WO2018168581A1 - Insect repellent and method for producing insect repellent - Google Patents

Insect repellent and method for producing insect repellent Download PDF

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Publication number
WO2018168581A1
WO2018168581A1 PCT/JP2018/008629 JP2018008629W WO2018168581A1 WO 2018168581 A1 WO2018168581 A1 WO 2018168581A1 JP 2018008629 W JP2018008629 W JP 2018008629W WO 2018168581 A1 WO2018168581 A1 WO 2018168581A1
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Prior art keywords
pest repellent
fatty acid
producing
acid
metabolism
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PCT/JP2018/008629
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French (fr)
Japanese (ja)
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大野　勝也
久美子 高田
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イビデン株式会社
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Priority to JP2019505910A priority Critical patent/JPWO2018168581A1/en
Publication of WO2018168581A1 publication Critical patent/WO2018168581A1/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
    • 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
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria

Definitions

  • the present invention relates to a pest repellent and a method for producing the pest repellent.
  • drugs such as pyrethrin, which is an active ingredient of pesticide chrysanthemums, and synthetic pyrethroids such as allethrin derived from pyrethrin derivatives have been used. While pyrethroids have low toxicity to mammals and birds, they act strongly on insect nerve cells to produce neurotoxins and kill insects. In recent years, neonicotinoid insecticides that kill insects by binding to the insect neurotransmitter acetylcholine receptor have been widely used as insecticides.
  • insecticidal method in which insects are directly killed using components that are generally toxic to such insects has a problem that even beneficial insects and natural enemy insects necessary for pollination are uniformly killed.
  • insecticides that have a long residual effect due to their ability to penetrate into plants such as neonicotinoids, may affect beneficial insects over a long period of time, and there is a concern about disruption of the ecosystem.
  • insecticide-resistant insects due to excessive application of agricultural chemicals is also a problem. From the viewpoint of environmental impact such as soil contamination, reduction of the amount of chemically synthesized pesticides is desired.
  • Patent Document 1 describes a microbial biosurfactant.
  • Patent Document 1 discloses a method for controlling pests using a fermentation broth filtrate containing rhamnolipid as a biosurfactant, which can be used as an insecticide for controlling house flies and nematodes. Are listed. However, in the method for controlling pests described in Patent Document 1, a drug must be applied directly to kill the pest, and therefore, it is necessary to apply the drug every time the pest is generated.
  • the conventional method of repelling pests is effective by gradually evaporating the drug, and has the advantage of widening the range of repelling pests, but the drug disappears due to volatilization. Therefore, there is a problem that the sustainability of the effect is short.
  • fatty acid oxides including lipid peroxides exhibit antibacterial action. It is also known that jasmonic acid and the like are biosynthesized in plants using fatty acids such as linolenic acid as raw materials. However, it is not known that fatty acid metabolites obtained by oxidizing fatty acids by microbial metabolism have an effect of inducing repellent effects in plants.
  • an object of the present invention is to provide a pest repellent that has low soil contamination and toxicity and can exhibit a long-term repellent effect in plants, and a method for producing the same.
  • the present invention relates to a pest repellent containing a fatty acid metabolite obtained by metabolizing fatty acids to proteobacteria.
  • the pest repellent is preferably a pest repellent which is a pest repellent secretion promoter in plants.
  • a pest repellent in which the fatty acid is a fatty acid having 4 to 30 carbon atoms is preferred.
  • a pest repellent in which the fatty acid is a fatty acid that is liquid at 20 ° C. is preferred.
  • a pest repellent is preferred in which the metabolism is metabolism in a dissolved oxygen concentration environment of 0.1 to 8 mg / l (liter).
  • a pest repellent in which the metabolism is metabolism in the presence of at least one mineral selected from Mg, P, Na and K is preferred.
  • a pest repellent is preferred in which the proteobacteria are pre-cultured proteobacteria.
  • Pest repellents are preferred wherein the pre-cultured proteobacteria are proteobacteria pre-cultured to 1 ⁇ 10 8 to 9 ⁇ 10 10 cells / ml (milliliter).
  • the pest repellent is preferably a pest repellent containing a biosurfactant.
  • a pest repellent in which the metabolism is metabolism under conditions of 20 to 30 ° C. is preferable.
  • the pest repellent is preferably a pest repellent that is used so as to come into contact with the foliage or roots of a plant.
  • the present invention also relates to a method for producing a pest repellent containing a fatty acid metabolite, which includes a fatty acid metabolism step of metabolizing fatty acid to proteobacteria.
  • a method for producing a pest repellent, wherein the pest repellent is a pest repellent secretion promoter in plants, is preferred.
  • a method for producing a pest repellent in which the fatty acid is a fatty acid having 4 to 30 carbon atoms is preferred.
  • a method for producing a pest repellent in which the fatty acid is a liquid fatty acid at 20 ° C. is preferred.
  • a method for producing a pest repellent in which the fatty acid metabolism step is performed in an environment of dissolved oxygen concentration of 0.1 to 8 mg / l is preferable.
  • a method for producing a pest repellent in which the fatty acid metabolism step is carried out in the presence of at least one mineral selected from Mg, P, Na and K is preferred.
  • a method for producing a pest repellent wherein the proteobacteria are pre-cultured proteobacteria is preferred.
  • a method for producing a pest repellent wherein the pre-cultured proteobacteria are pre-cultured to 1 ⁇ 10 8 to 9 ⁇ 10 10 cells / ml, is preferred.
  • a method for producing a pest repellent which is a method for producing a pest repellent containing a biosurfactant is preferred.
  • a method for producing a pest repellent in which the fatty acid metabolism step is carried out at 20 to 30 ° C. is preferable.
  • a method for producing a pest repellent which is a method for producing a pest repellent applied to plants is preferred.
  • the pest repellent of the present invention has low soil contamination and toxicity and is excellent in long-term repellent effect in plants. Moreover, according to the method for producing a pest repellent of the present invention, a pest repellent having low soil contamination and toxicity and excellent in long-term repellent effect in plants can be produced.
  • Pest repellent of the present invention is characterized by containing a fatty acid metabolite obtained by metabolizing fatty acids to proteobacteria.
  • resistance to the plant can be induced in addition to a direct pest repellent effect by a repellent substance that can be contained in the fatty acid metabolite.
  • a repellent substance that can be contained in the fatty acid metabolite.
  • the fatty acid metabolite contains, in addition to the repellent substance, a salicylic acid pathway related to systemic acquired resistance, a substance that activates the jasmonic acid pathway, or a precursor of this substance.
  • the plant itself can produce and / or secrete repellents, and the functions of the plant relating to the inherent insect repellent can be further developed.
  • the pest repellent of the present invention is a pest repellent secretion promoter containing a substance capable of promoting the secretion of a pest repellent from a plant.
  • the pest repellent of the present invention may further contain a repellent substance having a direct pest repellent effect in addition to the pest repellent secretion promoting substance.
  • the pest repellent of the present invention can exhibit a long-term repellent effect as compared with conventional pest repellents, and is also considered to be effective for pests that have developed drug resistance against conventional pesticides.
  • fatty acid metabolites are much less toxic than chemically synthesized pesticides.
  • Metabolism in the present invention means that fatty acids are decomposed by an enzyme or the like secreted by proteobacteria.
  • a method of culturing proteobacteria in a medium containing fatty acid can be mentioned.
  • Proteobacteria have a gene that produces lipoxygenase (LOX), an enzyme involved in fatty acid metabolism, and can produce fatty acid metabolites.
  • LOX lipoxygenase
  • the carbon number of the fatty acid used in the present invention is 4 to 30, and preferably 10 to 20.
  • the melting point / boiling point is low, and therefore, the volatility tends to increase at the temperature at the time of culturing and hardly remain in the medium.
  • the number of carbons exceeds 30, the melting point / boiling point becomes high, so that it becomes a solid at the temperature at the time of culturing and tends to be separated without being mixed with the medium.
  • the melting point may not depend only on the number of carbons depending on the number of hydrogen bonds.
  • the fatty acid used in the present invention is preferably a liquid at 20 to 30 ° C. and more preferably a liquid at 20 ° C. from the viewpoint of metabolic efficiency and suppression of solidification in the medium.
  • the fatty acid of the present invention can be a saturated fatty acid, an unsaturated fatty acid, or a mixture containing both.
  • a free fatty acid (monocarboxylic acid) is preferable because it is excellent in a decomposition rate.
  • Free fatty acids having 4 to 30 carbon atoms include butyric acid (butyric acid), valeric acid (valeric acid), caproic acid, enanthic acid (heptylic acid), caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, pentadecyl Acid, palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, vaccenic acid, linoleic acid, ⁇ -linolenic acid, ⁇ -linolenic acid, eleostearic acid, arachidic acid, mead acid, arachidonic acid, behenic acid, Examples include lignoceric acid, nervonic acid, serotic acid, montanic acid, and melicic acid.
  • capric acid having 10 to 20 carbon atoms, lauric acid, myristic acid, pentadecylic acid, palmitic acid, palmitoleic acid, margaric acid, stearin Acid, oleic acid, vaccenic acid, linoleic acid, ⁇ -linolenic acid, ⁇ Linolenic acid, eleostearic acid, arachidic acid, mead acid, arachidonic acid are preferred, oleic acid having 18 carbon atoms, linoleic acid, alpha-linolenic acid, .gamma.-linolenic acid are more preferred.
  • the content of fatty acid is preferably 120 g / l or less, more preferably 100 g / l or less, and even more preferably 60 g / l or less. If it exceeds 120 g / l, emulsification with water in the medium becomes difficult, which may deteriorate metabolic efficiency and inhibit the growth of proteobacteria.
  • the lower limit of the fatty acid content is not particularly limited, but is preferably 1.0 g / l or more.
  • the medium containing fatty acid contains other mineral components.
  • a mineral component it is not specifically limited, The mineral component normally used for microorganism culture can be mentioned.
  • the component which has magnesium (Mg), phosphorus (P), sodium (Na), or potassium (K) is mentioned. These components can be used alone or in combination. Preferably two of these components may be used, more preferably three or more.
  • the content of the mineral component in the medium is not particularly limited, and can be the amount used in the conventional aerobic bacterium culture method, but since salt damage may occur during application to plants, preferably It can be used at 15 g / l or less, more preferably at 10 g / l or less.
  • proteobacteria used in the present invention is not particularly limited as long as the effects of the present invention are not impaired. From the viewpoint of fatty acid metabolism efficiency and growth efficiency, proteobacteria having a temperature suitable for growth (optimum temperature) of 10 to 40 ° C. are preferred, and proteobacteria having 20 to 30 ° C. are more preferred.
  • the proteobacteria are preferably pre-cultured proteobacteria because they are excellent in fatty acid metabolic efficiency, and the number of bacteria is preferably pre-cultured to 1 ⁇ 10 8 to 9 ⁇ 10 10 cells / ml. preferable.
  • the metabolism is preferably performed in a predetermined dissolved oxygen concentration environment.
  • the dissolved oxygen concentration in metabolism is preferably 0.1 mg / l or more.
  • a dissolved oxygen concentration is 8 mg / l or less, for example.
  • the dissolved oxygen concentration exceeds 8 mg / l, in parallel with the metabolic process by proteobacteria, decomposition of the fatty acid, which is a substrate, by oxygen in the medium proceeds, resulting in a decrease in metabolic efficiency and, consequently, an active ingredient. Metabolite production may be reduced.
