JP7429968B2 - Pest resistance inducer and plant pest control method - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act (1) Website publication date: December 19, 2019, website address: https://www. mdpi. com/1420-3049/25/1/17/htm

The present invention relates to a pest resistance inducer and a method for controlling pests in plants.

The occurrence of pests and diseases that reduce crop yields is a serious problem in the agricultural field. Pest control is important, as more than half of the damage caused by designated pests is caused by pests. There are various techniques for controlling pests that affect crops, but the main ones are chemical control methods that use chemical pesticides such as insecticides, cultivation control methods that use resistant varieties, and methods that use natural enemies. Biological control methods such as Among them, chemical pesticides are widely used because they are quick-acting and have high pesticidal effects. However, there are concerns about chemical pesticides, such as the impact on the ecosystem due to residue in the environment due to improper use, and the emergence of chemical-resistant pests due to continuous use of the same pesticide. Furthermore, from the viewpoint of environmental conservation and consumer safety, there is a need to reduce the amount of chemical pesticides used. In response to these needs, agriculture with reduced pesticide use has been implemented, but the occurrence of pests and diseases that would not have manifested under normal usage has also become a problem.

Biological control methods do not have these problems with chemical pesticides, but there are other problems. For example, when resistant varieties are grown instead of normal varieties, the emergence of pests that overcome the resistance becomes a problem. For this reason, in recent years there has been a demand for varieties that have the characteristic of stably maintaining resistance without collapse. Furthermore, with the method of using natural enemies, it is difficult to constantly procure natural enemies that are sufficiently effective.

In recent years, disease resistance inducers have attracted attention as an environmentally friendly disease control method. Unlike chemical pesticides, disease resistance inducers do not directly kill pathogens, but control diseases by inducing and strengthening the plant's inherent disease resistance. It is effective against a wide range of pathogens, and its effects last for a long time. Since the mechanism of plant disease resistance is conserved across species, there is a wide range of plant species in which resistance-inducing substances exhibit physiological activity.

Research using tomato, a plant belonging to the Solanaceae family, and Arabidopsis thaliana, a plant belonging to the Brassicaceae family, has revealed that the disease resistance pathway in plants is the salicylic acid pathway, which is effective against living parasitic pathogens such as viruses that obtain nutrients from living plant cells. It has been shown that they can be roughly divided into two types: jasmonic acid-ethylene, which is effective against biocidal pathogens such as gray mold fungus, which kills host cells and provides nutrients (for example, see Non-Patent Document 1). . However, while jasmonic acid and its analogs exhibit extremely strong ability to induce resistance against a wide range of insect pests, when treated with plants, they cause significant growth inhibition and acceleration of aging (for example, Non-Patent Document 2 reference). Therefore, its practical use as a lead compound for a resistance inducer is difficult.

The inventors have previously revealed that loliolide induces resistance in plants to pests such as Occidental thrips through a jasmonic acid-independent signal transduction pathway (for example, Patent Document 1 reference).

Patent No. 6108548

Glazebrook J, Annual Review of Phytopathology, 2005, vol. 43, p.205-227. Erb et al., Trends in Plant Science, 2012, vol. 17(5), p.250-259. Kawazu et. al., Arthropod-Plant Interactions, 2012, vol. 6(2), p.221-230. Murata et al., Plant Physiology, 2019, Vol. 179, p.1822-1833.

Loliolide has problems in that it is not commercially available and is difficult to obtain, and the cost of organic synthesis is high.

The present invention has been made in view of the above circumstances, and provides a new resistance inducer that is effective against pests and useful as a pest control agent, and pest control of plants using the resistance inducer. provide a method.

In order to find a substance that induces resistance to pests, the inventors focused on natural resources and tried to search using plants in which pest resistance had been induced. The present inventors have discovered that ionon induces excellent resistance to pests such as Thripsidae and Noctuidae insects in plants, even though it does not have direct insecticidal activity, leading to the completion of the present invention.

