行うことができる。本発明において付与対象とされるダイズは、発根した種子の状態であってもよく、発根していない種子の状態であってもよいが、発根していない種子の状態であることが好ましい。なお、既にダイズが生育した状態である場合は、ダイズの根、葉、主茎、又は分枝等のいずれの部位に対してもグルタミン酸及びシュードモナス属細菌の付与を行うことができる。 The state of soybean to be imparted in the present invention is not particularly limited and may be a state in which seeds or seedlings or soybeans have already grown, but is preferably in the state of seeds or seedlings, and more preferably in the state of seeds. Is. By being in the early stage of growth such as seeds and seedlings, it is possible to suppress the onset of stem epidemic and more effectively control stem epidemic in soybean. In addition, since the tissue of soybean is young, glutamic acid and Pseudomonas bacteria easily permeate the inside of soybean, and in that respect, the control of stem epidemic can be performed more effectively. The soybean to be imparted in the present invention may be in the state of rooted seeds or in the state of non-rooted seeds, but may be in the state of non-rooted seeds. preferable. When soybean is already in a grown state, glutamic acid and Pseudomonas bacteria can be applied to any part of the root, leaf, main stem, branch, etc. of soybean.
　上述したグルタミン酸及びシュードモナス属細菌はダイズ又はダイズを含む土に対して付与されるが、その付与に関する方法及び手段は、本発明の効果が得られる限りにおいて特に限定されない。例えば、グルタミン酸及びシュードモナス属細菌を共に、又は別々に、懸濁又は溶解した水（水溶液）を準備して、その準備した水（水溶液）をダイズ又はダイズを含む土に接触させることにより付与操作を行うことができる。 The above-mentioned glutamic acid and Pseudomonas bacteria are applied to soybean or soil containing soybean, but the method and means for the application are not particularly limited as long as the effects of the present invention can be obtained. For example, glutamic acid and Pseudomonas bacteria are suspended or dissolved together or separately, and water (aqueous solution) is prepared, and the prepared water (aqueous solution) is brought into contact with soybean or soil containing soybean to carry out the donation operation. It can be carried out.
　グルタミン酸及びシュードモナス属細菌の形態は、液体であってもよいし、固体であってもよい。当該形態が液体である場合は、吹き付け、滴下、又は浸漬等の操作によって、グルタミン酸及びシュードモナス属細菌をダイズ又はダイズを含む土に付与することができる。浸漬操作の場合には、容器を別途準備して、当該液体が入った容器の中に、ダイズ又はダイズを含む土が入った穴あき容器を入れて作業を行ってもよい。グルタミン酸及びシュードモナス属細菌の形態が固体である場合は、土の上又は土の中に当該固体を置くことによって付与してもよいし、或いはダイズの表面に当該固体を接触させることによって付与してもよい。 The morphology of glutamic acid and Pseudomonas bacteria may be liquid or solid. When the form is a liquid, glutamic acid and Pseudomonas bacteria can be applied to soybean or soil containing soybean by an operation such as spraying, dropping, or dipping. In the case of the dipping operation, a container may be prepared separately, and a perforated container containing soybean or soil containing soybean may be placed in the container containing the liquid to perform the work. If the morphology of glutamic acid and Pseudomonas is solid, it may be imparted by placing the solid on or in the soil, or by contacting the solid with the surface of soybean. May be good.
　ダイズ又はダイズを含む土に付与するグルタミン酸及びシュードモナス属細菌の量は、特に限定されず、付与形態等に応じて適宜設定することができる。例えば、ダイズに対してグルタミン酸を付与する場合は、ダイズ１個体に対して１回あたり０．０１ｍＭ～１Ｍ、好ましくは０．１ｍＭ～１００ｍＭの量で付与することができる。また、ダイズに対してシュードモナス属細菌を付与する場合は、ダイズ１個体に対して１回あたり１０４～１０２０ＣＦＵ、好ましくは１０６～１０１０ＣＦＵの細菌数で付与することができる。グルタミン酸及びシュードモナス属細菌の付与量は、これらを含む製剤中の濃度と当該製剤の付与量によって調整することができる。なお、グルタミン酸が塩である場合は、その遊離体（グルタミン酸イオン）に相当する分子量をもってグルタミン酸の量を計算することができる。 The amount of glutamic acid and Pseudomonas bacteria to be added to soybean or soil containing soybean is not particularly limited and can be appropriately set according to the form of application and the like. For example, when glutamic acid is given to soybean, it can be given in an amount of 0.01 mM to 1 M, preferably 0.1 mM to 100 mM per soybean. When Pseudomonas bacteria are given to soybean, the number of bacteria can be 10 4 to 10 20 CFU, preferably 10 6 to 10 10 CFU per soybean. The amount of glutamic acid and Pseudomonas bacteria to be added can be adjusted according to the concentration in the preparation containing these and the amount of the preparation. When glutamic acid is a salt, the amount of glutamic acid can be calculated based on the molecular weight corresponding to its free form (glutamic acid ion).
　土に対してグルタミン酸を付与する場合は、例えば、１ｃｍ３の土に対して１回あたり０．０１ｍＭ～１Ｍ、好ましくは０．１ｍＭ～１００ｍＭの量で付与することができる。また、土に対してシュードモナス属細菌を付与する場合は、１ｃｍ３の土に対して１回あたり１０４～１０２０ＣＦＵ、好ましくは１０６～１０１０ＣＦＵの細菌数で付与することができる。上記と同様に、グルタミン酸及びシュードモナス属細菌の付与量は、これらを含む製剤中の濃度と当該製剤の付与量によって調整することができる。 To grant glutamate against soil, for example, per once for soil 1 cm 3 0.01 mM ~ 1M, preferably it is applied in an amount of 0.1 mM ~ 100 mM. Also, when granting Pseudomonas bacteria against soil, 1 cm 3 of 10 4 ~ 10 20 CFU per once for soil, preferably it can be applied in the number of bacteria 10 6 ~ 10 10 CFU. Similar to the above, the amount of glutamic acid and Pseudomonas bacteria to be added can be adjusted according to the concentration in the preparation containing these and the amount of the preparation.