  • the dissolved oxygen concentration is 0.1 to 8 mg / l, more preferably 0.1 to 5 mg / l, and still more preferably 0.1 to 4 mg / l.
  • the dissolved oxygen concentration is a value measured by a diaphragm galvanic electrode method or a diaphragm polarographic method on a PO electrode with a dissolved oxygen meter manufactured by HORIBA, Ltd.
  • the temperature in metabolism can be appropriately adjusted according to the proteobacteria used, but 20-30 ° C. is preferable from the viewpoint of fatty acid metabolic efficiency.
  • the insect repellent may contain a biosurfactant in addition to the fatty acid metabolite.
  • Fatty acid metabolites are easily dispersed in water, which is considered preferable from the viewpoint of handling properties of pest repellents.
  • the biosurfactant according to the present invention means a surfactant-like substance that is produced by a microorganism to take up a highly hydrophobic substance and secreted outside the cell.
  • the biosurfactant secreted by proteobacteria facilitates the dispersion of fatty acid metabolites in water so that spray treatment and irrigation of pest repellents containing fatty acid metabolites can be performed efficiently and easily. Become.
  • biosurfactant not only the biosurfactant produced by the proteobacteria of the present invention at the time of fatty acid degradation, but also biosurfactants produced by other microorganisms may be used, that is, the pest repellent of the present invention. Biosurfactants produced by other microorganisms may be further added. Compared to artificially synthesized surfactants, biosurfactants are less toxic to living organisms and have higher biodegradability, which is thought to provide a more environmentally friendly pest repellent. In addition, in order to promote fatty acid degradation by proteobacteria, biosurfactants produced by other microorganisms may be added during fatty acid degradation by proteobacteria. Fatty acid uptake by proteobacteria may be promoted.
  • the pest repellent of the present invention has an excellent effect as a pest repellent that is low in soil contamination and toxicity and can induce a long-term repellent effect in plants.
  • the pest repellent of the present invention activates a group of genes involved in the production of infection-specific proteins (pathogenesis-related (PR) protein) or a group of genes involved in the production of enzymes producing salicylic acid or jasmonic acid in the applied plant.
  • PR pathogenesis-related
  • enzymes producing salicylic acid or jasmonic acid in the applied plant To induce systemic acquired resistance. For example, it is estimated that the biosynthesis of secondary metabolites such as anti-pesticide protease inhibitors and insect repellents induced by jasmonic acid is increased.
  • the pest repellent of the present invention can express both upstream and downstream genes in both the salicylic acid pathway and the jasmonic acid pathway, resistance induction persists and a continuous repellent effect is obtained. It is advantageous.
  • the pest repellent of the present invention can induce the development of organs related to resistance to plant pests.
  • organ development is the increased formation of trichomes (trichomes).
  • Tricombs are known to develop by the activation of jasmonic acid signaling in response to injury, and the development of trichomes indicates that resistance has been induced in plants. Tricomb can release volatile pest repellent components from its tip. Therefore, by developing trichomes, volatile pest repellent components can be more efficiently diffused and the defense function of the plant can be enhanced. It is also known that some trichomes have a function of catching and catching insects depending on the species. Furthermore, since the increase in trichome density can also increase the insect repellent effect as a physical barrier against herbivorous insects, the pest repellent effect of the plant itself can be more activated.
  • the pest repellent of the present invention may further contain these precursors in addition to volatile aldehydes, alcohols and esters thereof, which are herbivore-induced volatile substances, in fatty acid metabolites.
  • Precursors can be converted to volatile aldehydes, alcohols and their ester forms by being absorbed by plants or decomposed in the natural environment. Therefore, the persistence of the repellent effect is thought to be due to the delay in the time from the application of the pest repellent to the plant until the precursor is converted to volatile aldehydes, alcohols and their esters. It is done.
  • the pest repellent of the present invention can be used to repel agricultural pests.
  • agricultural pests include, but are not limited to, pests such as aphids such as cotton aphids, spider mites, scarab beetles, stag beetles such as scallops, and bees such as chrysanthemum.
  • the pest repellent of the present invention exhibits a repellent effect even against the larvae of Spodoptera litura, which is known to have low susceptibility to insecticides, and is therefore effective against pests that have been conventionally difficult to control. It is excellent in that it can demonstrate.
  • the method for applying the pest repellent of the present invention to plants is not particularly limited.
  • it can be applied by spraying on the foliage or roots of the plant, dipping the foliage or roots of the plant, and / or soil irrigation on the soil in which the plant is growing.
  • the plant to be applied is not limited, and it can be used favorably for plants in general. Examples thereof include dicotyledonous plants such as eggplant, cucurbitaceae, and roseceae, and monocotyledonous plants such as gramineous.
  • the manufacturing method of the pest repellent containing the fatty acid metabolite of this invention is characterized by including the fatty acid metabolism process which metabolizes a fatty acid to proteobacteria.
  • the pest repellent obtained by the production method of the present invention is a pest repellent secretion promoter in plants. That is, a substance capable of promoting the secretion of a pest repellent substance from a plant can be produced by the fatty acid metabolism process of the present invention.
  • the fatty acid metabolism step of the present invention may be a step in which a repellent substance having a direct pest repellent effect is produced in addition to such a pest repellent secretion promoting substance.
  • the fatty acid metabolism step is a step in which fatty acids are decomposed by an enzyme or the like that is secreted or secreted by proteobacteria.
  • an enzyme or the like that is secreted or secreted by proteobacteria.
  • cultivating proteobacteria with the culture medium containing a fatty acid is mentioned.
  • the fatty acid metabolism step is preferably performed in a predetermined dissolved oxygen concentration environment.
  • the dissolved oxygen concentration in the fatty acid metabolism step is preferably 0.1 mg / l or more.
  • the dissolved oxygen concentration is less than 0.1 mg / l, the fatty acid resolution of proteobacteria tends to decrease and the metabolic efficiency of fatty acids tends to be extremely low.
  • it is preferable that a dissolved oxygen concentration is 8 mg / l or less, for example.
  • the dissolved oxygen concentration exceeds 8 mg / l, in parallel with the metabolic process by proteobacteria, decomposition of the fatty acid, which is a substrate, by oxygen in the medium proceeds, resulting in a decrease in metabolic efficiency and, consequently, an active ingredient.
  • the dissolved oxygen concentration is 0.1 to 8 mg / l, more preferably 0.1 to 5 mg / l, and still more preferably 0.1 to 4 mg / l.
  • the dissolved oxygen concentration is a value measured by a diaphragm galvanic electrode method or a diaphragm polarographic method on a PO electrode with a dissolved oxygen meter manufactured by HORIBA, Ltd.
  • the dissolved oxygen concentration can be adjusted by the culture vessel, the number of shakes, the amount of aeration, etc.
  • the culture conditions in the fatty acid metabolism step can be the same as the conventional conditions for culturing aerobic bacteria except that the dissolved oxygen concentration is within a predetermined range. Examples thereof include a method of culturing for 3 to 7 days by shaking with a flask and aeration culture with a spinner flask or a jar fermenter.
  • the number of days of culture is preferably the number of days during which the fatty acid is sufficiently emulsified and decomposed, but the number of days of culture varies depending on the agitation and the amount of bacteria.
  • the fatty acid decomposition state is determined by measuring the absorbance at a wavelength of 230 nm, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC / MS), liquid chromatography. It is preferable to confirm by graph mass spectrometry (LC / MS) or the like.
  • the temperature in the fatty acid metabolism step can be appropriately adjusted according to the proteobacteria used, and it is preferable to carry out the treatment at 20 to 30 ° C. from the viewpoint of fatty acid metabolic efficiency.
  • fatty acid and proteobacteria in the fatty acid metabolism step those described above in the description of the pest repellent of the present invention can be used.
  • the pre-culturing step for proteobacteria is not particularly limited, and a normal method for culturing aerobic bacteria can be used. It is preferable that only the cells are collected from the preculture solution by centrifugation or the like and used in the fatty acid metabolism step.
  • the pest repellent obtained by the production method of the present invention can contain a biosurfactant in addition to a fatty acid metabolite.
  • the biosurfactant according to the present invention means a surfactant-like substance that is produced by a microorganism to take up a highly hydrophobic substance and secreted outside the cell. Biosurfactants secreted by proteobacteria also facilitate the dispersion of fatty acid metabolites in water. It is thought that the handleability of the pest repellent of the present invention is improved.
  • the pest repellent of the present invention is obtained as a culture solution that is a mixture with a culture medium, a proteobacterium exocrine product containing a biosurfactant, microbial cells, and the like.
  • the culture solution may be used as it is as the pest repellent of the present invention, or the supernatant obtained by removing the cells from the culture solution by centrifugation or the like may be used as the pest repellent.
  • the pest repellent obtained by the production method of the present invention is applied to plants.
  • the culture solution can be used as it is. However, in the case of the stock solution, the treated portion of the plant at high temperatures may be condensed due to the evaporation of minerals and the effect of osmotic pressure. It may be desirable to do so.
  • the dilution factor is not particularly limited as long as the effect of the present invention is exhibited, but is preferably 10 to 1000 times diluted, more preferably 10 to 100 times diluted.
  • test pest repellent ⁇ pre-culture process 20 g of peptone (Difco protein enzyme hydrolyzate), 1.5 g of magnesium sulfate heptahydrate and 1.5 g of dipotassium hydrogen phosphate in 1 l (liter) of water in a glass Erlenmeyer flask, or 1 l (liter) ) 10 g of peptone (Difco protein enzyme hydrolyzate), 5 g of yeast extract and 10 g of sodium chloride are dissolved in water, autoclaved at 121 ° C. for 20 minutes, and cooled to room temperature. Inoculated. The mouth of the culture vessel was sealed with a silicon stopper.
  • the inoculated container was cultured for 24 hours under the conditions of 25 ° C. ⁇ 5 ° C. and 120 rpm using a bioshaker (BR-23UM manufactured by Taitec Corporation).
  • the number of bacteria in the culture was 5 ⁇ 10 8 cells / ml.
  • the cells were collected from the culture solution by centrifuging the culture solution under conditions of 15,000 ⁇ G and 20 ° C.
  • ⁇ Fatty acid metabolism process In 1 l (liter) of sterilized water in a glass Erlenmeyer flask, 12 g of linoleic acid (primary linoleic acid manufactured by Wako Pure Chemical Industries, Ltd.), 1.5 g of magnesium sulfate heptahydrate and / or dipotassium hydrogen phosphate 1 .5 g and the total amount of the cells obtained from the pre-culture step were added. This was cultured for 4 days under the conditions of 20 ° C., 120 rpm, and dissolved oxygen concentration of 4 mg / l using a bioshaker (BR-23UM manufactured by Taitec Corporation).
  • a bioshaker BR-23UM manufactured by Taitec Corporation
  • the degradation of linoleic acid involves measuring the concentration of oxidized lipid, which is one of linoleic acid intermediate products, in the culture solution using a spectrophotometer BioSpec-mini manufactured by Shimadzu Corporation at a wavelength of 230 nm. Confirmed by. After the culture, the following evaluation was performed using a culture solution containing the cells as a test pest repellent.
  • Examples 1-2 Cucumber true leaves having a length of about 10 cm at the longest part were cut into 2 cm squares and immersed in a test pest repellent (stock solution) and a 10-fold diluted solution thereof (Examples 1 and 2).