That is, the present invention includes the following aspects.
[1] An insect resistance inducing agent containing α-ionone as an active ingredient and inducing resistance to thripsid insects in plants.
[2] A pest resistance inducer containing α-ionone as an active ingredient and inducing resistance to Noctuaceae insects in plants.
[3] A method for controlling pests in plants, which comprises contacting or absorbing the pest resistance inducer according to [1] or [2] into the plant body.
[4] The method for controlling plant pests according to [3], wherein the plant is a dicotyledonous plant.
[5] The method for controlling plant pests according to [3] or [4], wherein the plant is a Solanaceae plant, a Cruciferae plant, or a Cucurbitaceae plant.

The pest resistance inducer according to the present invention exhibits a pest control effect by inducing resistance to pests in plants. Therefore, the pest resistance inducer and the plant pest control method using the same have very little risk of the appearance of resistant strains, and are preferable pest control methods from the viewpoint of environmental protection.

FIG. 2 is a diagram showing the effect of 24-hour treatment with α-ionone on the number of O. occidentalis thrips survivors in tomatoes in Example 1. FIG. 2 is a diagram showing the effect of 24-hour treatment with β-ionone on the survival number of Occidentalis thrips in tomatoes in Comparative Example 1. FIG. 2 is a diagram showing the effect of α-ionone dripping treatment on the survival rate of Occidental thrips in Example 2. FIG. 2 is a diagram showing the effect of 24-hour α-ionone treatment on the induction of expression of each gene in tomato in Example 3. 3 is a diagram showing the effect of 24-hour β-ionone treatment on the induction of expression of each gene in tomato in Comparative Example 2. FIG. FIG. 4 is a diagram showing the effect of α-ionone treatment on salicylic acid accumulation in Example 4. FIG. 4 is a diagram showing the effect of α-ionone treatment on jasmonic acid accumulation in Example 4. FIG. 3 is a diagram showing the effect of 48-hour treatment with α-ionone on the number of occidental thrips eggs laid in Arabidopsis thaliana in Example 5. FIG. 6 is a diagram showing the effect of a 48-hour treatment with α-ionone on the number of Spodoptera survives in tomatoes in Example 6. FIG. 6 is a diagram showing the effect of 48-hour treatment with α-ionone on the body weight of Spodoptera in tomatoes in Example 6. FIG. 7 is a diagram showing the effect of 24-hour treatment with α-ionone on the number of surviving southern thrips in cucumbers in Example 7.

The pest resistance inducer according to the present invention is α-ionone (“α-ionone”, “(E)-4-(2,6,6-trimethylcyclohex-2-enyl)but-3-ene-2 -on) as an active ingredient, and induces resistance to Thripsidae or Noctuidae insects in plants. α-Yonone is a compound represented by the following formulas (1-1) and (1-2) (hereinafter sometimes referred to as "compound (1-1)" and "compound (1-2)") It is. Compound (1-1) is the (R)-(+) form of α-ionone, and compound (1-2) is the (S)-(-) form of α-ionone. The α-ionone used in the present invention may be in the (R)-(+) form, the (S)-(-) form, or a mixture thereof. Hereinafter, in the present invention and the present specification, "α-ionone" includes the (R)-(+) and (S)-(-) forms. α-Yonone CAS No. is "127-41-3". Although α-ionone does not exhibit insecticidal activity, it can enhance the pest control activity of plants.

The α-ionone used in the present invention may be an extract from a natural product. α-Yonone is a carotenoid metabolite and is abundantly contained in plants and algae that have a carotenoid metabolic pathway.

Among plants and algae that have a carotenoid metabolic pathway, α-ionone is particularly abundant in carrots, pumpkins, tobacco, tea, laver, amanita, violets, and the like. α-ionone can be extracted from these plants and algae.

α-ionone can be extracted from plants and the like by a conventional method using an organic solvent. Specifically, for example, it is extracted from fresh or dried products such as leaves and stems of plants containing α-ionone using an organic solvent such as methanol, ethanol, acetone, or hexane. The dried material such as a plant may be one that has been subjected to a drying process such as sun drying or natural drying, or may be one that has been rapidly frozen using liquid nitrogen. Furthermore, it is also preferable to shred or crush the material in advance before adding the organic solvent. The obtained extract is concentrated by vacuum distillation or the like, and then the organic solvent layer is separated using a separating funnel to obtain a crude extract containing α-ionone. This crude extract can be used as an active ingredient of the pest resistance inducer according to the present invention, either as it is or after being concentrated if necessary.