　グルタミン酸及びシュードモナス属細菌についてはいずれも、それらのダイズ又はダイズを含む土への付与時期は、ダイズの播種前、播種時、及び播種後（育苗期、定植期等を含む）のいずれであってもよく、特に限定されないが、好ましくはダイズの播種前又は播種後３日以内である。ここで、本明細書において「播種」とは、生育の出発点としてダイズを土壌に植えることを意味し、例えば、土壌への種まきや土壌に苗を植えること等が包含される。 For both glutamate and Pseudomonas spp., The soybeans or the soil containing the soybeans should be applied at any time before, at the time of sowing, and after sowing (including the seedling raising period, the planting period, etc.). It is also good, and is not particularly limited, but preferably within 3 days before or after sowing of soybean. Here, “sowing” in the present specification means planting soybeans in soil as a starting point for growth, and includes, for example, sowing seeds in soil and planting seedlings in soil.
　グルタミン酸及びシュードモナス属細菌について、ダイズの播種前に付与する場合、その具体的な時期は特に限定されないが、例えば、播種前７日以内、播種前６日以内、播種前５日以内、播種前４日以内、播種前３日以内、播種前２日以内、播種前１日以内、播種前１２時間以内、播種前６時間以内、播種前３時間以内、播種前１時間以内、播種前３０分以内、播種前１０分以内、播種前５分以内、播種前１分以内、又は播種前３０秒以内である。また、ダイズの播種後に付与する場合、その付与時期は、播種後７日以内、播種後６日以内、播種後５日以内、又は播種後４日以内としてもよく、或いは、播種後２日以内、播種後１日以内、播種後１２時間以内、播種後６時間以内、播種後３時間以内、播種後１時間以内、播種後３０分以内、播種後１０分以内、播種後５分以内、播種後１分以内、又は播種後３０秒以内としてもよい。上記の付与時期は、典型的には播種前後７日以内である。本発明では、グルタミン酸及びシュードモナス属細菌はいずれもダイズの生育初期段階でダイズと接触することが好ましいため、そのような観点から上記の付与時期とダイズ播種時期との間の期間は短い方が好ましく、例えば、少なくとも播種前後３日以内、或いは少なくとも播種前後１日以内に付与を行うのが好ましい。 When glutamate and Pseudomonas bacteria are given before sowing, the specific timing is not particularly limited, but for example, within 7 days before sowing, within 6 days before sowing, within 5 days before sowing, and 4 before sowing. Within days, within 3 days before sowing, within 2 days before sowing, within 1 day before sowing, within 12 hours before sowing, within 6 hours before sowing, within 3 hours before sowing, within 1 hour before sowing, within 30 minutes before sowing , Within 10 minutes before sowing, within 5 minutes before sowing, within 1 minute before sowing, or within 30 seconds before sowing. When the soybean is given after sowing, it may be given within 7 days after sowing, within 6 days after sowing, within 5 days after sowing, or within 4 days after sowing, or within 2 days after sowing. , Within 1 day after sowing, within 12 hours after sowing, within 6 hours after sowing, within 3 hours after sowing, within 1 hour after sowing, within 30 minutes after sowing, within 10 minutes after sowing, within 5 minutes after sowing, within 5 minutes after sowing It may be within 1 minute after sowing or within 30 seconds after sowing. The above-mentioned granting time is typically within 7 days before and after sowing. In the present invention, it is preferable that both glutamic acid and Pseudomonas bacteria come into contact with soybean in the early stage of soybean growth. Therefore, from such a viewpoint, it is preferable that the period between the above-mentioned feeding time and soybean sowing time is short. For example, it is preferable to apply at least within 3 days before and after sowing, or at least within 1 day before and after sowing.
　本発明の方法においては、グルタミン酸及びシュードモナス属細菌は同時に付与されてもよいし、別々に付与されてもよい。本発明の方法では、好ましくは、グルタミン酸及びシュードモナス属細菌は同時に付与される。両成分が同時に付与されるといっても、ある程度の時間間隔は許容される。その場合の時間間隔としては、例えば、１０分以内、５分以内、３分以内、１分以内、又は３０秒以内である。 In the method of the present invention, glutamic acid and Pseudomonas bacteria may be added simultaneously or separately. In the method of the present invention, preferably, glutamic acid and Pseudomonas bacteria are simultaneously applied. Even if both components are applied at the same time, some time interval is allowed. In that case, the time interval is, for example, 10 minutes or less, 5 minutes or less, 3 minutes or less, 1 minute or less, or 30 seconds or less.