  • a petri dish (9 cm in diameter) was prepared by placing leaves immersed in a test pest repellent (test group) and leaves immersed in water (control group) one by one at intervals of 3 cm.
  • a filter paper (1 cm ⁇ 1 cm) was placed between the leaves in each petri dish, and 20 cotton aphids were placed on the filter paper.
  • Repellency (%) (1 ⁇ number of accumulated insects in test group / number of accumulated insects in control group) ⁇ 100 The results are shown in FIG.
  • Comparative examples 1 and 2 In place of the test pest repellent, a 0.1% solution of surfactin sodium (Comparative Example 1), which is a representative biosurfactant, and 0.4% of citronella, a citrus aroma component that is considered to have a repellent effect Treatment was observed in the same manner as in Examples 1 and 2 except that the solution (Comparative Example 2) was used. The results are shown in FIG.
  • the test group treated with the test pest repellent has a higher repellent rate compared to Surfactin sodium in Comparative Example 1 and Citronella in Comparative Example 2. Indicated. In particular, in Example 1 using the stock solution of the test pest repellent, the cotton aphid repellent rate reached 100%, and a strong cotton aphid repellent effect was confirmed. On the other hand, in the comparative example 1 using the biosurfactant, the repelling rate was only 68%, and in the comparative example 2 using the citronella, the repelling effect was 30%. Therefore, it can be seen that the pest repellent of the present invention has an excellent repellent effect.
  • Pest repellent effect in mini roses Example 3
  • a dilute solution of test pest repellent diluted 100 times with water is sprayed with leaves once a week (about 30 ml / strain) and soil Treated by irrigation (about 50 ml / strain).
  • the treatment was carried out for 6 to 7 months. After the treatment, naturally occurring pest parasites and food damage were examined. An untreated section was provided as a control. Pest infestation was evaluated by the average number of parasitic heads per strain or the number of parasitic leaves per strain. The eating damage was evaluated by the degree of insect damage.
  • the degree of insect damage is evaluated by five levels of 0 to 4 for the damage of each strain to be measured, the number of strains at each stage of damage is measured, and the numerical value (0 to 4) indicating the stage of damage to the number of strains is measured.
  • the total number multiplied by the number is divided by the total number of shares to be measured multiplied by 4 and multiplied by 100.
  • the stage of damage is defined as follows.
  • the number of leaves is 100 or more.
  • the stage 0 has no damaged leaves
  • the stage 1 has 3 damaged leaves
  • the stage 2 has 10 damaged leaves.
  • stage 3 is defined as a case where 30 or less leaves are damaged
  • stage 4 is a case where 30 or more leaves are damaged.
  • the pest repellent of the present invention has an excellent pest repellent effect.
  • the pest repellent of the present invention and the pest repellent produced by the production method of the present invention are pest repellents having low soil contamination and toxicity and excellent pest repellent effect.

Abstract

Provided are: an insect repellent which exhibits an excellent insect-repelling effect and resistance-inducing effect, and also exhibits low toxicity and soil contamination; and a method for producing the same. The insect repellent contains a fatty acid metabolite obtained by causing proteobacteria to metabolize a fatty acid. The method for producing the insect repellent containing the fatty acid metabolite includes a fatty acid metabolism step for causing proteobacteria to metabolize a fatty acid.

Description

害虫忌避剤および害虫忌避剤の製造方法Pest repellent and method for producing pest repellent
　本発明は、害虫忌避剤および害虫忌避剤の製造方法に関する。 The present invention relates to a pest repellent and a method for producing the pest repellent.
　従来より、植物害虫に対し殺虫を目的として、除虫菊の有効成分であるピレトリン、そしてピレトリンの誘導体由来のアレスリンなどの合成ピレスロイドなどをはじめとした薬剤が使用されてきた。ピレスロイド類は、哺乳類や鳥類に対する毒性は低い一方、昆虫類の神経細胞に強力に作用して神経毒をもたらし殺虫する。また、近年では昆虫類の神経伝達物質アセチルコリン受容体に結合することで殺虫するネオニコチノイド系殺虫剤も広範囲に殺虫剤として使用されている。 Conventionally, for the purpose of insecticide against plant pests, drugs such as pyrethrin, which is an active ingredient of pesticide chrysanthemums, and synthetic pyrethroids such as allethrin derived from pyrethrin derivatives have been used. While pyrethroids have low toxicity to mammals and birds, they act strongly on insect nerve cells to produce neurotoxins and kill insects. In recent years, neonicotinoid insecticides that kill insects by binding to the insect neurotransmitter acetylcholine receptor have been widely used as insecticides.
　しかしながら、このような虫にとって一般的に毒となる成分を用いて直接殺虫する殺虫方法では、受粉などに必要な有益昆虫や天敵虫も一様に殺虫してしまうという問題がある。また、ネオニコチノイドのように植物への浸透移行性などにより残効が長い殺虫剤では、有益昆虫が長期にわたって影響を受けてしまう恐れもあり、生態系の乱れが懸念される。さらに、過多の農薬散布による殺虫剤耐性虫の発生も問題となっている。土壌汚染などの環境への負荷の観点からも、化学合成農薬の使用量の削減が望まれている。 However, the insecticidal method in which insects are directly killed using components that are generally toxic to such insects has a problem that even beneficial insects and natural enemy insects necessary for pollination are uniformly killed. In addition, insecticides that have a long residual effect due to their ability to penetrate into plants, such as neonicotinoids, may affect beneficial insects over a long period of time, and there is a concern about disruption of the ecosystem. Furthermore, the occurrence of insecticide-resistant insects due to excessive application of agricultural chemicals is also a problem. From the viewpoint of environmental impact such as soil contamination, reduction of the amount of chemically synthesized pesticides is desired.
　そこで、害虫を忌避する方法や化学合成農薬を使用せずに天然物質に由来する薬剤を用いて植物を病害虫から防除する方法などの対策が報告されている。天然物質に由来する薬剤としては、例えば、特許文献１には、微生物バイオサーファクタントが記載されている。 Therefore, measures such as a method for repelling pests and a method for controlling plants from pests using chemicals derived from natural substances without using chemically synthesized pesticides have been reported. As a drug derived from a natural substance, for example, Patent Document 1 describes a microbial biosurfactant.
　また、虫に対する毒ではなく、オレイン酸や脂肪酸エステル、合成でんぷんなどを有効成分とした気門封鎖系の殺虫剤を使用する方法も知られている。気門封鎖型殺虫剤は、害虫の気門を塞ぎ、窒息させることで殺虫効果を発揮する。大型の有益昆虫類を殺虫するリスクや薬剤耐性虫を発生させるリスクが少ないと考えられている。 In addition, there is also known a method using an air-sealed insecticide containing oleic acid, fatty acid ester, synthetic starch and the like as an active ingredient instead of a poison for insects. The air gate-sealing insecticide exerts an insecticidal effect by closing the air gate of the pest and suffocating it. The risk of killing large beneficial insects and the risk of generating drug-resistant insects are considered to be low.
特表２００８－５０１０３９号公報Special table 2008-501039 gazette
　特許文献１には、バイオサーファクタントとして、ラムノリピドを含有させた発酵ブロス濾液を用いた病害虫を防除する方法が開示されており、これをイエバエや線虫を防除するための殺虫剤として使用することが記載されている。しかしながら、特許文献１記載の病害虫を防除する方法では、病害虫を死滅させるために直接薬剤を適用しなければならないため、病害虫が発生するごとに薬剤を施用する必要性がある。 Patent Document 1 discloses a method for controlling pests using a fermentation broth filtrate containing rhamnolipid as a biosurfactant, which can be used as an insecticide for controlling house flies and nematodes. Are listed. However, in the method for controlling pests described in Patent Document 1, a drug must be applied directly to kill the pest, and therefore, it is necessary to apply the drug every time the pest is generated.
　従来の気門封鎖型殺虫剤もまた、植物害虫に対して直接噴霧しなければならないため、発生の度に噴霧する必要がある。とりわけ、世代交代の早い高温期等においては、頻繁に噴霧しなければならない。 Since conventional air gate-sealing insecticides must also be sprayed directly against plant pests, they must be sprayed each time they occur. In particular, it must be frequently sprayed, such as in a high temperature period where the generation changes quickly.
　また、従来の害虫を忌避する方法は、薬剤が徐々に揮散することで効力を発揮するものであって、害虫を忌避させる範囲を広くできる利点を有しているが、揮発によって薬剤が消失していくため、効果の持続性が短いという問題点がある。 In addition, the conventional method of repelling pests is effective by gradually evaporating the drug, and has the advantage of widening the range of repelling pests, but the drug disappears due to volatilization. Therefore, there is a problem that the sustainability of the effect is short.
　過酸化脂質をはじめ脂肪酸酸化物が抗菌作用を示すことは知られている。また、リノレン酸などの脂肪酸を原料にし、植物体内でジャスモン酸などが生合成されることも知られている。しかしながら、脂肪酸が微生物代謝によって酸化された脂肪酸代謝物に植物における忌避効果を誘導する作用があることは知られていない。 It is known that fatty acid oxides including lipid peroxides exhibit antibacterial action. It is also known that jasmonic acid and the like are biosynthesized in plants using fatty acids such as linolenic acid as raw materials. However, it is not known that fatty acid metabolites obtained by oxidizing fatty acids by microbial metabolism have an effect of inducing repellent effects in plants.
　本発明は、前記課題に鑑み、土壌汚染や毒性が低く、植物における忌避効果を長期的に発揮することのできる害虫忌避剤、およびその製造方法を提供することを目的とする。 In view of the above problems, an object of the present invention is to provide a pest repellent that has low soil contamination and toxicity and can exhibit a long-term repellent effect in plants, and a method for producing the same.
　本発明は、脂肪酸をプロテオバクテリアに代謝させることで得られる脂肪酸代謝物を含む害虫忌避剤に関する。 The present invention relates to a pest repellent containing a fatty acid metabolite obtained by metabolizing fatty acids to proteobacteria.
　前記害虫忌避剤が、植物における害虫忌避物質分泌促進剤である害虫忌避剤が好ましい。 The pest repellent is preferably a pest repellent which is a pest repellent secretion promoter in plants.
　前記脂肪酸が、炭素数４～３０の脂肪酸である害虫忌避剤が好ましい。 A pest repellent in which the fatty acid is a fatty acid having 4 to 30 carbon atoms is preferred.
　前記脂肪酸が、２０℃で液体の脂肪酸である害虫忌避剤が好ましい。 A pest repellent in which the fatty acid is a fatty acid that is liquid at 20 ° C. is preferred.
　前記代謝が、０．１～８ｍｇ／ｌ（リットル）の溶存酸素濃度環境下での代謝である害虫忌避剤が好ましい。 A pest repellent is preferred in which the metabolism is metabolism in a dissolved oxygen concentration environment of 0.1 to 8 mg / l (liter).
　前記代謝が、Ｍｇ、Ｐ、ＮａおよびＫから選ばれる少なくとも１種以上のミネラルの存在下での代謝である害虫忌避剤が好ましい。 A pest repellent in which the metabolism is metabolism in the presence of at least one mineral selected from Mg, P, Na and K is preferred.
　前記プロテオバクテリアが、前培養されたプロテオバクテリアである害虫忌避剤が好ましい。 A pest repellent is preferred in which the proteobacteria are pre-cultured proteobacteria.