As the active ingredient of the pest resistance inducer according to the present invention, it is also preferable to use a purified extract obtained by refining the crude extract by chromatography. For example, the crude extract may be subjected to normal phase silica gel column chromatography to separate a fraction containing α-ionone. The obtained fraction can be used as an active ingredient of the pest resistance inducer according to the present invention, either as it is or after being concentrated if necessary. Further, α-ionone can be purified to a higher purity by further subjecting the fraction containing α-ionone obtained by silica gel column chromatography to reversed phase chromatography.

The α-ionone used in the present invention may be a synthetic product. α-ionone can be synthesized relatively easily using known synthesis methods. α-ionone can be synthesized from pseudoionone, for example. Specifically, for example, pseudoionone is cyclized by adding dilute acid and heating to obtain a mixture of α-ionone and β-ionone. At this time, by appropriately adjusting the reaction conditions such as the type of dilute acid used, the ratio of the α-form and the β-form to be produced can be changed. For example, α-ionone is mainly obtained by carrying out the reaction using phosphoric acid as a dilute acid. Note that pseudoionone can be produced by condensing citral with acetone through an aldol reaction using a base catalyst. In addition, it can be synthesized from carotene such as α-carotene by oxidoreductase or photooxidation. Further, α-ionone used in the present invention may be a commercially available product. Commercially available products of α-ionone include, for example, α-ionone (product code: 093-00692) manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.

α-ionone, which is an active ingredient of the pest resistance inducer according to the present invention, may be a solvate or may be contained as a salt. Examples of the solvate include solvates of water, methanol, ethanol, ethyl acetate, and the like. Examples of salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts, iron salts, zinc salts, copper salts, nickel salts, etc. metal salts; inorganic salts such as acetates and ammonium salts; organic amine salts such as dibenzylamine salts, glucosamine salts, ethylenediamine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, diethanolamine salts, and tetramethylammonium salts; glycine salts , amino acid salts such as lysine salt, arginine salt, ornithine salt, and asparagine salt.

The concentration of α-ionone in the pest resistance inducer according to the present invention may be sufficient as long as it induces pest control activity in plants, and the concentration of α-ionone in the pest resistance inducer according to the present invention may be sufficient to induce pest control activity in plants. It is appropriately determined in consideration of the type of target plant to be used, the amount of spraying at one time, the frequency of spraying, etc. For example, in consideration of the pest control effect and the amount of one-time application, the concentration of α-ionone in the pest resistance inducer of the present invention when applied (sprayed) to plants is 10 μM (mol/L) or more. is preferable, 100 μM or more is more preferable, and even more preferably 300 μM or more. When the concentration of α-ionone is 10 μM or more, the pest control effect can be more fully exhibited. On the other hand, α-ionone is a substance produced in almost all species of organisms that perform photosynthesis and is included in many foods, so it is presumed that it has little impact on the environment and the human body. Therefore, the upper limit of the concentration of α-ionone in the pest resistance inducer according to the present invention is not particularly defined, but from the viewpoint of cost etc., it is preferably 2000 μM, more preferably 1000 μM, even more preferably 700 μM, and 500 μM. Particularly preferred.

The pest resistance inducer according to the present invention may be a concentrate that is diluted and used when applied to plants. For example, when the concentration of α-ionone in the pest resistance inducer according to the present invention is 1mM or more and 1M or less, it is effective to dilute it 100 times or more and 100,000 times or less with an appropriate solvent such as water or ethanol before use. The component concentration can be adjusted to a desired concentration (for example, about 10 μM) before use.

The dosage form of the pest resistance inducer according to the present invention is not particularly limited, and examples thereof include powders, granules, tablets, wettable powders, emulsions, aqueous solutions, oils, aerosols, and the like. When using the pest resistance inducer according to the present invention for agricultural and horticultural purposes, wettable powders, emulsions, water-solvents, oils, aerosols, powders, etc. are preferable because they can be easily sprayed onto plants. From the viewpoint of absorbability into plants, wettable powders, emulsions, aqueous solutions, and aerosols are more preferred. These formulations can be manufactured according to conventional methods. For example, various preparations are produced by diluting the active ingredient (α-ionone) with an inert liquid (solvent) or solid carrier, and adding other additives as necessary. Examples of such additives include surfactants, excipients, binders, disintegrants, lubricants, colorants, flavoring agents, pH adjusters, and the like.