　また、本発明の方法では、特に限定されないが、グルタミン酸及びシュードモナス属細菌のうちいずれか一方を付与した後でその３日以内に他方を付与することができる。双方の付与時期の間隔は、２日以内、１日以内、１２時間以内、６時間以内、３時間以内、又は１時間以内であってもよい。このようにグルタミン酸及びシュードモナス属細菌を別々に付与する場合、双方の付与時期の間隔は、例えば３０分以上、１時間以上、６時間以上、又は１２時間以上とすることができる。本発明においてシュードモナス属細菌は、グルタミン酸のダイズ茎疫病防除作用を増強させる働きを有していると考えられることから、双方の付与時期の間隔は長くならない方が好ましい。そのような観点から、グルタミン酸とシュードモナス属細菌とを付与する場合の双方の付与時期の間隔は、少なくとも１日以内であることが好ましい。なお、上述したダイズの播種に対するグルタミン酸及びシュードモナス属細菌の付与時期は、いずれか一方が付与される時期として適用され、他方は当該付与後３日以内に付与することができる。 Further, in the method of the present invention, although not particularly limited, it is possible to add one of glutamic acid and Pseudomonas bacteria within 3 days after the addition. The interval between the two grant periods may be within 2 days, within 1 day, within 12 hours, within 6 hours, within 3 hours, or within 1 hour. When glutamic acid and Pseudomonas bacteria are separately applied in this way, the interval between the application periods of both can be, for example, 30 minutes or more, 1 hour or more, 6 hours or more, or 12 hours or more. In the present invention, the Pseudomonas bacterium is considered to have a function of enhancing the soybean stalk epidemic control effect of glutamic acid, and therefore it is preferable that the interval between the two application periods is not long. From such a viewpoint, it is preferable that the interval between the application times of both glutamic acid and Pseudomonas bacteria is at least one day or less. The above-mentioned soybean seeding can be applied with glutamic acid and Pseudomonas bacteria as the time when either one is given, and the other can be given within 3 days after the soybean seeding.
　グルタミン酸とシュードモナス属細菌とは、いずれを先に付与してもよい。すなわち、グルタミン酸を先に付与し、次いでシュードモナス属細菌を付与してもよいし、或いはシュードモナス属細菌を先に付与し、次いでグルタミン酸を付与してもよい。 Glutamic acid and Pseudomonas bacteria may be given first. That is, glutamic acid may be given first, and then Pseudomonas bacteria may be given, or Pseudomonas bacteria may be given first, and then glutamic acid may be given.
　本発明の方法はまた、グルタミン酸及びシュードモナス属細菌を付与した後でさらにグルタミン酸を付与する工程を含むことができる。グルタミン酸の更なる付与により、グルタミン酸及びシュードモナス属細菌のダイズ茎疫病防除作用の増強効果を持続できると考えられ、さらに効果的にダイズ茎疫病を防除できることが期待される。グルタミン酸の更なる付与は１回以上行うことができ、２回以上、３回以上、４回以上、又は５回以上としてもよい。グルタミン酸の更なる付与は、典型的には１～２０回、好ましくは２～１０回、より好ましくは３～８回である。 The method of the present invention can also include the step of adding glutamic acid and Pseudomonas bacteria and then further adding glutamic acid. Further addition of glutamic acid is considered to be able to sustain the enhancing effect of glutamic acid and Pseudomonas bacteria for controlling soybean stalk epidemic, and is expected to be able to more effectively control soybean stalk epidemic. Further application of glutamic acid can be performed once or more, and may be performed twice or more, three times or more, four times or more, or five times or more. Further addition of glutamic acid is typically 1 to 20 times, preferably 2 to 10 times, more preferably 3 to 8 times.
　グルタミン酸の更なる付与は継続的に行うことができる。グルタミン酸をさらに付与する時期の間隔は、特に限定されないが、例えば、６時間以上、１２時間以上、１日以上、２日以上、３日以上、又は４日以上とすることができ、また、１０日以下、７日以下、６日以下、５日以下、４日以下、３日以下、２日以下、１日以下、又は１２時間以下としてもよい。グルタミン酸をさらに付与する時期の間隔は、典型的には６時間～１０日、好ましくは１２時間～７日、より好ましくは１～３日である。 Further addition of glutamic acid can be continued. The interval at which glutamic acid is further applied is not particularly limited, but may be, for example, 6 hours or more, 12 hours or more, 1 day or more, 2 days or more, 3 days or more, or 4 days or more, and 10 It may be less than a day, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, or less than 12 hours. The interval at which glutamic acid is further applied is typically 6 hours to 10 days, preferably 12 hours to 7 days, and more preferably 1 to 3 days.
　グルタミン酸をさらに付与する形態や手段は特に限定されず、上述した内容と同様にすることができる。また、グルタミン酸をさらに付与する量も特に限定されず、上記と同様にすることができる。 The form and means for further adding glutamic acid are not particularly limited, and the same contents as described above can be used. Further, the amount of glutamic acid to be further added is not particularly limited, and the same can be applied as described above.
　ダイズにおける茎疫病は、通常、ファイトフトラ（Ｐｈｙｔｏｐｈｔｈｏｒａ）属の菌類により引き起こされる。ファイトフトラ属菌は、真菌の中の卵菌綱に属する菌類であり、感染後植物組織の中で成長する土壌真菌であることが知られている。ダイズにおける茎疫病を引き起こすファイトフトラ属菌の代表例は、Ｐｈｙｔｏｐｈｔｈｏｒａ　ｓｏｊａｅ（より具体的には、Ｐｈｙｔｏｐｈｔｈｏｒａ　ｓｏｊａｅ　Ｎ１）である。 Stem plague in soybean is usually caused by fungi of the genus Phytophthora. The genus Oomycete is a fungus belonging to the class Oomycete among fungi, and is known to be a soil fungus that grows in plant tissues after infection. A typical example of Phytophthora spp. That causes stalk epidemics in soybean is Phytophthora sojae (more specifically, Phytophthora sojae N1).