　前記前培養されたプロテオバクテリアが、菌数１×１０8～９×１０10ｃｅｌｌｓ／ｍｌ（ミリリットル）に前培養されたプロテオバクテリアである害虫忌避剤が好ましい。 Pest repellents are preferred wherein the pre-cultured proteobacteria are proteobacteria pre-cultured to 1 × 10 8 to 9 × 10 10 cells / ml (milliliter).
　前記害虫忌避剤が、バイオサーファクタントを含む害虫忌避剤であることが好ましい。 The pest repellent is preferably a pest repellent containing a biosurfactant.
　前記代謝が、２０～３０℃の条件下での代謝である害虫忌避剤が好ましい。 A pest repellent in which the metabolism is metabolism under conditions of 20 to 30 ° C. is preferable.
　前記害虫忌避剤が、植物の茎葉または根に接触されるように用いられる害虫忌避剤であることが好ましい。 The pest repellent is preferably a pest repellent that is used so as to come into contact with the foliage or roots of a plant.
　また、本発明は、脂肪酸をプロテオバクテリアに代謝させる脂肪酸代謝工程を含む、脂肪酸代謝物を含む害虫忌避剤の製造方法に関する。 The present invention also relates to a method for producing a pest repellent containing a fatty acid metabolite, which includes a fatty acid metabolism step of metabolizing fatty acid to proteobacteria.
　前記害虫忌避剤が、植物における害虫忌避物質分泌促進剤である害虫忌避剤の製造方法が好ましい。 A method for producing a pest repellent, wherein the pest repellent is a pest repellent secretion promoter in plants, is preferred.
　前記脂肪酸が、炭素数４～３０の脂肪酸である害虫忌避剤の製造方法が好ましい。 A method for producing a pest repellent in which the fatty acid is a fatty acid having 4 to 30 carbon atoms is preferred.
　前記脂肪酸が、２０℃で液体の脂肪酸である害虫忌避剤の製造方法が好ましい。 A method for producing a pest repellent in which the fatty acid is a liquid fatty acid at 20 ° C. is preferred.
　前記脂肪酸代謝工程を、０．１～８ｍｇ／ｌの溶存酸素濃度環境下で実施する害虫忌避剤の製造方法が好ましい。 A method for producing a pest repellent in which the fatty acid metabolism step is performed in an environment of dissolved oxygen concentration of 0.1 to 8 mg / l is preferable.
　前記脂肪酸代謝工程を、Ｍｇ、Ｐ、ＮａおよびＫから選ばれる少なくとも１種以上のミネラルの存在下で実施する害虫忌避剤の製造方法が好ましい。 A method for producing a pest repellent in which the fatty acid metabolism step is carried out in the presence of at least one mineral selected from Mg, P, Na and K is preferred.
　前記プロテオバクテリアが、前培養されたプロテオバクテリアである害虫忌避剤の製造方法が好ましい。 A method for producing a pest repellent wherein the proteobacteria are pre-cultured proteobacteria is preferred.
　前記前培養されたプロテオバクテリアが、菌数１×１０8～９×１０10ｃｅｌｌｓ／ｍｌに前培養されたプロテオバクテリアである害虫忌避剤の製造方法が好ましい。 A method for producing a pest repellent, wherein the pre-cultured proteobacteria are pre-cultured to 1 × 10 8 to 9 × 10 10 cells / ml, is preferred.
　バイオサーファクタントを含む害虫忌避剤の製造方法である害虫忌避剤の製造方法が好ましい。 A method for producing a pest repellent which is a method for producing a pest repellent containing a biosurfactant is preferred.
　前記脂肪酸代謝工程を、２０～３０℃の条件下で実施する害虫忌避剤の製造方法が好ましい。 A method for producing a pest repellent in which the fatty acid metabolism step is carried out at 20 to 30 ° C. is preferable.
　植物に施用される害虫忌避剤の製造方法である害虫忌避剤の製造方法が好ましい。 A method for producing a pest repellent which is a method for producing a pest repellent applied to plants is preferred.
　本発明の害虫忌避剤は、土壌汚染や毒性が低く、植物における長期的な忌避効果に優れる。また、本発明の害虫忌避剤の製造方法によれば、土壌汚染や毒性が低く、植物における長期的な忌避効果に優れる害虫忌避剤を製造することができる。 The pest repellent of the present invention has low soil contamination and toxicity and is excellent in long-term repellent effect in plants. Moreover, according to the method for producing a pest repellent of the present invention, a pest repellent having low soil contamination and toxicity and excellent in long-term repellent effect in plants can be produced.
ワタアブラムシの忌避効果を示すグラフである。It is a graph which shows the repellent effect of cotton aphid.
害虫忌避剤
　本発明の害虫忌避剤は、脂肪酸をプロテオバクテリアに代謝させることで得られる脂肪酸代謝物を含むことを特徴とする。 Pest repellent The pest repellent of the present invention is characterized by containing a fatty acid metabolite obtained by metabolizing fatty acids to proteobacteria.
　脂肪酸代謝物を植物の茎葉または根の一部に接触させることで、脂肪酸代謝物中に含まれ得る忌避物質による直接の害虫忌避効果に加えて、植物に抵抗性が誘導され得る。これは、脂肪酸代謝物中に、忌避物質に加えて、全身獲得抵抗性に関わるサリチル酸経路や、ジャスモン酸経路を活性化する物質またはこの物質の前駆体が含まれていることによると考えられる。抵抗性誘導の結果、植物自身に忌避物質を産生および／または分泌させたり、本来備わっている防虫に関する植物の機能をより発達させたりすることができる。したがって、本発明の害虫忌避剤は、植物からの害虫忌避物質の分泌を促進させることのできる物質を含む害虫忌避物質分泌促進剤である。そして、本発明の害虫忌避剤は、害虫忌避物質分泌促進物質に加えて、直接的な害虫忌避効果を有する忌避物質をさらに含んでいてもよい。 By bringing a fatty acid metabolite into contact with a part of a plant's foliage or root, resistance to the plant can be induced in addition to a direct pest repellent effect by a repellent substance that can be contained in the fatty acid metabolite. This is considered to be because the fatty acid metabolite contains, in addition to the repellent substance, a salicylic acid pathway related to systemic acquired resistance, a substance that activates the jasmonic acid pathway, or a precursor of this substance. As a result of the induction of resistance, the plant itself can produce and / or secrete repellents, and the functions of the plant relating to the inherent insect repellent can be further developed. Therefore, the pest repellent of the present invention is a pest repellent secretion promoter containing a substance capable of promoting the secretion of a pest repellent from a plant. The pest repellent of the present invention may further contain a repellent substance having a direct pest repellent effect in addition to the pest repellent secretion promoting substance.
　本発明の害虫忌避剤は、従来の害虫忌避剤と比べてより長期的な忌避効果を発揮でき、また従来の殺虫剤に対する薬剤抵抗性を発達させた害虫にも有効であると考えられる。かつ、脂肪酸代謝物は、化学合成農薬と比べその毒性が遥かに低い。 The pest repellent of the present invention can exhibit a long-term repellent effect as compared with conventional pest repellents, and is also considered to be effective for pests that have developed drug resistance against conventional pesticides. In addition, fatty acid metabolites are much less toxic than chemically synthesized pesticides.
　本発明における代謝とは、プロテオバクテリアが外分泌または内分泌する酵素等により脂肪酸の分解が行われることをいう。例えば、脂肪酸を含有する培地でプロテオバクテリアを培養する方法が挙げられる。 Metabolism in the present invention means that fatty acids are decomposed by an enzyme or the like secreted by proteobacteria. For example, a method of culturing proteobacteria in a medium containing fatty acid can be mentioned.
　プロテオバクテリアは、脂肪酸の代謝に関わる酵素であるリポキシゲナーゼ（lipoxygenase：ＬＯＸ）を産生する遺伝子を持っており、脂肪酸代謝物を生成できる。 Proteobacteria have a gene that produces lipoxygenase (LOX), an enzyme involved in fatty acid metabolism, and can produce fatty acid metabolites.
　本発明において用いられる脂肪酸の炭素数は４～３０であり、１０～２０が好ましい。炭素数が４未満の場合は、融点・沸点が低いため、培養時の温度で揮発性が高まり培地中に残存しにくくなる傾向がある。また、炭素数が３０を超える場合は、融点・沸点が高くなるため、培養時の温度で固体となり培地と混合できず分離してしまう傾向がある。ただし、融点は水素結合の数によって炭素数のみに依存しない場合もある。 The carbon number of the fatty acid used in the present invention is 4 to 30, and preferably 10 to 20. When the number of carbon atoms is less than 4, the melting point / boiling point is low, and therefore, the volatility tends to increase at the temperature at the time of culturing and hardly remain in the medium. Further, when the number of carbons exceeds 30, the melting point / boiling point becomes high, so that it becomes a solid at the temperature at the time of culturing and tends to be separated without being mixed with the medium. However, the melting point may not depend only on the number of carbons depending on the number of hydrogen bonds.
　本発明において用いられる脂肪酸は、代謝効率の観点や培地中で固化することを抑制する観点から、２０～３０℃で液体であることが好ましく、２０℃で液体であることがより好ましい。 The fatty acid used in the present invention is preferably a liquid at 20 to 30 ° C. and more preferably a liquid at 20 ° C. from the viewpoint of metabolic efficiency and suppression of solidification in the medium.
　本発明の脂肪酸は、飽和脂肪酸もしくは不飽和脂肪酸のいずれか、または両方を含む混合物とすることができる。また、植物油やグリセリドの形態や遊離脂肪酸を用いることができるが、分解速度に優れるという理由から遊離脂肪酸（モノカルボン酸）が好ましい。 The fatty acid of the present invention can be a saturated fatty acid, an unsaturated fatty acid, or a mixture containing both. Moreover, although the form of vegetable oil and a glyceride, and a free fatty acid can be used, a free fatty acid (monocarboxylic acid) is preferable because it is excellent in a decomposition rate.
　炭素数４～３０の遊離脂肪酸としては、酪酸（ブチル酸）、吉草酸（バレリアン酸）、カプロン酸、エナント酸（ヘプチル酸）、カプリル酸、ペラルゴン酸、カプリン酸、ラウリン酸、ミリスチン酸、ペンタデシル酸、パルミチン酸、パルミトレイン酸、マルガリン酸、ステアリン酸、オレイン酸、バクセン酸、リノール酸、α－リノレン酸、γ－リノレン酸、エレオステアリン酸、アラキジン酸、ミード酸、アラキドン酸、ベヘン酸、リグノセリン酸、ネルボン酸、セロチン酸、モンタン酸、メリシン酸などが挙げられ、なかでも炭素数が１０～２０のカプリン酸、ラウリン酸、ミリスチン酸、ペンタデシル酸、パルミチン酸、パルミトレイン酸、マルガリン酸、ステアリン酸、オレイン酸、バクセン酸、リノール酸、α－リノレン酸、γ－リノレン酸、エレオステアリン酸、アラキジン酸、ミード酸、アラキドン酸が好ましく、炭素数が１８のオレイン酸、リノール酸、α－リノレン酸、γ－リノレン酸がより好ましい。 Free fatty acids having 4 to 30 carbon atoms include butyric acid (butyric acid), valeric acid (valeric acid), caproic acid, enanthic acid (heptylic acid), caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, pentadecyl Acid, palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, vaccenic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, eleostearic acid, arachidic acid, mead acid, arachidonic acid, behenic acid, Examples include lignoceric acid, nervonic acid, serotic acid, montanic acid, and melicic acid. Among them, capric acid having 10 to 20 carbon atoms, lauric acid, myristic acid, pentadecylic acid, palmitic acid, palmitoleic acid, margaric acid, stearin Acid, oleic acid, vaccenic acid, linoleic acid, α-linolenic acid, γ Linolenic acid, eleostearic acid, arachidic acid, mead acid, arachidonic acid are preferred, oleic acid having 18 carbon atoms, linoleic acid, alpha-linolenic acid, .gamma.-linolenic acid are more preferred.