When the pest resistance inducer according to the present invention is in a liquid form such as an aqueous solution or a wettable powder, the solvent for dissolving or dispersing α-ionone must be inert to α-ionone, and It is preferable to use a solvent that is unlikely to have an adverse effect on the target plant. As the solvent, methanol, ethanol, water, etc. are used as appropriate. In the case of an aqueous solvent, it can be prepared, for example, by dissolving α-ionone in methanol or ethanol and diluting the resulting solution with water.

The pest resistance inducer according to the present invention may contain other insecticides, fungicides, herbicides, plant growth regulators, fertilizers, etc. within the range that does not inhibit the pest control activity-inducing effect of α-ionone. Good too.

α-ionone can induce pest control activity in plants, particularly against Thripsidae insects or Noctuidae insects, and can enhance resistance to these pests. Therefore, in plants treated with the pest resistance inducer according to the present invention, damage caused by suction by Thripsidae insects or feeding damage by larvae of Noctuidae insects is reduced. Furthermore, due to this insect resistance-inducing effect, α-ionone can be expected to be used as a lead compound for resistance-inducing agents against Thripsidae insects or Noctuidae insects.

The pests to be controlled by the insect resistance inducer of the present invention may be those belonging to the Thripidae family or the Noctuidae family.
Examples of Thripidae insects include Frankliniella occidentalis, Thrips palmi, Scirtothrips dorsalis, Thrips tabaci, Frankliniella intonsa, etc. It will be done. Among these, preferred is Frankliniella occidentalis or Thrips palmi.
There are many genera of Noctuidae insects, such as Spodoptera, Athetis, Polyphaenis, Trachea, Orthogonia, Actinotia, Phlogophora, Apamea, Sapporia, Bambusiphila, and Atrachea. , Capsula, Sesamia, Dryobotodes, Xylena, Lithophane, Eupsilia, Rhynchaglaea, Mesorhynchaglaea, Sugitania, Incertobole, Telorta, Antivaleria, Nyctycia, Cosmia, etc. Those belonging to the genus Spodoptera are more preferred. Examples of those belonging to the genus Spodoptera include Spodoptera litura, Spodoptera exigua, Spodoptera mauritia acronyctoides, and Spodoptera frugiperda. is particularly preferred.

The plant pest control method according to the present invention includes contacting or absorbing the pest resistance inducer according to the present invention into a plant body. By contacting or absorbing the pest resistance inducer according to the present invention into a plant body, α-ionone as an active ingredient acts on the plant body, and the disease resistance of the plant body is enhanced, resulting in sap-sucking damage caused by Thripsidae insects. Alternatively, feeding damage caused by larvae of insects of the family Noctuidae can be reduced.

In the plant pest control method according to the present invention, the method of bringing the pest resistance inducer according to the present invention into contact with or absorbed into the plant body is not particularly limited, and is the same as the method of spraying other agricultural chemicals. It can be done by When the pest resistance inducer according to the present invention is in liquid form, it is applied to the surfaces of leaves, stems, etc. of plants, either as is or after diluting with an appropriate solvent such as water or ethanol. or spraying is preferred. Application and spraying can be carried out in the same manner as normal foliar spraying. Alternatively, a liquid pest resistance inducer or a diluted solution thereof may be sprinkled on the soil where the plants are planted. When the pest resistance inducer is sprinkled on soil, the active ingredient, α-ionone, is absorbed into the plant body through the roots and stems at the ground level. Alternatively, a liquid pest resistance inducer or a diluted solution thereof can be brought into contact with plants in the form of vapor by slow release or the like. Alternatively, seedlings or the like before planting may be directly immersed in a liquid pest resistance inducer or a diluted solution thereof, so that the plant can absorb the insect resistance inducer. In addition, a liquid pest resistance inducer or its diluted solution can be mixed into a nutrient solution for hydroponic cultivation or hydroponic cultivation, and thereby absorbed through roots, stems, etc. at the ground level. When the pest resistance inducer according to the present invention is a powder, it can be used as it is or as a dispersion in an appropriate solvent such as water or ethanol, by coating, spraying, steaming, dipping, or nutrient solution. Contamination can be treated. When the pest resistance inducing agent according to the present invention is in a relatively large solid form such as granules, it can be sprinkled on the soil where plants are planted.