　（２）ダイズにおける茎疫病の防除用組成物
　本発明の一態様は、グルタミン酸を含む、ダイズにおける茎疫病の防除用組成物である。本発明の組成物は、シュードモナス属細菌をさらに含むこともできる。 (2) Composition for Controlling Stem Epidemic in Soybean One aspect of the present invention is a composition for controlling stem epidemic in soybean containing glutamic acid. The composition of the present invention may further include Pseudomonas bacteria.
　本発明の組成物に用いられるグルタミン酸及びシュードモナス属細菌については、上述した通りである。 The glutamic acid and Pseudomonas bacteria used in the composition of the present invention are as described above.
　本発明の組成物に含まれるグルタミン酸の量は、剤形の種類等に応じて適宜設定することができる。本発明の組成物中のグルタミン酸の含有量は、例えば、組成物１００ｇ当たり０．０１ｍＭ～１Ｍ、好ましくは０．１ｍＭ～１００ｍＭであるが、特に限定されるわけではない。 The amount of glutamic acid contained in the composition of the present invention can be appropriately set according to the type of dosage form and the like. The content of glutamic acid in the composition of the present invention is, for example, 0.01 mM to 1 M, preferably 0.1 mM to 100 mM per 100 g of the composition, but is not particularly limited.
　本発明の組成物に含まれるシュードモナス属細菌の量は、細菌種、細菌の性質（耐乾燥性など）、剤形の種類等に応じて適宜設定することができる。本発明の組成物中のシュードモナス属細菌の含有量は、例えば、組成物１００ｇ当たり１０４～１０２０ＣＦＵ、好ましくは１０６～１０１２ＣＦＵであるが、特に限定されるわけではない。 The amount of Pseudomonas bacteria contained in the composition of the present invention can be appropriately set according to the bacterial species, the properties of the bacteria (drought resistance, etc.), the type of dosage form, and the like. The content of Pseudomonas bacteria in the composition of the present invention is, for example, 10 4 to 10 20 CFU, preferably 10 6 to 10 12 CFU per 100 g of the composition, but is not particularly limited.
　本発明の組成物へのシュードモナス属細菌の添加方法は特に限定されず、そのまま添加してもよい。例えば、当業者に公知の方法でシュードモナス属細菌を培養し、液体培地であれば遠心分離等で回収し、固体培地であれば形成コロニーを白金耳等で回収して、本発明の組成物に添加することができる。或いは、液体中に保存していた状態から自体公知の方法で凍結乾燥処理を行い、シュードモナス属細菌を固体物として本発明の組成物に添加してもよい。 The method of adding Pseudomonas bacteria to the composition of the present invention is not particularly limited, and may be added as it is. For example, bacteria of the genus Pseudomonas are cultivated by a method known to those skilled in the art, and if it is a liquid medium, it is recovered by centrifugation or the like, and if it is a solid medium, the formed colonies are recovered by a loop loop or the like to obtain the composition of the present invention. Can be added. Alternatively, the bacterium of the genus Pseudomonas may be added to the composition of the present invention as a solid by freeze-drying the product while it is stored in a liquid by a method known per se.
　本発明の組成物は、賦形剤、増粘剤、結合剤、安定化剤、防腐剤、ｐＨ調整剤、着色剤、着香剤等の添加剤を含んでいてもよい。各種の添加剤は、特に限定されないが、生物農薬の技術分野において公知の材料を用いることができ、その配合量は、当業者の公知技術に基づいて適宜調整することができる。また、本発明の組成物の形態は、液体、固体、ゲル、ペースト等のいずれの形態であってもよく、使用状況等に応じて適宜設定することができる。 The composition of the present invention may contain additives such as excipients, thickeners, binders, stabilizers, preservatives, pH adjusters, colorants, and flavoring agents. The various additives are not particularly limited, but materials known in the technical field of biopesticide can be used, and the blending amount thereof can be appropriately adjusted based on the known techniques of those skilled in the art. In addition, the form of the composition of the present invention may be any form such as liquid, solid, gel, paste, etc., and can be appropriately set according to the usage situation and the like.
　本発明の組成物は、濃縮物の状態であってもよい。その場合の濃縮倍率は特に限定されず、例えば、２～１０００倍、５～１００倍、又は１０～５０倍とすることができる。本発明の組成物が濃縮物である場合は、水等の溶媒を用いて適宜希釈を行い、その希釈物をダイズ又はダイズを含む土に付与することができる。グルタミン酸及びシュードモナス属細菌について、本発明の組成物におけるそれらの含有量は、濃縮倍率に応じて設定することができる。 The composition of the present invention may be in the form of a concentrate. In that case, the concentration ratio is not particularly limited, and can be, for example, 2 to 1000 times, 5 to 100 times, or 10 to 50 times. When the composition of the present invention is a concentrate, it can be appropriately diluted with a solvent such as water, and the diluted product can be applied to soybean or soil containing soybean. For glutamic acid and Pseudomonas bacteria, their content in the composition of the present invention can be set according to the concentration ratio.
　シュードモナス属細菌を用いる場合、本発明の組成物は、グルタミン酸及びシュードモナス属細菌が含まれる単一の製剤として構成されていてもよく、或いは、グルタミン酸及びシュードモナス属細菌とが別々に含まれる二つの製剤として構成されていてもよい。本発明の組成物が二つの製剤として構成される場合は、当該二つの製剤は併用されること（即ち、併用剤であること）が好ましい。また、本発明の組成物が二つの製剤として構成される場合、当該二つの製剤は異なる種類の剤形であってもよいし、同一種類の剤形であってもよい。 When using Pseudomonas bacteria, the composition of the present invention may be configured as a single preparation containing glutamic acid and Pseudomonas bacteria, or two preparations containing glutamic acid and Pseudomonas bacteria separately. It may be configured as. When the composition of the present invention is composed of two preparations, it is preferable that the two preparations are used in combination (that is, they are concomitant agents). When the composition of the present invention is composed of two formulations, the two formulations may be in different types of dosage forms or may be in the same type of dosage form.