　脂肪酸を含有する培地を使用する場合の脂肪酸の含有量は、１２０ｇ／ｌ以下が好ましく、１００ｇ／ｌ以下がより好ましく、６０ｇ／ｌ以下がさらに好ましい。１２０ｇ／ｌを超える場合は、培地の水分との乳化が困難となり、代謝効率が悪化する恐れやプロテオバクテリアの生育を阻害する恐れがある。また、脂肪酸の含有量の下限は特に限定されないが、１．０ｇ／ｌ以上が好ましい。 When using a medium containing fatty acid, the content of fatty acid is preferably 120 g / l or less, more preferably 100 g / l or less, and even more preferably 60 g / l or less. If it exceeds 120 g / l, emulsification with water in the medium becomes difficult, which may deteriorate metabolic efficiency and inhibit the growth of proteobacteria. The lower limit of the fatty acid content is not particularly limited, but is preferably 1.0 g / l or more.
　脂肪酸を含有する培地は、他にミネラル成分を含有することが好ましい。ミネラル成分としては、特に限定されず微生物培養に通常用いられるミネラル成分を挙げることができる。例えば、マグネシウム（Ｍｇ）、リン（Ｐ）、ナトリウム（Ｎａ）またはカリウム（Ｋ）を有する成分が挙げられる。これらの成分は単独で使用することも、複数を併用することもできる。好ましくはこれらの成分のうちの２種類、さらに好ましくは３種類以上が使用され得る。培地中のミネラル成分の含有量は特に限定されず、従来の好気性細菌の培養方法で使用される量とすることができるが、植物への施用時に塩害が発生する恐れがあるため、好ましくは１５ｇ／ｌ以下、より好ましくは１０ｇ／ｌ以下で使用され得る。 It is preferable that the medium containing fatty acid contains other mineral components. As a mineral component, it is not specifically limited, The mineral component normally used for microorganism culture can be mentioned. For example, the component which has magnesium (Mg), phosphorus (P), sodium (Na), or potassium (K) is mentioned. These components can be used alone or in combination. Preferably two of these components may be used, more preferably three or more. The content of the mineral component in the medium is not particularly limited, and can be the amount used in the conventional aerobic bacterium culture method, but since salt damage may occur during application to plants, preferably It can be used at 15 g / l or less, more preferably at 10 g / l or less.
　本発明にて用いられるプロテオバクテリアは、本発明の効果を損なわない限り特に限定されない。好ましくは脂肪酸の代謝効率や生育効率の観点から、増殖に適した温度（至適温度）が１０～４０℃のプロテオバクテリアが好ましく、２０～３０℃のプロテオバクテリアがより好ましい。 The proteobacteria used in the present invention is not particularly limited as long as the effects of the present invention are not impaired. From the viewpoint of fatty acid metabolism efficiency and growth efficiency, proteobacteria having a temperature suitable for growth (optimum temperature) of 10 to 40 ° C. are preferred, and proteobacteria having 20 to 30 ° C. are more preferred.
　プロテオバクテリアは、脂肪酸の代謝効率に優れるという理由から、前培養されたプロテオバクテリアであることが好ましく、菌数が１×１０8～９×１０10ｃｅｌｌｓ／ｍｌまで前培養されていることがより好ましい。 The proteobacteria are preferably pre-cultured proteobacteria because they are excellent in fatty acid metabolic efficiency, and the number of bacteria is preferably pre-cultured to 1 × 10 8 to 9 × 10 10 cells / ml. preferable.
　本発明においては、代謝が、所定の溶存酸素濃度環境下で行われることが好ましい。代謝における溶存酸素濃度は、０．１ｍｇ／ｌ以上であることが好ましい。溶存酸素濃度が０．１ｍｇ／ｌ未満の場合は、プロテオバクテリアの脂肪酸分解能が低下し脂肪酸の代謝効率が極めて低くなる傾向がある。また、溶存酸素濃度は例えば、８ｍｇ／ｌ以下であることが好ましい。溶存酸素濃度が８ｍｇ／ｌを超える場合は、プロテオバクテリアによる代謝工程と並行して、基質である脂肪酸の培地中の酸素による分解が進行してしまい、代謝効率が低下し、ひいては有効成分である代謝産物の産生量が低下してしまう恐れがある。好ましくは、溶存酸素濃度は、０．１～８ｍｇ／ｌであり、より好ましくは０．１～５ｍｇ／ｌであり、さらに好ましくは０．１～４ｍｇ／ｌである。なお、溶存酸素濃度は株式会社堀場製作所製の溶存酸素計でＰＯ電極に隔膜ガルバニ電極法または隔膜ポーラログラフ法により測定される値とする。 In the present invention, the metabolism is preferably performed in a predetermined dissolved oxygen concentration environment. The dissolved oxygen concentration in metabolism is preferably 0.1 mg / l or more. When the dissolved oxygen concentration is less than 0.1 mg / l, the fatty acid resolution of proteobacteria tends to decrease and the metabolic efficiency of fatty acids tends to be extremely low. Moreover, it is preferable that a dissolved oxygen concentration is 8 mg / l or less, for example. When the dissolved oxygen concentration exceeds 8 mg / l, in parallel with the metabolic process by proteobacteria, decomposition of the fatty acid, which is a substrate, by oxygen in the medium proceeds, resulting in a decrease in metabolic efficiency and, consequently, an active ingredient. Metabolite production may be reduced. Preferably, the dissolved oxygen concentration is 0.1 to 8 mg / l, more preferably 0.1 to 5 mg / l, and still more preferably 0.1 to 4 mg / l. The dissolved oxygen concentration is a value measured by a diaphragm galvanic electrode method or a diaphragm polarographic method on a PO electrode with a dissolved oxygen meter manufactured by HORIBA, Ltd.
　代謝における温度は使用するプロテオバクテリアに応じて適宜調整することができるが、脂肪酸の代謝効率の観点から、２０～３０℃が好ましい。 The temperature in metabolism can be appropriately adjusted according to the proteobacteria used, but 20-30 ° C. is preferable from the viewpoint of fatty acid metabolic efficiency.
　本発明において、害虫忌避剤は、脂肪酸代謝物に加えバイオサーファクタントを含有し得る。脂肪酸代謝物が水に分散されやすくなり、害虫忌避剤の取扱性の観点から好ましいと考えられる。本発明に係るバイオサーファクタントとは、微生物が疎水性の高い物質を取り込むために産生し、細胞外へと分泌する界面活性剤様の物質を意味する。本発明において、プロテオバクテリアによって分泌されたバイオサーファクタントは、脂肪酸代謝物の水への分散も容易にするため、脂肪酸代謝物を含む害虫忌避剤の噴霧処理や灌注などが効率よく簡単に行えるようになる。しかしながら、バイオサーファクタントとしては、脂肪酸の分解時に本発明のプロテオバクテリアによって産生されたバイオサーファクタントだけではなく、他の微生物が産生したバイオサーファクタントが使用されてもよく、すなわち、本発明の害虫忌避剤には他の微生物によって産生されたバイオサーファクタントがさらに添加されてもよい。人工的に合成された界面活性剤と比較して、バイオサーファクタントは生物に関する毒性が低く、また、生分解性も高いため、より環境に優しい害虫忌避剤が得られると考えられる。また、プロテオバクテリアによる脂肪酸分解を促進させるために、他の微生物が産生したバイオサーファクタントが、プロテオバクテリアによる脂肪酸分解において添加されてもよい。プロテオバクテリアによる脂肪酸の取り込みが促進される可能性がある。 In the present invention, the insect repellent may contain a biosurfactant in addition to the fatty acid metabolite. Fatty acid metabolites are easily dispersed in water, which is considered preferable from the viewpoint of handling properties of pest repellents. The biosurfactant according to the present invention means a surfactant-like substance that is produced by a microorganism to take up a highly hydrophobic substance and secreted outside the cell. In the present invention, the biosurfactant secreted by proteobacteria facilitates the dispersion of fatty acid metabolites in water so that spray treatment and irrigation of pest repellents containing fatty acid metabolites can be performed efficiently and easily. Become. However, as the biosurfactant, not only the biosurfactant produced by the proteobacteria of the present invention at the time of fatty acid degradation, but also biosurfactants produced by other microorganisms may be used, that is, the pest repellent of the present invention. Biosurfactants produced by other microorganisms may be further added. Compared to artificially synthesized surfactants, biosurfactants are less toxic to living organisms and have higher biodegradability, which is thought to provide a more environmentally friendly pest repellent. In addition, in order to promote fatty acid degradation by proteobacteria, biosurfactants produced by other microorganisms may be added during fatty acid degradation by proteobacteria. Fatty acid uptake by proteobacteria may be promoted.
　本発明の害虫忌避剤は、土壌汚染や毒性が低く、かつ植物において長期的な忌避効果を誘導し得るという害虫忌避剤としての優れた効果を有する。本発明の害虫忌避剤は、施用される植物において感染特異的タンパク質（pathogenesis-related (PR) protein）の生成に関与する遺伝子群やサリチル酸またはジャスモン酸を生成する酵素産生に関わる遺伝子群などを活性化して全身獲得抵抗性を誘導することができる。例えば、ジャスモン酸により誘導される抗害虫性のプロテアーゼ・インヒビターや昆虫忌避物質などの二次代謝産物等の生合成が増加されると推定している。しかも、本発明の害虫忌避剤は、サリチル酸経路およびジャスモン酸経路ともに、上流および下流の遺伝子の両方を発現させることができるので、抵抗性誘導が持続し、持続的な忌避効果が得られるため特に有利である。 The pest repellent of the present invention has an excellent effect as a pest repellent that is low in soil contamination and toxicity and can induce a long-term repellent effect in plants. The pest repellent of the present invention activates a group of genes involved in the production of infection-specific proteins (pathogenesis-related (PR) protein) or a group of genes involved in the production of enzymes producing salicylic acid or jasmonic acid in the applied plant. To induce systemic acquired resistance. For example, it is estimated that the biosynthesis of secondary metabolites such as anti-pesticide protease inhibitors and insect repellents induced by jasmonic acid is increased. Moreover, since the pest repellent of the present invention can express both upstream and downstream genes in both the salicylic acid pathway and the jasmonic acid pathway, resistance induction persists and a continuous repellent effect is obtained. It is advantageous.