In the plant pest control method according to the present invention, the application amount of the pest resistance inducer according to the present invention to the target plant is determined based on the type of target plant, the type of pest to be controlled, the degree of damage, the environmental conditions, It is appropriately determined in consideration of the active ingredient content, dosage form, etc. of the pest resistance inducer. In the present invention, in order to easily obtain a sufficient pest control effect, the concentration of α-ionone at the time of application to plants is 10 μM or more, preferably 100 μM or more, more preferably 300 μM or more. It is preferable to apply such pest resistance inducers.

In the plant pest control method according to the present invention, the target plant is not particularly limited as long as it is a plant that can be harmed by Thripsidae insects or Noctuidae insects, and dicotyledonous plants are preferable; It can also be a monocotyledonous plant. In the method for controlling pests of plants according to the present invention, the plants to be controlled are preferably plants traded in the market, and more preferably agricultural products such as vegetables, flowers, fruit trees, tea, and ornamental plants. preferable. Among these, vegetables are preferred, such as tomatoes, eggplants, green peppers, paprika, potatoes, plants of the nightshade family such as chili peppers, cabbage, broccoli, Brussels sprouts, Japanese radish, turnips, radish, cauliflower, Komatsuna, Japanese cabbage, Bok choy, Nabana, Chinese cabbage, and Mizuna. More preferred are plants of the Cruciferae family such as , radish, and plants of the Cucurbitaceae family such as cucumbers, pumpkins, zucchini, gourds, loofahs, chives, vines, watermelons, and melons, and tomatoes are particularly preferred.

Hereinafter, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to the following examples unless the gist thereof is changed.

[Example 1]
<Examination of the control effect on the survival number of occidental thrips in tomatoes treated with α-ionone for 24 hours>
As α-ionone, a commercially available product (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was used. α-ionone was diluted with ethanol to predetermined concentrations (1 μM, 10 μM, 100 μM, and 300 μM (μmol/L)). As a control, a solution (0 μM) consisting only of ethanol was also prepared.

Next, tomatoes grown in pots that were 3 weeks old or more and 4 weeks old or less were placed in a transparent resin cylindrical container (volume of about 1000 mL; 14 cm height x 10 cm diameter). Next, a 1.5 mL microtube containing 100 μL of α-ionone solution at a predetermined concentration and ethanol (0 μM) was fixed to the inner wall of the container with tape, and a 7 cm x 5 cm cutout was placed in a lid with a nylon mesh pasted on it. To prevent α-ionone vapor from leaking from the container, the entire lid was covered with food-grade cling film, and the container was placed in a thermostatic chamber controlled at 24°C ± 1°C under 16-hour light/8-hour dark conditions. I placed it on. After 24 hours, the 1.5 mL microtube containing the α-ionone solution was taken out, and 20 adult female occidental thrips per tomato plant were released.The tube was immediately covered and cultivated under the same conditions as above. After 14 days, the number of survivors was counted.

Figure 1 shows the effect of 24-hour treatment with α-ionone on the number of Occidentalis thrips survivors in tomatoes. In FIG. 1, different alphabets indicate significant differences (Tukey Kramer HSD test p<0.05; n=11-15). In the α-ionone treated area, a decrease in the number of surviving O. citrus thrips was observed at concentrations of 10 μM or higher compared to the control area.

[Comparative example 1]
<Examination of the control effect on the survival number of occidental thrips in tomatoes treated with β-ionone for 24 hours>
The control effect of treatment with β-ionone, which is an isomer of α-ionone, was tested according to the method described in Example 1. β-ionone (“β-ionone”, “(E)-4-(2,6,6-trimethylcyclohex-1-enyl)but-3-en-2-one”) is represented by the following formula (2) This is a compound represented by In the assay, the method described in Example 1 was used for ethanol solutions containing β-ionone (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) at final concentrations of 1 μM, 10 μM, 100 μM, and 300 μM, and a solution consisting only of ethanol (0 μM). After treating tomatoes with the tomato, 20 adult female occidental thrips were released per tomato plant, and the number of survivors was counted. Note that tomatoes treated with a solution consisting only of ethanol (0 μM) were used as a control.