　本発明の組成物は、ダイズにおける茎疫病の防除用として用いられる。シュードモナス属細菌を用いる場合、本発明の組成物は、生物農薬又は微生物農薬としても使用することができる。そのため、シュードモナス属細菌を用いる場合、本発明の組成物は農薬組成物と称することもできる。 The composition of the present invention is used for controlling stalk epidemics in soybean. When Pseudomonas bacteria are used, the composition of the present invention can also be used as a biopesticide or a microbial pesticide. Therefore, when Pseudomonas bacteria are used, the composition of the present invention can also be referred to as a pesticide composition.
　（３）ダイズにおける茎疫病の防除用キット
　本発明の一態様は、グルタミン酸を含む、ダイズにおける茎疫病の防除用キットである。本発明のキットは、シュードモナス属細菌をさらに含むこともできる。 (3) Kit for controlling stem epidemic in soybean One aspect of the present invention is a kit for controlling stem epidemic in soybean containing glutamate. The kit of the present invention may further include Pseudomonas bacteria.
　本発明のキットに用いられるグルタミン酸及びシュードモナス属細菌については、上述した通りである。 The glutamic acid and Pseudomonas bacteria used in the kit of the present invention are as described above.
　グルタミン酸及びシュードモナス属細菌について、それらの形態は試薬であってもよいし、或いは調製物であってもよく、特に限定されない。また、シュードモナス属細菌を用いる場合、グルタミン酸及びシュードモナス属細菌は、双方が同一の製剤に含まれていてもよいし、別個の製剤にそれぞれ分けて含まれていてもよい。グルタミン酸及びシュードモナス属細菌が別個の製剤に分けられている場合、当該製剤は異なる種類の剤形であってもよいし、同一種類の剤形であってもよい。 Regarding glutamic acid and Pseudomonas bacteria, their forms may be reagents or preparations, and are not particularly limited. When Pseudomonas bacteria are used, both glutamic acid and Pseudomonas bacteria may be contained in the same preparation, or may be separately contained in separate preparations. When glutamic acid and Pseudomonas bacteria are separated into separate formulations, the formulations may be in different dosage forms or in the same dosage form.
　本発明のキットにおいて用いられる製剤の形態は特に限定されず、液体、固体、ゲル、ペースト等のいずれの形態であってもよい。また、グルタミン酸及びシュードモナス属細菌について、製剤中のそれらの含有量も特に限定されず、上述した本発明の組成物に準じて任意に設定することができる。 The form of the pharmaceutical product used in the kit of the present invention is not particularly limited, and may be any form such as liquid, solid, gel, and paste. In addition, the contents of glutamic acid and Pseudomonas bacteria in the preparation are not particularly limited, and can be arbitrarily set according to the composition of the present invention described above.
　本発明のキットにおいて用いられる製剤は、シングルユースとして一回当たりの使用量が個別に包装されていてもよいし、複数回（例えば、２回、３回、４回、５回、１０回、又はそれ以上）の使用量を含んだ形態で包装されていてもよい。使用される容器も特に限定されず、製剤の使用量等に応じて適宜設定することができる。 The formulation used in the kit of the present invention may be individually packaged in a single-use amount for single use, or may be used a plurality of times (for example, 2 times, 3 times, 4 times, 5 times, 10 times, etc.). Or more) may be packaged in a form containing the usage amount. The container to be used is not particularly limited, and can be appropriately set according to the amount of the pharmaceutical product used and the like.
　本発明のキットにおいてグルタミン酸及びシュードモナス属細菌が別個の試薬又は製剤に分けられている場合、双方は同一のパッケージに組み込まれている必要はなく、別々にパッケージングされていて使用時に併用されるものであってもよい。また、本発明のキットは、グルタミン酸及びシュードモナス属細菌の使用に関する取扱説明書を含んでいてもよい。 When glutamic acid and Pseudomonas bacteria are separated into separate reagents or formulations in the kit of the present invention, they do not have to be packaged in the same package, but are packaged separately and used together at the time of use. It may be. The kit of the present invention may also include an instruction manual for the use of glutamic acid and Pseudomonas bacteria.
　本発明のキットは、ダイズにおける茎疫病の防除用として用いられる。シュードモナス属細菌を用いる場合、本発明のキットは、生物農薬又は微生物農薬としても使用することができる。 The kit of the present invention is used for controlling stalk epidemics in soybean. When using Pseudomonas bacteria, the kit of the present invention can also be used as a biopesticide or a microbial pesticide.
　以下、実施例によって本発明を具体的に説明するが、これらは本発明の技術的範囲を限定するためのものではない。当業者は本明細書の記載に基づいて容易に本発明に修飾及び変更を加えることができ、それらも本発明の技術的範囲に含まれる。 Hereinafter, the present invention will be specifically described with reference to Examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can readily modify and modify the invention based on the description herein, which are also within the technical scope of the invention.