　本発明の害虫忌避剤は、植物の病害虫への抵抗性に関わる器官の発達を誘導し得る。器官の発達の具体例としては、毛状突起（トライコーム）の増加した形成が挙げられる。トライコームは、傷害に応答したジャスモン酸シグナル伝達の活性化により発達することが知られており、トライコームの発達は、植物に抵抗性が誘導されたことを示すものである。トライコームは、その先端から揮発性の害虫忌避成分を放出し得る。したがって、トライコームを発達させることにより、揮発性害虫忌避成分のより効率的な拡散が可能となり、植物の防御機能を高めることができる。また、種によっては、トライコーム自身に虫を絡め捕る機能があることも知られている。さらに、トライコーム密度の増加は、植食性昆虫に対する物理的な障壁としての防虫効果も高め得るため、植物自身の害虫忌避効果がより活性化され得る。 The pest repellent of the present invention can induce the development of organs related to resistance to plant pests. A specific example of organ development is the increased formation of trichomes (trichomes). Tricombs are known to develop by the activation of jasmonic acid signaling in response to injury, and the development of trichomes indicates that resistance has been induced in plants. Tricomb can release volatile pest repellent components from its tip. Therefore, by developing trichomes, volatile pest repellent components can be more efficiently diffused and the defense function of the plant can be enhanced. It is also known that some trichomes have a function of catching and catching insects depending on the species. Furthermore, since the increase in trichome density can also increase the insect repellent effect as a physical barrier against herbivorous insects, the pest repellent effect of the plant itself can be more activated.
　さらに、本発明の害虫忌避剤は、脂肪酸代謝物中に、植食者誘導性揮発性物質である揮発性アルデヒド、アルコールおよびそれらのエステル体に加え、これらの前駆物質をさらに含有していてもよい。前駆物質は、植物に吸収されたり、自然環境下で分解されたりすることにより、揮発性アルデヒド、アルコールおよびそれらのエステル体に変換され得る。したがって、害虫忌避剤の植物への適用時から、前駆物質が揮発性アルデヒド、アルコールおよびそれらのエステル体へと変換されるまでの時間の遅れによって、忌避効果に持続性がさらに付与されると考えられる。 Furthermore, the pest repellent of the present invention may further contain these precursors in addition to volatile aldehydes, alcohols and esters thereof, which are herbivore-induced volatile substances, in fatty acid metabolites. Good. Precursors can be converted to volatile aldehydes, alcohols and their ester forms by being absorbed by plants or decomposed in the natural environment. Therefore, the persistence of the repellent effect is thought to be due to the delay in the time from the application of the pest repellent to the plant until the precursor is converted to volatile aldehydes, alcohols and their esters. It is done.
　本発明の害虫忌避剤は、農業害虫を忌避するために用いられ得る。農業害虫としては、これらに限定される訳ではないが、ワタアブラムシなどのアブラムシ科、ハダニ科、コガネムシ科、ハスモンヨトウなどのヤガ科、チュウレンジハバチなどのハバチ科などの害虫が例示される。本発明の害虫忌避剤は、殺虫剤に対する感受性が低いことが知られているハスモンヨトウの幼虫などに対しても忌避効果を示すため、従来防除が困難であるとされてきた害虫に対しても効果を発揮できるという点で優れている。 The pest repellent of the present invention can be used to repel agricultural pests. Examples of agricultural pests include, but are not limited to, pests such as aphids such as cotton aphids, spider mites, scarab beetles, stag beetles such as scallops, and bees such as chrysanthemum. The pest repellent of the present invention exhibits a repellent effect even against the larvae of Spodoptera litura, which is known to have low susceptibility to insecticides, and is therefore effective against pests that have been conventionally difficult to control. It is excellent in that it can demonstrate.
　本発明の害虫忌避剤の植物への施用方法としては特に限定されない。例えば、植物の茎葉もしくは根への噴霧、植物の茎葉もしくは根の浸漬、および／または、植物の生育環境である土壌に対する土壌灌注によって施用され得る。また、施用される植物も限定されるものではなく、植物一般に対して良好に用いることができる。例えば、ナス科、ウリ科、バラ科などの双子葉類植物、イネ科などの単子葉類植物が挙げられる。 The method for applying the pest repellent of the present invention to plants is not particularly limited. For example, it can be applied by spraying on the foliage or roots of the plant, dipping the foliage or roots of the plant, and / or soil irrigation on the soil in which the plant is growing. Moreover, the plant to be applied is not limited, and it can be used favorably for plants in general. Examples thereof include dicotyledonous plants such as eggplant, cucurbitaceae, and roseceae, and monocotyledonous plants such as gramineous.
製造方法
　本発明の脂肪酸代謝物を含む害虫忌避剤の製造方法は、脂肪酸をプロテオバクテリアに代謝させる脂肪酸代謝工程を含むことを特徴とする。 Manufacturing method The manufacturing method of the pest repellent containing the fatty acid metabolite of this invention is characterized by including the fatty acid metabolism process which metabolizes a fatty acid to proteobacteria.
　本発明の製造方法により得られる害虫忌避剤は、植物における害虫忌避物質分泌促進剤である。すなわち、本発明の脂肪酸代謝工程により、植物からの害虫忌避物質の分泌を促進させることのできる物質が製造され得る。本発明の脂肪酸代謝工程は、このような害虫忌避物質分泌促進物質に加えて、直接的な害虫忌避効果を有する忌避物質が製造される工程であってもよい。 The pest repellent obtained by the production method of the present invention is a pest repellent secretion promoter in plants. That is, a substance capable of promoting the secretion of a pest repellent substance from a plant can be produced by the fatty acid metabolism process of the present invention. The fatty acid metabolism step of the present invention may be a step in which a repellent substance having a direct pest repellent effect is produced in addition to such a pest repellent secretion promoting substance.
　本発明における脂肪酸代謝工程は、プロテオバクテリアが外分泌または内分泌する酵素等により脂肪酸の分解が行われる工程である。例えば、脂肪酸を含有する培地でプロテオバクテリアを培養することにより行われる工程が挙げられる。 In the present invention, the fatty acid metabolism step is a step in which fatty acids are decomposed by an enzyme or the like that is secreted or secreted by proteobacteria. For example, the process performed by culture | cultivating proteobacteria with the culture medium containing a fatty acid is mentioned.
　脂肪酸代謝工程は、所定の溶存酸素濃度環境下で行われることが好ましい。例えば、脂肪酸代謝工程における溶存酸素濃度は、０．１ｍｇ／ｌ以上であることが好ましい。溶存酸素濃度が０．１ｍｇ／ｌ未満の場合は、プロテオバクテリアの脂肪酸分解能が低下し脂肪酸の代謝効率が極めて低くなる傾向がある。また、溶存酸素濃度は例えば、８ｍｇ／ｌ以下であることが好ましい。溶存酸素濃度が８ｍｇ／ｌを超える場合は、プロテオバクテリアによる代謝工程と並行して、基質である脂肪酸の培地中の酸素による分解が進行してしまい、代謝効率が低下し、ひいては有効成分である代謝産物の産生量が低下してしまう恐れがある。好ましくは、溶存酸素濃度は、０．１～８ｍｇ／ｌであり、より好ましくは０．１～５ｍｇ／ｌであり、さらに好ましくは０．１～４ｍｇ／ｌである。なお、溶存酸素濃度は株式会社堀場製作所製の溶存酸素計でＰＯ電極に隔膜ガルバニ電極法または隔膜ポーラログラフ法により測定される値とする。 The fatty acid metabolism step is preferably performed in a predetermined dissolved oxygen concentration environment. For example, the dissolved oxygen concentration in the fatty acid metabolism step is preferably 0.1 mg / l or more. When the dissolved oxygen concentration is less than 0.1 mg / l, the fatty acid resolution of proteobacteria tends to decrease and the metabolic efficiency of fatty acids tends to be extremely low. Moreover, it is preferable that a dissolved oxygen concentration is 8 mg / l or less, for example. When the dissolved oxygen concentration exceeds 8 mg / l, in parallel with the metabolic process by proteobacteria, decomposition of the fatty acid, which is a substrate, by oxygen in the medium proceeds, resulting in a decrease in metabolic efficiency and, consequently, an active ingredient. Metabolite production may be reduced. Preferably, the dissolved oxygen concentration is 0.1 to 8 mg / l, more preferably 0.1 to 5 mg / l, and still more preferably 0.1 to 4 mg / l. The dissolved oxygen concentration is a value measured by a diaphragm galvanic electrode method or a diaphragm polarographic method on a PO electrode with a dissolved oxygen meter manufactured by HORIBA, Ltd.
　溶存酸素濃度は、培養容器、振とう数、通気量などによって、調整することができる。 The dissolved oxygen concentration can be adjusted by the culture vessel, the number of shakes, the amount of aeration, etc.
　脂肪酸代謝工程における培養条件は、溶存酸素濃度を所定の範囲とすること以外は、従来の好気性細菌を培養する条件と同様の条件とすることができる。例えば、フラスコによる振とうや、スピナーフラスコまたはジャーファメンターによる通気培養により３～７日間培養する方法が挙げられる。 The culture conditions in the fatty acid metabolism step can be the same as the conventional conditions for culturing aerobic bacteria except that the dissolved oxygen concentration is within a predetermined range. Examples thereof include a method of culturing for 3 to 7 days by shaking with a flask and aeration culture with a spinner flask or a jar fermenter.
　培養日数は、脂肪酸の乳化、分解等が充分に行われる日数とすることが好ましいが、撹拌や菌量によって培養日数は変化する。なお、脂肪酸代謝工程の終了は、脂肪酸の分解状態を、波長２３０ｎｍにおける吸光度の測定、薄層クロマトグラフィー（ＴＬＣ）、高速液体クロマトグラフィー（ＨＰＬＣ）、ガスクロマトグラフ質量分析（ＧＣ／ＭＳ）、液体クロマトグラフ質量分析（ＬＣ／ＭＳ）等で確認することが好ましい。 The number of days of culture is preferably the number of days during which the fatty acid is sufficiently emulsified and decomposed, but the number of days of culture varies depending on the agitation and the amount of bacteria. At the end of the fatty acid metabolism step, the fatty acid decomposition state is determined by measuring the absorbance at a wavelength of 230 nm, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC / MS), liquid chromatography. It is preferable to confirm by graph mass spectrometry (LC / MS) or the like.
　脂肪酸代謝工程における温度は使用するプロテオバクテリアに応じて適宜調整することができ、脂肪酸の代謝効率の観点から、２０～３０℃の条件下で実施することが好ましい。 The temperature in the fatty acid metabolism step can be appropriately adjusted according to the proteobacteria used, and it is preferable to carry out the treatment at 20 to 30 ° C. from the viewpoint of fatty acid metabolic efficiency.
　脂肪酸代謝工程における脂肪酸およびプロテオバクテリアは、本発明の害虫忌避剤の説明で前述したものを使用することができる。 As the fatty acid and proteobacteria in the fatty acid metabolism step, those described above in the description of the pest repellent of the present invention can be used.
　なお、プロテオバクテリアの前培養工程としては、特に限定されず通常の好気性細菌の培養方法とすることができる。前培養液から遠心分離等により菌体のみを回収し、脂肪酸代謝工程に用いることが好ましい。 In addition, the pre-culturing step for proteobacteria is not particularly limited, and a normal method for culturing aerobic bacteria can be used. It is preferable that only the cells are collected from the preculture solution by centrifugation or the like and used in the fatty acid metabolism step.
　本発明の製造方法により得られる害虫忌避剤は、脂肪酸代謝物に加えバイオサーファクタントを含有し得る。本発明に係るバイオサーファクタントとは、微生物が疎水性の高い物質を取り込むために産生し、細胞外へと分泌する界面活性剤様の物質を意味する。プロテオバクテリアによって分泌されたバイオサーファクタントは、脂肪酸代謝物の水への分散も容易にする。本発明の害虫忌避剤の取扱性が向上すると考えられる。 The pest repellent obtained by the production method of the present invention can contain a biosurfactant in addition to a fatty acid metabolite. The biosurfactant according to the present invention means a surfactant-like substance that is produced by a microorganism to take up a highly hydrophobic substance and secreted outside the cell. Biosurfactants secreted by proteobacteria also facilitate the dispersion of fatty acid metabolites in water. It is thought that the handleability of the pest repellent of the present invention is improved.