Figure 2 shows the effect of 24-hour treatment with β-ionone on the number of Occidentalis thrips survivors in tomatoes. In FIG. 2, "n.s." is an abbreviation for "Not Significant" and indicates that there is no statistically significant difference. The same applies thereafter.

As shown in FIG. 2, there was no significant difference between the number of surviving O. citrus thrips in the β-ionone treatment area and that in the control, and no decrease in the number of survivors was observed. This result shows that it is important for the pesticidal effect of α-ionone that the double bond in the ring structure of α-ionone be located between the 2nd and 3rd carbon positions.

[Example 2]
<Toxicity assay of α-ionone against occidental thrips>
Place tomato leaves on absorbent cotton pre-soaked with water on a glass plate (7.5 cm x 2.5 cm), and then place an acrylic manger cell (7.5 cm x 2.5 cm, cell (inner diameter: 1.5 cm) was placed. Drop 0.5 μL of α-ionone (stock solution, 300 μM) into a Petri dish, place one 2nd instar larva of Occidental Thrips on top of it, roll the larva on the liquid for 5 seconds with a face brush, and remove excess with filter paper. I sucked up the liquid. Thereafter, it was placed on a tomato leaf in a manger cell and placed in a thermostatic chamber controlled at 24°C ± 1°C under 16-hour light/8-hour dark conditions. After 48 hours, the survival rate (percentage of the number of surviving larvae after the assay to the number of larvae at the start of the assay) was examined. A control group was prepared using 0.5 μL of ethanol instead of the α-ionone solution.

FIG. 3 shows the effect of the α-ionone drop treatment on the survival rate of Occidental thrips. There was no difference in the survival rate of O. citrus thrips between the α-ionone treatment area and the control area, indicating that α-ionone does not have direct toxicity (occidental thripsicidal activity) against O. citrus thrips. It was done.

[Example 3]
<Examination of the effect of α-ionone treatment on tomato gene expression induction>
Tomatoes grown in pots that were 3 weeks old or more and 4 weeks old or less were placed in a transparent resin cylindrical container (volume of about 1000 mL; 14 cm height x 10 cm diameter). Next, a 1.5 mL microtube containing 100 μL of α-ionone solution at a predetermined concentration (1 μM, 10 μM, 100 μM, and 300 μM) and ethanol (0 μM) was fixed to the inner wall of the container with tape and cut into a size of 7 cm x 5 cm. Place a lid with a nylon mesh pasted on the container, cover the entire lid with food-grade cling film to prevent α-ionone vapor from leaking from the container, and store at 24°C ± 1°C, 16 hours light/8 hours dark. The cells were placed in a thermostatic chamber controlled under period conditions. After 24 hours, tomato leaves were collected, immediately frozen in liquid nitrogen, and used for total RNA extraction. Extraction of total RNA from tomato leaves, synthesis of cDNA from RNA, and real-time PCR using the SYBR method using cDNA were conducted in accordance with previous reports (Non-patent Documents 3 and 4). Salicylic acid-responsive genes SlGLuB (tomato β-glucanase gene) and SlPR3a (tomato acid chitinase III), loliolide-responsive gene SlLin5 (tomato invertase), and jasmonic acid-responsive gene SlPR3b (tomato The effect of α-ionone on the induction of expression of basic chitinase III), SlLapA1 (tomato leucine aminopeptidase), and SlPin2 (tomato proteinase inhibitor II) was investigated. Note that the expression level of each gene was corrected by the expression level of the tomato actin gene (Accession number: BT012695).

FIG. 4 shows the effect of α-ionone treatment for 24 hours on the induction of expression of each gene in tomato. α-ionone induced the expression of SlGLuB and SlPR3b at concentrations of 100 μM or higher. On the other hand, no expression-inducing effect of α-ionone on SlPR3a, SlLapA1, SlPin2, and SlLin5 was observed.