　＜材料＞
　供試植物：ダイズの種子（品種：エンレイ）
　ダイズ茎疫病の感染菌（ファイトフトラ属菌）：Ｐｈｙｔｏｐｈｔｈｏｒａ　ｓｏｊａｅ　Ｎ１
　シュードモナス属細菌：Ｐｓｅｕｄｏｍｏｎａｓ　ｐｒｏｔｅｇｅｎｓ　Ｃａｂ５７
　土：育苗培土（プランテーションイワモト） <Material>
Test plant: Soybean seed (variety: Enrei)
Infectious bacteria of soybean stem epidemic (Phytophthora genus): Phytophthora sojae N1
Pseudomonas bacteria: Pseudomonas protegens Cab57
Soil: Raising seedling cultivation soil (plantation Iwamoto)
　Ｖ８培地：
　（培地組成）
　　Ｖ８野菜ジュースＯｒｉｇｉｎａｌ（キャンベル）　４０ｍＬ
　　ＣａＣＯ３　　　　　　　　　　　　　　　　　　　０．６ｇ
　　Ｈ２Ｏ　　Ｖ８野菜ジュースとＣａＣＯ３の混合物の上清３５ｍＬに対し３１５ｍＬ
　　Ａｇａｒ，Ｐｏｗｄｅｒ（Ｗａｋｏ）　　　　　　　４．３６ｇ
　（調製方法）
１）１００ｍＬ容ビーカーにＶ８野菜ジュースＯｒｉｇｉｎａｌ　４０ｍＬとＣａＣＯ３　０．６ｇとを投入し、５～１０分スターラーで撹拌した。
２）１）で得られた溶液を５０ｍＬ容ファルコンチューブに移し、遠心分離した（３，０００ｒｐｍ、３分間）。
３）５００ｍＬ容ビーカーに上清を３５ｍＬ回収し、純水３１５ｍＬを加えた。
４）２００ｍＬ容三角フラスコ４個にＡｇａｒ，Ｐｏｗｄｅｒ　１．０９ｇをそれぞれはかりとり、３）で得られた調製液を８７．５ｍＬずつ加え、フラスコの口をアルミホイルで覆ってオートクレーブした（１２１℃、２０分間）。
５）シャーレに分注した。 V8 medium:
(Medium composition)
V8 Vegetable Juice Original (Campbell) 40mL
CaCO 3 0.6g
315mL respect supernatant 35mL of H 2 O V8 vegetable juice and mixtures CaCO 3
Agar, Powder (Wako) 4.36g
(Preparation method)
1) 40 mL of V8 vegetable juice Original and 0.6 g of CaCO 3 were put into a 100 mL beaker, and the mixture was stirred with a stirrer for 5 to 10 minutes.
2) The solution obtained in 1) was transferred to a 50 mL Falcon tube and centrifuged (3,000 rpm, 3 minutes).
3) 35 mL of the supernatant was collected in a 500 mL beaker, and 315 mL of pure water was added.
4) Weigh 1.09 g of Agar and Powder into four 200 mL Erlenmeyer flasks, add 87.5 mL each of the preparation solution obtained in 3), cover the mouth of the flask with aluminum foil, and autoclave (121 ° C., 20 minutes).
5) Dispensed into a petri dish.
　Ｎｕｔｒｉｅｎｔ　Ａｇａｒ（ＮＡ）培地：
　　Ｂｌｏｏｄ　ａｇａｒ　ｂａｓｅ（Ｏｘｏｉｄ）　　　４０ｇ
　　Ｙｅａｓｔ　ｅｘｔｒａｃｔ（Ｏｘｏｉｄ）　　　　　５ｇ
　　Ｈ２Ｏ　　　　　　　　　　　　　　　　　　　　　　１０００ｍＬ
　　（培地調製後、オートクレーブを行い、シャーレに分注した。）
　Ｎｕｔｒｉｅｎｔ　ｙｅａｓｔ　ｂｒｏｔｈ（ＮＹＢ）培地：
　　Ｎｕｔｒｉｅｎｔ　ｂｒｏｔｈ（Ｏｘｏｉｄ）　　　　２５ｇ
　　Ｙｅａｓｔ　ｅｘｔｒａｃｔ（Ｏｘｏｉｄ）　　　　　５ｇ
　　Ｈ２Ｏ　　　　　　　　　　　　　　　　　　　　　　１０００ｍＬ
　　（培地調製後、オートクレーブを行った。） Nutrient Agar (NA) medium:
Blood agar base (Oxoid) 40g
Yeast extract (Oxoid) 5g
H 2 O 1000mL
(After preparing the medium, autoclave was performed and dispensed into a petri dish.)
Nutrient yeast bouillon (NYB) medium:
Nutrient bouillon (Oxoid) 25g
Yeast extract (Oxoid) 5g
H 2 O 1000mL
(After preparing the medium, autoclaving was performed.)
　＜方法＞
　試験を行う前に、使用する育苗培土をオートクレーブにより滅菌処理し、ファイトフトラ属菌はＶ８培地を用いて培養しておいた。シュードモナス属細菌については、ＮＡ培地で得られたシングルコロニーをＮＹＢ培地で液体培養し、培養液を遠心分離処理した後の沈殿物を滅菌水で希釈して、ＯＤ６００＝０．１の細菌懸濁液を調製しておいた。また、試験に供するダイズの種子は保湿箱に入れて、４℃で２４時間、暗黒下で保管した。 <Method>
Before conducting the test, the seedling raising soil to be used was sterilized by an autoclave, and the genus Fightoftra was cultivated in V8 medium. For Pseudomonas bacteria, single colonies obtained in NA medium were liquid-cultured in NYB medium, and the precipitate after centrifugation of the culture solution was diluted with sterile water to suspend the bacteria with OD600 = 0.1. The liquid was prepared. The soybean seeds to be tested were placed in a moisturizing box and stored at 4 ° C. for 24 hours in the dark.