　本発明の害虫忌避剤は、培地、バイオサーファクタントを含むプロテオバクテリアの外分泌物、菌体などとの混合物である培養液として得られる。当該培養液をそのまま本発明の害虫忌避剤としてもよく、培養液から遠心分離などにより菌体を除去した上澄み液を害虫忌避剤としてもよい。本発明の製造方法により得られる害虫忌避剤は、植物に施用される。培養液は原液のままでも使用することができるが、原液の場合は高温時に植物への処理部分が、ミネラル分が蒸発濃縮され浸透圧の影響で縮む恐れがあるため、原液を希釈して使用することが望ましい場合がある。希釈倍率としては本発明の効果を発揮する限り特に限定されないが、１０～１０００倍希釈が好ましく、１０～１００倍希釈がさらに好ましい。なお、除去した菌体を再度、脂肪酸を含有する培地で培養することにより本発明の脂肪酸代謝工程を繰り返し行うことが可能である。 The pest repellent of the present invention is obtained as a culture solution that is a mixture with a culture medium, a proteobacterium exocrine product containing a biosurfactant, microbial cells, and the like. The culture solution may be used as it is as the pest repellent of the present invention, or the supernatant obtained by removing the cells from the culture solution by centrifugation or the like may be used as the pest repellent. The pest repellent obtained by the production method of the present invention is applied to plants. The culture solution can be used as it is. However, in the case of the stock solution, the treated portion of the plant at high temperatures may be condensed due to the evaporation of minerals and the effect of osmotic pressure. It may be desirable to do so. The dilution factor is not particularly limited as long as the effect of the present invention is exhibited, but is preferably 10 to 1000 times diluted, more preferably 10 to 100 times diluted. In addition, it is possible to repeat the fatty acid metabolism process of this invention by cultivating the removed microbial cell again in the culture medium containing a fatty acid.
　本発明を実施例に基づいて説明するが、本発明は実施例のみに限定されるものではない。 The present invention will be described based on examples, but the present invention is not limited to the examples.
試験用害虫忌避剤の調製
＜前培養工程＞
　ガラス製三角フラスコ内の１ｌ（リットル）の水にペプトン（Ｄｉｆｃｏ製のタンパク質酵素加水分解物）２０ｇ、硫酸マグネシウム七水和物１．５ｇおよびリン酸水素二カリウム１．５ｇ、または、１ｌ（リットル）の水にペプトン（Ｄｉｆｃｏ製のタンパク質酵素加水分解物）１０ｇ、イーストエキストラクト５ｇおよび塩化ナトリウム１０ｇを溶解させ、１２１℃、２０分間オートクレーブ滅菌を行い、室温まで冷却後、プロテオバクテリアの菌液を植菌した。なお、培養容器の口はシリコン栓で密栓した。植菌後の容器をバイオシェーカー（タイテック株式会社製のＢＲ－２３ＵＭ）を用い、２５℃±５℃、１２０ｒｐｍの条件下で、２４時間培養を行った。培養液中の菌数は５×１０8ｃｅｌｌｓ／ｍｌであった。培養後、培養液を１５，０００×Ｇ、２０℃の条件で遠心分離することで培養液から菌体を回収した。 Preparation of test pest repellent <pre-culture process>
20 g of peptone (Difco protein enzyme hydrolyzate), 1.5 g of magnesium sulfate heptahydrate and 1.5 g of dipotassium hydrogen phosphate in 1 l (liter) of water in a glass Erlenmeyer flask, or 1 l (liter) ) 10 g of peptone (Difco protein enzyme hydrolyzate), 5 g of yeast extract and 10 g of sodium chloride are dissolved in water, autoclaved at 121 ° C. for 20 minutes, and cooled to room temperature. Inoculated. The mouth of the culture vessel was sealed with a silicon stopper. The inoculated container was cultured for 24 hours under the conditions of 25 ° C. ± 5 ° C. and 120 rpm using a bioshaker (BR-23UM manufactured by Taitec Corporation). The number of bacteria in the culture was 5 × 10 8 cells / ml. After culturing, the cells were collected from the culture solution by centrifuging the culture solution under conditions of 15,000 × G and 20 ° C.
＜脂肪酸代謝工程＞
　ガラス製三角フラスコ内の１ｌ（リットル）の滅菌水に、リノール酸（和光純薬工業株式会社製の一級リノール酸）１２ｇ、硫酸マグネシウム七水和物１．５ｇおよび／またはリン酸水素二カリウム１．５ｇ、および前培養工程から得られた菌体の全量を加えた。これを、バイオシェーカー（タイテック株式会社製のＢＲ－２３ＵＭ）を用い、２０℃、１２０ｒｐｍ、溶存酸素濃度４ｍｇ／ｌの条件下で、４日間培養を行った。なお、リノール酸の分解は、リノール酸中間生成物の１つである酸化脂質の培養液中の濃度を株式会社島津製作所製の分光光度計ＢｉｏＳｐｅｃ－ｍｉｎｉを用いて波長２３０ｎｍにおける吸光度を測定することによって、確認した。培養後、菌体を含む培養液を試験用害虫忌避剤とし、下記の評価を行った。 <Fatty acid metabolism process>
In 1 l (liter) of sterilized water in a glass Erlenmeyer flask, 12 g of linoleic acid (primary linoleic acid manufactured by Wako Pure Chemical Industries, Ltd.), 1.5 g of magnesium sulfate heptahydrate and / or dipotassium hydrogen phosphate 1 .5 g and the total amount of the cells obtained from the pre-culture step were added. This was cultured for 4 days under the conditions of 20 ° C., 120 rpm, and dissolved oxygen concentration of 4 mg / l using a bioshaker (BR-23UM manufactured by Taitec Corporation). The degradation of linoleic acid involves measuring the concentration of oxidized lipid, which is one of linoleic acid intermediate products, in the culture solution using a spectrophotometer BioSpec-mini manufactured by Shimadzu Corporation at a wavelength of 230 nm. Confirmed by. After the culture, the following evaluation was performed using a culture solution containing the cells as a test pest repellent.
ワタアブラムシの忌避効果
・実施例１～２
　最も長い部分の長さが１０ｃｍ程度のキュウリの本葉を２ｃｍ四方に切り取り、試験用害虫忌避剤（原液）およびその１０倍希釈液に１０秒間浸漬させた（実施例１および２）。シャーレ（径９ｃｍ）内に、試験用害虫忌避剤に浸漬した葉（試験区）と水に浸漬した葉（対照区）とを３ｃｍ間隔で１枚ずつ置いたものを準備した。それぞれのシャーレ内の葉と葉との間に、ろ紙（１ｃｍ×１ｃｍ)を置き、ろ紙上にワタアブラムシ成虫を２０匹置いた。シャーレの蓋を閉じて暗所で静置し、２４時間後に、試験区および対照区の葉上に集積したワタアブラムシの数を計測し忌避率を求めた。忌避（％）は以下の式に基づいて求めた。
　忌避率（％）＝（１－試験区の集積虫数／対照区の集積虫数）×１００
結果を図１に示す。 Repellent effect of cotton aphids, Examples 1-2
Cucumber true leaves having a length of about 10 cm at the longest part were cut into 2 cm squares and immersed in a test pest repellent (stock solution) and a 10-fold diluted solution thereof (Examples 1 and 2). A petri dish (9 cm in diameter) was prepared by placing leaves immersed in a test pest repellent (test group) and leaves immersed in water (control group) one by one at intervals of 3 cm. A filter paper (1 cm × 1 cm) was placed between the leaves in each petri dish, and 20 cotton aphids were placed on the filter paper. The petri dish lid was closed and allowed to stand in the dark, and 24 hours later, the number of cotton aphids accumulated on the leaves of the test group and the control group was measured to determine the repelling rate. Repellency (%) was calculated based on the following formula.
Repelling rate (%) = (1−number of accumulated insects in test group / number of accumulated insects in control group) × 100
The results are shown in FIG.
・比較例１～２
　試験用害虫忌避剤に替えて、代表的なバイオサーファクタントであるサーファクチンナトリウムの０．１％溶液（比較例１）、および、忌避効果があるとされる柑橘系香気成分シトロネラの０．４％溶液（比較例２）を用いたこと以外は実施例１～２と同様に処理して観察した。結果を図１に示す。 Comparative examples 1 and 2
In place of the test pest repellent, a 0.1% solution of surfactin sodium (Comparative Example 1), which is a representative biosurfactant, and 0.4% of citronella, a citrus aroma component that is considered to have a repellent effect Treatment was observed in the same manner as in Examples 1 and 2 except that the solution (Comparative Example 2) was used. The results are shown in FIG.
　図１に示されるように、試験用害虫忌避剤（原液あるいはその１０倍希釈液）で処理した試験区は、比較例１のサーファクチンナトリウムや比較例２のシトロネラと比較して高い忌避率を示した。特に、試験用害虫忌避剤の原液を用いた実施例１では、ワタアブラムシの忌避率が１００％に達し、強力なワタアブラムシ忌避効果が確認された。一方、バイオサーファクタントを使用した比較例１では忌避率は６８％にとどまり、また、シトロネラを使用した比較例２では忌避効果は３０％となり、不十分な結果となった。したがって、本発明の害虫忌避剤が優れた忌避効果を有していることがわかる。 As shown in FIG. 1, the test group treated with the test pest repellent (stock solution or a 10-fold dilution thereof) has a higher repellent rate compared to Surfactin sodium in Comparative Example 1 and Citronella in Comparative Example 2. Indicated. In particular, in Example 1 using the stock solution of the test pest repellent, the cotton aphid repellent rate reached 100%, and a strong cotton aphid repellent effect was confirmed. On the other hand, in the comparative example 1 using the biosurfactant, the repelling rate was only 68%, and in the comparative example 2 using the citronella, the repelling effect was 30%. Therefore, it can be seen that the pest repellent of the present invention has an excellent repellent effect.