[Comparative example 2]
<Examination of the effect of β-ionone treatment on tomato gene expression induction>
The effect of treatment with β-ionone, which is an optical isomer of α-ionone, on induction of tomato gene expression was determined according to the method described in Example 3. In the assay, tomatoes were incubated for 24 hours using the method described in Example 3 for an ethanol solution containing β-ionone (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) at a final concentration of 300 μM and a solution consisting only of ethanol (0 μM). After the treatment, tomato leaves were collected and the expression levels of SlGLuB and SlPR3b in the tomato leaves were examined.

FIG. 5 shows the effect of β-ionone treatment for 24 hours on the induction of expression of each gene in tomato. 300 μM β-ionone did not induce the expression of SlGLuB and SlPR3b.
β-ionone is said to have insecticidal and repellent effects against thrips. However, as is clear from the results of Comparative Examples 1 and 2, β-ionone is not effective in inducing resistance to thripsid insects or the expression of marker genes such as SlGLuB and SlPR3b in tomatoes. Ta.

[Example 4]
<Examination of the effect of α-ionone treatment on the accumulation of plant hormones>
Tomatoes grown in pots that were 3 weeks old or more and 4 weeks old or less were placed in a transparent resin cylindrical container (volume of about 1000 mL; 14 cm height x 10 cm diameter). Next, a 1.5 mL microtube containing 100 μL of α-ionone solution at a predetermined concentration (1 μM, 10 μM, 100 μM, and 300 μM) and ethanol (0 μM) was fixed to the inner wall of the container with tape and cut into a size of 7 cm x 5 cm. Place a lid with a nylon mesh pasted on the container, cover the entire lid with food-grade cling film to prevent α-ionone vapor from leaking from the container, and store at 24°C ± 1°C, 16 hours light/8 hours dark. The cells were placed in a thermostatic chamber controlled under period conditions. After 24 hours, tomato leaves were collected, immediately frozen in liquid nitrogen, and used for plant hormone extraction. Quantification of salicylic acid and jasmonic acid, which are plant hormones, was carried out in accordance with previous reports (Non-patent Documents 3 and 4). Note that tomatoes treated with a solution consisting only of ethanol (0 μM) were used as a control.

FIG. 6A shows the effect of α-ionone treatment on the accumulation of salicylic acid, and FIG. 6B shows the effect of α-ionone treatment on the accumulation of jasmonic acid. No difference was observed in the endogenous amounts of salicylic acid and jasmonic acid between the α-ionone treated plot and the control plot, indicating that α-ionone did not cause an increase in the endogenous amount of salicylic acid and jasmonic acid.

[Example 5]
<Examination of the control effect on the survival number of O. citrus thrips in Arabidopsis thaliana treated with α-ionone for 48 hours>
Leaf pieces (1 cm diameter) punched from 2-month-old Arabidopsis leaves were placed in a 48-well microtiter plate to which 0.8 mL of 300 μM α-ionone solution had been added per well in advance, and incubated at 24°C ± 1°C for 16 hours. It was placed in a thermostatic chamber controlled under light/8 hour dark conditions. After 48 hours, one female adult of Occidental thrips was released per leaf, immediately covered with a lid, and cultivated under the same conditions as above. Three days later, the number of eggs laid was measured. Note that tomatoes treated with a solution consisting only of ethanol (0 μM) were used as a control.

FIG. 7 shows the effect of 48-hour treatment with α-ionone on O. citrus thrips egg production in Arabidopsis. Note that in FIG. 7, different alphabets indicate significant differences (p<0.01; n=16 to 18). In the α-ionone treated area, a decrease in the number of eggs laid by Occidental thrips was observed compared to the control area.

α-ionone did not show any direct insecticidal effect against occidental thrips. This suggests that the decrease in the number of survivors observed in tomatoes and Arabidopsis thaliana treated with α-ionone is due to increased pest resistance of the plants. Although α-ionone showed an effect of increasing the expression of SlGLuB and SlPR3b, it did not show an expression-inducing effect on other salicylic acid-responsive genes, jasmonic acid-responsive genes, and loliolide-responsive genes. Furthermore, α-ionone did not show any effect on increasing the endogenous amounts of salicylic acid and jasmonic acid. From these facts, it is inferred that the induction of pest resistance by α-ionone is not mediated by salicylic acid or jasmonic acid.