　ファイトフトラ属菌を培養したＶ８培地を１０ｍＬシリンジ（注射針（１８Ｇ）付き）に通して細かくつぶし、当該培養培地を育苗培土に添加して十分に混合した。ファイトフトラ属菌培養Ｖ８培地の添加量は、育苗培土１Ｌに対して４．６３ｇの量とした。Ｖ８培地を添加した後、育苗培土を、底部に穴があって水が透過できる育苗用トレイ（１区画あたり６ｃｍ四方、深さ５．５ｃｍ）に均等に１８０ｍＬずつ投入した。それから、ダイズの種子を５粒ずつ、胚軸の部分を下にして育苗培土の上に播種した。 The V8 medium in which the genus Fightoftra was cultured was passed through a 10 mL syringe (with an injection needle (18 G)) and crushed into small pieces, and the culture medium was added to the seedling culture soil and mixed thoroughly. The amount of the Fightoftra genus culture V8 medium added was 4.63 g per 1 L of the nursery soil. After adding the V8 medium, 180 mL of the seedling raising soil was evenly poured into a seedling raising tray (6 cm square, 5.5 cm deep) having a hole at the bottom and allowing water to permeate. Then, five soybean seeds were sown on the nursery soil with the hypocotyl part facing down.
　試験区としては、（ａ）水のみの付与、（ｂ）シュードモナス属細菌のみの付与、（ｃ）グルタミン酸のみの付与、ならびに（ｄ）グルタミン酸及びシュードモナス属細菌の付与、の４種類を設定した。具体的な手順は以下の通りである。 Four types of test plots were set: (a) water only, (b) Pseudomonas bacteria only, (c) glutamic acid only, and (d) glutamic acid and Pseudomonas bacteria. The specific procedure is as follows.
　バットに水または１０ｍＭグルタミン酸水溶液を入れ、その中に、ダイズの種子が播種された育苗用トレイを浸した。なお、水または１０ｍＭグルタミン酸水溶液の量は、育苗用トレイ１区画につき５０ｍＬとした。育苗用トレイを浸すとほぼ同時に、滅菌水またはシュードモナス属細菌の細菌懸濁液をダイズの種子にめがけて添加した。滅菌水および細菌懸濁液の添加量は、育苗用トレイ１区画につき５ｍＬとした。上記のグルタミン酸及びシュードモナス属細菌のダイズ又はダイズを含む土への付与は、ダイズの播種後１時間以内に行い、グルタミン酸とシュードモナス属細菌とを付与する間隔は５分以内であった。 Water or a 10 mM glutamic acid aqueous solution was put in a bat, and a tray for raising seedlings in which soybean seeds were sown was immersed in the bat. The amount of water or 10 mM glutamic acid aqueous solution was 50 mL per section of the nursery tray. Almost at the same time as the seedling tray was soaked, sterile water or a bacterial suspension of Pseudomonas bacteria was added to the soybean seeds. The amount of sterilized water and bacterial suspension added was 5 mL per section of the nursery tray. The above-mentioned application of glutamic acid and Pseudomonas bacteria to soybean or soil containing soybean was performed within 1 hour after soybean sowing, and the interval of application of glutamic acid and Pseudomonas bacteria was within 5 minutes.
　その後、育苗用トレイをラップフィルムで覆い、植物用インキュベーター内に静置した。明期１６時間、暗期８時間に設定し、２５℃で２週間、ダイズの栽培を行った。栽培期間中は５日おきに、バット中の水または１０ｍＭグルタミン酸水溶液を補給した。なお、補給する水または１０ｍＭグルタミン酸水溶液の量は、上記と同様に、育苗用トレイ１区画につき５０ｍＬとした。補給時に水または１０ｍＭグルタミン酸水溶液がバット中に残っている場合は廃棄し、新たな水または１０ｍＭグルタミン酸水溶液を補給した。 After that, the seedling raising tray was covered with plastic wrap and left in the plant incubator. Soybeans were cultivated at 25 ° C. for 2 weeks at 16 hours in the light period and 8 hours in the dark period. Water in the vat or a 10 mM glutamic acid aqueous solution was replenished every 5 days during the cultivation period. The amount of water to be replenished or the 10 mM glutamic acid aqueous solution was set to 50 mL per section of the seedling raising tray in the same manner as described above. If water or a 10 mM glutamic acid aqueous solution remained in the vat at the time of replenishment, it was discarded and a new water or 10 mM glutamic acid aqueous solution was replenished.
　２週間の栽培後、根に付着した育苗培土を十分に洗い流し、ダイズ植物体から双葉を除去して重量を測定し、平均値を算出した。また、栽培期間中は経時的にダイズの生存数も記録した。 After cultivating for 2 weeks, the seedling raising soil adhering to the roots was thoroughly washed away, Futaba was removed from the soybean plant, the weight was measured, and the average value was calculated. In addition, the number of surviving soybeans was recorded over time during the cultivation period.