ミニバラにおける害虫忌避効果
・実施例３
　屋外で栽培中のミニバラ（品種：ルージュ）６株に対して、試験用害虫忌避剤を水で１００倍に希釈した希釈液を週一回の頻度で葉茎散布（３０ｍｌ／株程度）および土壌灌注（５０ｍｌ／株程度）することで処理した。処理は、６～７月の間実施し、処理後、自然発生した害虫寄生および食害を調べた。対照として無処理区を設けた。害虫寄生は、１株あたりの寄生頭数または１株あたりの寄生葉数の平均値で評価した。食害は、虫害度で評価した。ここで、虫害度とは、計測対象の各株の食害を０～４の５段階で評価し、各食害段階の株数を計測し、その株数に食害の段階を表す数値（０～４）を乗じて合計した数値を計測対象の全株数に４を乗じた数字で除して、１００倍したものをいう。なお、食害の段階は、以下のように定義する。葉の枚数が１００枚以上の株を計測対象とし、段階０は、食害を受けた葉がない、段階１が食害を受けた葉が３枚以下、段階２は食害を受けた葉が１０枚以下、段階３は食害を受けた葉が３０枚以下、段階４が食害を受けた葉が３０枚を超える場合とそれぞれ定義する。例えば、１０本の株を例にして説明すると、食害の段階が０（数値０）の株が２本、段階１（数値１）の株が２本、段階２（数値２）の株が１本、段階３（数値３）の株が２本、段階４（数値４）の株が３本の場合は、虫害度は（（０×２本＋1×２本＋２×１本＋３×２本＋４×３本）／４×１０本）×１００で計算され、虫害度は５５となる。結果を表１に示す。また、評価の判定は下記の基準に基づき判断した。
×：無処理区と比較して、抑制効果なし
○：無処理区と比較して、抑制効果あり Pest repellent effect in mini roses, Example 3
For 6 mini roses (variety: rouge) currently cultivated outdoors, a dilute solution of test pest repellent diluted 100 times with water is sprayed with leaves once a week (about 30 ml / strain) and soil Treated by irrigation (about 50 ml / strain). The treatment was carried out for 6 to 7 months. After the treatment, naturally occurring pest parasites and food damage were examined. An untreated section was provided as a control. Pest infestation was evaluated by the average number of parasitic heads per strain or the number of parasitic leaves per strain. The eating damage was evaluated by the degree of insect damage. Here, the degree of insect damage is evaluated by five levels of 0 to 4 for the damage of each strain to be measured, the number of strains at each stage of damage is measured, and the numerical value (0 to 4) indicating the stage of damage to the number of strains is measured. The total number multiplied by the number is divided by the total number of shares to be measured multiplied by 4 and multiplied by 100. The stage of damage is defined as follows. The number of leaves is 100 or more. The stage 0 has no damaged leaves, the stage 1 has 3 damaged leaves, the stage 2 has 10 damaged leaves. Hereinafter, stage 3 is defined as a case where 30 or less leaves are damaged, and stage 4 is a case where 30 or more leaves are damaged. For example, taking 10 stocks as an example, 2 stocks at the stage of eating damage 0 (numerical value 0), 2 stocks at stage 1 (numerical value 1), 1 stock at stage 2 (numerical value 2) If there are 2 strains at stage 3 (numerical value 3) and 3 strains at stage 4 (numerical value 4), the degree of insect damage is ((0 × 2 + 1 × 2 + 2 × 1 + 3 × 2) + 4 × 3) / 4 × 10)) × 100, and the insect damage level is 55. The results are shown in Table 1. The evaluation was determined based on the following criteria.
×: No inhibitory effect compared to untreated zone ○: Suppressive effect compared to untreated zone
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表１に示されるように、試験用害虫忌避剤の１００倍希釈液で処理した試験区においては、無処理区と比較して、害虫寄生および食害ともに低かった。したがって、本発明の害虫忌避剤が優れた害虫忌避効果を有していることがわかる。 As shown in Table 1, in the test group treated with a 100-fold diluted solution of the test pest repellent, both the pest parasitism and the food damage were lower than those in the non-treated group. Therefore, it can be seen that the pest repellent of the present invention has an excellent pest repellent effect.
　上記の結果より、本発明の害虫忌避剤および本発明の製造方法により製造される害虫忌避剤が、土壌汚染や毒性が低く、害虫忌避効果に優れた害虫忌避剤であることがわかる。 From the above results, it can be seen that the pest repellent of the present invention and the pest repellent produced by the production method of the present invention are pest repellents having low soil contamination and toxicity and excellent pest repellent effect.

Claims (22)

  1. 脂肪酸をプロテオバクテリアに代謝させることで得られる脂肪酸代謝物を含む害虫忌避剤。 A pest repellent containing fatty acid metabolites obtained by metabolizing fatty acids to proteobacteria.
  2. 前記害虫忌避剤が、植物における害虫忌避物質分泌促進剤である請求項１記載の害虫忌避剤。 The pest repellent according to claim 1, wherein the pest repellent is a pest repellent secretion promoter in plants.
  3. 前記脂肪酸が、炭素数４～３０の脂肪酸である請求項１または２に記載の害虫忌避剤。 The pest repellent according to claim 1 or 2, wherein the fatty acid is a fatty acid having 4 to 30 carbon atoms.
  4. 前記脂肪酸が、２０℃で液体の脂肪酸である請求項１～３のいずれか１項に記載の害虫忌避剤。 The pest repellent according to any one of claims 1 to 3, wherein the fatty acid is a liquid fatty acid at 20 ° C.
  5. 前記代謝が、０．１～８ｍｇ／ｌの溶存酸素濃度環境下での代謝である請求項１～４のいずれか１項に記載の害虫忌避剤。 The pest repellent according to any one of claims 1 to 4, wherein the metabolism is metabolism under a dissolved oxygen concentration environment of 0.1 to 8 mg / l.
  6. 前記代謝が、Ｍｇ、Ｐ、ＮａおよびＫから選ばれる少なくとも１種以上のミネラルの存在下での代謝である請求項１～５のいずれか１項に記載の害虫忌避剤。 The pest repellent according to any one of claims 1 to 5, wherein the metabolism is metabolism in the presence of at least one mineral selected from Mg, P, Na and K.
  7. 前記プロテオバクテリアが、前培養されたプロテオバクテリアである請求項１～６のいずれか１項に記載の害虫忌避剤。 The pest repellent according to any one of claims 1 to 6, wherein the proteobacterium is a pre-cultured proteobacterium.
  8. 前記前培養されたプロテオバクテリアが、菌数１×１０8～９×１０10ｃｅｌｌｓ／ｍｌに前培養されたプロテオバクテリアである請求項７記載の害虫忌避剤。 The pest repellent according to claim 7, wherein the pre-cultured proteobacterium is a proteobacterium pre-cultured to 1 × 10 8 to 9 × 10 10 cells / ml.
  9. バイオサーファクタントを含む請求項１～８のいずれか１項に記載の害虫忌避剤。 The pest repellent according to any one of claims 1 to 8, comprising a biosurfactant.
  10. 前記代謝が、２０～３０℃の条件下での代謝である請求項１～９のいずれか１項に記載の害虫忌避剤。 The pest repellent according to any one of claims 1 to 9, wherein the metabolism is metabolism under a condition of 20 to 30 ° C.
  11. 植物の茎葉または根に接触されるように用いられる請求項１～１０のいずれか１項に記載の害虫忌避剤。 The pest repellent according to any one of claims 1 to 10, which is used so as to come into contact with a foliage or a root of a plant.
  12. 脂肪酸をプロテオバクテリアに代謝させる脂肪酸代謝工程を含む、脂肪酸代謝物を含む害虫忌避剤の製造方法。 A method for producing a pest repellent containing a fatty acid metabolite, comprising a fatty acid metabolism step of metabolizing fatty acid to proteobacteria.
  13. 前記害虫忌避剤が、植物における害虫忌避物質分泌促進剤である請求項１２記載の害虫忌避剤の製造方法。 The method for producing a pest repellent according to claim 12, wherein the pest repellent is a pest repellent secretion promoter in plants.
  14. 前記脂肪酸が、炭素数４～３０の脂肪酸である請求項１２または１３に記載の害虫忌避剤の製造方法。 The method for producing a pest repellent according to claim 12 or 13, wherein the fatty acid is a fatty acid having 4 to 30 carbon atoms.
  15. 前記脂肪酸が、２０℃で液体の脂肪酸である請求項１２～１４のいずれか１項に記載の害虫忌避剤の製造方法。 The method for producing a pest repellent according to any one of claims 12 to 14, wherein the fatty acid is a liquid fatty acid at 20 ° C.
  16. 前記脂肪酸代謝工程を、０．１～８ｍｇ／ｌの溶存酸素濃度環境下で実施する請求項１２～１５のいずれか１項に記載の害虫忌避剤の製造方法。 The method for producing a pest repellent according to any one of claims 12 to 15, wherein the fatty acid metabolism step is carried out in an environment of dissolved oxygen concentration of 0.1 to 8 mg / l.
  17. 前記脂肪酸代謝工程を、Ｍｇ、Ｐ、ＮａおよびＫから選ばれる少なくとも１種以上のミネラルの存在下で実施する請求項１２～１６のいずれか１項に記載の害虫忌避剤の製造方法。 The method for producing a pest repellent according to any one of claims 12 to 16, wherein the fatty acid metabolism step is carried out in the presence of at least one mineral selected from Mg, P, Na and K.
  18. 前記プロテオバクテリアが、前培養されたプロテオバクテリアである請求項１２～１７のいずれか１項に記載の害虫忌避剤の製造方法。 The method for producing a pest repellent according to any one of claims 12 to 17, wherein the proteobacteria are pre-cultured proteobacteria.
  19. 前記前培養されたプロテオバクテリアが、菌数１×１０8～９×１０10ｃｅｌｌｓ／ｍｌに前培養されたプロテオバクテリアである請求項１８記載の害虫忌避剤の製造方法。 19. The method for producing a pest repellent according to claim 18, wherein the pre-cultured proteobacterium is a proteobacterium pre-cultured to 1 × 10 8 to 9 × 10 10 cells / ml.
  20. バイオサーファクタントを含む害虫忌避剤の製造方法である請求項１２～１９のいずれか１項に記載の害虫忌避剤の製造方法。 The method for producing a pest repellent according to any one of claims 12 to 19, which is a method for producing a pest repellent containing a biosurfactant.
  21. 前記脂肪酸代謝工程を、２０～３０℃の条件下で実施する請求項１２～２０のいずれか１項に記載の害虫忌避剤の製造方法。 The method for producing a pest repellent according to any one of claims 12 to 20, wherein the fatty acid metabolism step is carried out under a condition of 20 to 30 ° C.
  22. 植物に施用される害虫忌避剤の製造方法である請求項１２～２１のいずれか１項に記載の害虫忌避剤の製造方法。 The method for producing a pest repellent according to any one of claims 12 to 21, which is a method for producing a pest repellent applied to a plant.
PCT/JP2018/008629 2017-03-14 2018-03-06 Insect repellent and method for producing insect repellent WO2018168581A1 (en)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
JPH06199616A (en) * 1992-12-29 1994-07-19 Tsurumi Soda Co Ltd Insect pest repellent
JP2002504141A (en) * 1997-06-16 2002-02-05 アグラクエスト，インコーポレイテッド Compositions containing flat breast oil or active fraction thereof and methods of using those compositions as insect repellents
WO2015029872A1 (en) * 2013-08-26 2015-03-05 学校法人上智学院 Repellant for repelling root-knot nematodes, method for manufacturing same and repelling method using repellant
WO2018047918A1 (en) * 2016-09-08 2018-03-15 イビデン株式会社 Plant activator and process for producing plant activator

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06199616A (en) * 1992-12-29 1994-07-19 Tsurumi Soda Co Ltd Insect pest repellent
JP2002504141A (en) * 1997-06-16 2002-02-05 アグラクエスト，インコーポレイテッド Compositions containing flat breast oil or active fraction thereof and methods of using those compositions as insect repellents
WO2015029872A1 (en) * 2013-08-26 2015-03-05 学校法人上智学院 Repellant for repelling root-knot nematodes, method for manufacturing same and repelling method using repellant
WO2018047918A1 (en) * 2016-09-08 2018-03-15 イビデン株式会社 Plant activator and process for producing plant activator

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OHNO, KATSUYA: "Approach of Plant Responses to Plant Disease Control by Bacterial Metabolites of Fatty Acid (BMFA) (1", ANNUAL MEETING OF THE JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY, 5 March 2017 (2017-03-05), pages 07 *

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