[Example 6]
<Examination of the control effect on the survival number and body weight of Spodoptera in tomatoes treated with α-ionone for 48 hours>
Tomato leaves from 3 weeks old to 4 weeks old were placed in a glass Petri dish (16 cm diameter) to which α-ionone solution of each concentration had been added in advance, and incubated at 24°C ± 1°C, 16 hours light/8 hours dark. The cells were placed in a thermostatic chamber controlled under period conditions. After 48 hours, the tomato leaves were removed and each leaf was placed in a transparent resin cylindrical container (approximately 1000 mL volume; 14 cm height x 10 cm diameter), and 10 Spodoptera larvae per leaf were released on the surface of the leaf. The plants were then immediately covered with lids and cultivated under the same conditions as above. To prevent the leaves from drying out during cultivation, the petiole was stuck into a 1.5 mL microtube to which 1 mL of distilled water was previously added. After 5 days, the number of survivors and body weight were measured. Note that tomatoes treated with a solution consisting only of ethanol (0 μM) were used as a control.

FIG. 8A shows the effect of 48-hour treatment with α-ionone on the number of Spodoptera viables in tomatoes. FIG. 8B shows the effect of 48-hour treatment with α-ionone on Spodoptera spp. body weight in tomatoes. Note that in FIGS. 8A and 8B, different alphabets indicate significant differences (p<0.05; n=10). In the α-ionone treated plot, a decrease in the number of surviving Spodoptera and the body weight was observed compared to the control plot.
From the above, it has been revealed that α-ionone can induce resistance in plants to the chewing larvae of Spodoptera japonica (Noctuidae), which has a feeding style different from that of Thripsidae.

[Example 7]
<Examination of the control effect on the survival number of southern yellow thrips in cucumbers treated with α-ionone for 24 hours>
A 3-week-old cucumber strain grown in a pot was placed in a transparent resin cylindrical container (approximately 1000 mL volume; 14 cm height x 10 cm diameter), and 300 μM α-ionone solution and ethanol ( (0 μM) was fixed in a container, and cucumbers were exposed to the vapor of each solution for 24 hours. The 1.5 mL microtubes containing each solution were taken out, and 10 female adult southern thrips per cucumber plant were released.The tubes were immediately covered and cultivated under the same conditions. After 14 days, the number of survivors was counted at 24°C±1°C under 16-hour light/8-hour dark conditions.

Figure 9 shows the effect of 24-hour treatment with α-ionone on the number of southern thrips survivors in cucumber. In addition, in FIG. 9, different alphabets indicate that there is a significant difference (t-test p<0.05; n=8). In the α-ionone treated area, a decrease in the number of surviving southern thrips was observed compared to the control area.
From the above, α-ionone is effective against southern yellow thrips, which belongs to the same insect family Thripsidae as occidental thrips, as well as on cucumbers (Cucurbitaceae), as well as tomatoes (Solanaceae) and Arabidopsis (Brassicaceae). It has become clear that resistance can be induced.

The pest resistance inducer and the method for controlling plant pests using the same according to the present invention are effective against insect damage caused by Thripsidae insects or Noctuidae insects, particularly sap-sucking damage or feeding damage, and cause the emergence of resistant strains. There is very little danger, and it is also safer for the environment and the human body than conventional pesticides. Therefore, the pest resistance inducer according to the present invention and the method for controlling pests of plants using the same are particularly applicable to the cultivation of agricultural crops and ornamental plants, particularly in the cultivation of solanaceous plants, cruciferous plants, and cucurbitaceous plants. It can be suitably used in

Claims (5)

A pest resistance inducer containing α-ionone as an active ingredient, which induces resistance to thripsidae insects in plants. A pest resistance inducer containing α-ionone as an active ingredient, which induces resistance to Noctuaceae insects in plants. A method for controlling pests in plants, which comprises contacting or absorbing the pest resistance inducer according to claim 1 or 2 into a plant body. The method for controlling pests of plants according to claim 3, wherein the plant is a dicotyledonous plant. The method for controlling pests of plants according to claim 3 or 4, wherein the plant is a plant of the family Solanaceae, a plant of the Brassicaceae family, or a plant of the Cucurbitaceae family.

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