　栽培後のダイズ植物体の重量は図１に示された通りであり、グルタミン酸を付与することによりダイズ茎疫病の防除効果が得られることがわかり、また、シュードモナス属細菌を併用させることによりその効果が高まることが明らかとなった。 The weight of the soybean plant after cultivation is as shown in FIG. 1, and it was found that the control effect of soybean stem epidemic can be obtained by adding glutamic acid, and the effect by using the Pseudomonas bacterium in combination. It became clear that
　また、栽培期間中のダイズの生存数は図２に示した通りである。栽培開始１３日後の時点で、水のみを付与した試験区とシュードモナス属細菌のみを付与した試験区ではダイズの生存数は７個となり、これに対してグルタミン酸のみを付与した試験区ではダイズの生存数は９個、グルタミン酸及びシュードモナス属細菌を付与した試験区ではダイズの生存数は１０個であった。この結果からも、ダイズ茎疫病の防除効果はグルタミン酸の付与によって得られ、その効果はシュードモナス属細菌を併用させることにより高まることが示された。 The number of surviving soybeans during the cultivation period is as shown in Fig. 2. As of 13 days after the start of cultivation, the number of surviving soybeans was 7 in the test plots to which only water was given and the test plots to which only Pseudomonas bacteria were given, whereas the number of soybeans alive in the test plots to which only glutamic acid was given. The number was 9, and the number of surviving soybeans was 10 in the test plots fed with glutamate and Pseudomonas bacteria. From this result, it was shown that the control effect of soybean stem epidemic was obtained by the addition of glutamic acid, and the effect was enhanced by the combined use of Pseudomonas bacteria.
　本発明により提供される技術は、ダイズの生産に関する農業分野において有用である。また、本発明により提供されるシュードモナス属細菌とグルタミン酸は、農薬分野、特に生物農薬の分野において利用することができる。 The technique provided by the present invention is useful in the agricultural field related to soybean production. In addition, the Pseudomonas bacterium and glutamic acid provided by the present invention can be used in the field of pesticides, particularly in the field of biopesticides.

Claims (11)

  1. 　グルタミン酸をダイズ又はダイズを含む土に付与する工程を含む、ダイズにおける茎疫病の防除方法。 A method for controlling stalk epidemics in soybean, which comprises a step of applying glutamic acid to soybean or soil containing soybean.
  2. 　シュードモナス属細菌をダイズ又はダイズを含む土に付与する工程をさらに含む、請求項１に記載の方法。 The method according to claim 1, further comprising a step of applying Pseudomonas bacteria to soybean or soil containing soybean.
  3. 　ダイズが種子又は幼苗の状態である、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the soybean is in the state of seeds or seedlings.
  4. 　ダイズの播種前又は播種後３日以内にグルタミン酸を付与する、請求項１～３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein glutamic acid is applied before sowing of soybean or within 3 days after sowing.
  5. 　ダイズの播種前又は播種後３日以内にシュードモナス属細菌を付与する、請求項２～４のいずれか１項に記載の方法。 The method according to any one of claims 2 to 4, wherein Pseudomonas bacteria are applied before sowing of soybean or within 3 days after sowing.
  6. 　グルタミン酸及びシュードモナス属細菌を同時に付与する、請求項２～５のいずれか１項に記載の方法。 The method according to any one of claims 2 to 5, wherein glutamic acid and Pseudomonas bacteria are simultaneously applied.
  7. 　グルタミン酸及びシュードモナス属細菌を付与した後でさらにグルタミン酸を付与する工程を含む、請求項２～６のいずれか１項に記載の方法。 The method according to any one of claims 2 to 6, which comprises a step of further adding glutamic acid after adding glutamic acid and Pseudomonas bacteria.
  8. 　グルタミン酸を含む、ダイズにおける茎疫病の防除用組成物。 A composition for controlling stem plague in soybean, which contains glutamic acid.
  9. 　シュードモナス属細菌をさらに含む、請求項８に記載の組成物。 The composition according to claim 8, further comprising a Pseudomonas bacterium.
  10. 　グルタミン酸を含む、ダイズにおける茎疫病の防除用キット。 A kit for controlling stem plague in soybeans, which contains glutamic acid.
  11. 　シュードモナス属細菌をさらに含む、請求項１０に記載のキット。 The kit according to claim 10, further comprising Pseudomonas bacteria.
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WO2023121329A1 (en) * 2021-12-22 2023-06-29 씨제이제일제당 (주) Composition for promoting plant disease resistance

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JPH09124427A (en) * 1995-10-27 1997-05-13 Norin Suisansyo Chugoku Nogyo Shikenjo Control agent for rice plant damping-off using new microorganismic strain and method for controlling the same
JP2000290117A (en) * 1999-04-01 2000-10-17 Natl Inst Of Sericultural & Entomological Science Microbial herbicide and weeding
JP2011200155A (en) * 2010-03-25 2011-10-13 Nisshin Oillio Group Ltd Method for cultivating soybean, and soybean processed food using soybean cultivated by the cultivation method
WO2019198735A1 (en) * 2018-04-10 2019-10-17 国立研究開発法人農業・食品産業技術総合研究機構 Method of controlling soil-borne diseases of plants

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Publication number Priority date Publication date Assignee Title
JPH09124427A (en) * 1995-10-27 1997-05-13 Norin Suisansyo Chugoku Nogyo Shikenjo Control agent for rice plant damping-off using new microorganismic strain and method for controlling the same
JP2000290117A (en) * 1999-04-01 2000-10-17 Natl Inst Of Sericultural & Entomological Science Microbial herbicide and weeding
JP2011200155A (en) * 2010-03-25 2011-10-13 Nisshin Oillio Group Ltd Method for cultivating soybean, and soybean processed food using soybean cultivated by the cultivation method
WO2019198735A1 (en) * 2018-04-10 2019-10-17 国立研究開発法人農業・食品産業技術総合研究機構 Method of controlling soil-borne diseases of plants

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023121329A1 (en) * 2021-12-22 2023-06-29 씨제이제일제당 (주) Composition for promoting plant disease resistance

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