JP2013249278A - Control agent and control method for pine wilt - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a control agent and a control method for pine wilt having a low environmental impact, superior safety to wildlife, and high controlling effect on Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, and Monochamus alternatus Hope.SOLUTION: A control agent for pine wilt, a control agent for Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, and a control agent for Monochamus alternatus Hope include an extract from a plant of the Bidens genus in the family Asteraceae as an active component. A control method for pine wilt includes application of the control agents to one or more among plants of the genus Pinus, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, and Monochamus alternatus Hope.

Description

  The present invention belongs to the technical field of plant disease control, and relates to a control agent and control method for pinewood nematode and pinewood beetle, and a control agent and control method for pine wilt disease caused by these organisms.

  It is the pine wood nematode that causes pine wilt disease (pine wilt) (Linear phylum, Phantom, Aferenchoides, Aferenchoides, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle) and its vectors The Japanese pinewood beetle (Arthropoda, Insecta, Coleoptera, Cerambycidae, Monochamus alternatus Hope) invaded Nagasaki in 1905 with wood imported from North America as a shipbuilding material, and subsequently excluding Hokkaido and Aomori The amount of damage caused by pine wilt disease in 2010 reaches approximately 580,000 cubic meters (volume of damaged material). Japanese red pine and black pine, endemic species of the Nansei Islands, Yakutanoeyo (endangered species) and Ryukyu pine are severely damaged, and Matsubara existing in various parts of Japan is under threat of this disease . In particular, Ryukyu pine distributed in the Ryukyu Islands is more susceptible to this disease than other species and suffers great damage. This disease has occurred not only in Japan but also in China, Taiwan, South Korea, and Europe. In addition to the above species, Pinus plants, including red pine, Korean pine, European red pine, and French silkworm (Hereinafter sometimes simply referred to as “pine”) is susceptible to this disease. European countries are quarantining pine timber imports to prevent the spread of infection.

  Pine wood nematode disease is caused by sexual maturation of the pine wood beetle that has emerged and escaped from the dead pine trunk in May to July (holding pine wood nematode in the body, generally 8,000 to tens of thousands) They fly to pine shoots and eat the epidermis. It is thought that chemical substances such as β-myrcene and ethanol that attract pinewood nematodes evaporate from the site where the epidermis peeled, and show positive chemotaxis to pinewood nematodes. This causes the pinewood nematode to infest the pine tissue (June to September). After parasitism, if it is fast, symptoms of pine wilt appear in about 2-3 weeks. A pine-nosed beetle female flies to a pine that died in July-August and lays eggs under the pine tree. The hatched pine beetle larva grows while feeding under the skin of the tree, forms a cocoon chamber in the 11th to May, and becomes a pupa, and emerges in May to June.

  Control of this disease is generally carried out using preventive agents against pinewood nematodes, insecticides against the vector insect, pinewood beetle, felling dead trees, and fumigation treatments, but these methods are effective. On the other hand, the effects on wildlife and the environment, the damage caused by the drifting of agricultural chemicals, labor and cost burden are regarded as problems. In particular, organophosphorus insecticides used for the pine beetle are feared to affect humans and other organisms, and the continuous use of pesticides is known to induce the emergence of drug-resistant insects. There is also a method using resistant pine, but there are drawbacks such as few resistant lines and difficulty in breeding by cutting, and foreign lines are not suitable for Japanese climate.

  In this way, the control of pine wilt disease (pine blight) currently depends on pesticides as much as possible, particularly from the viewpoint of biodiversity protection, because measures with a large environmental burden are being taken. A departure from control methods is desired.

  As a method for controlling pinewood nematode or pinewood beetle, there are methods using nets, chemical substances, enzymes, electric currents, microorganisms, etc. There is a method of using a product (Patent Document 1), Amaryllidaceae Bulb Press (Patent Document 2), African Marigold Oil, Sweet Basil Oil and Palmarosa Oil (Patent Document 3). There is no known way to do it. The plants used in Patent Documents 1 to 3 are already widely used as wood, fuel, foodstuffs, essential oil raw materials, ornamental plants, soil improvement crops, etc., and procured in large quantities as raw materials for pest control agents. It may not be easy to do. Moreover, when these plants are used for pest control, there is a problem that, for example, resources useful as food may be consumed.

  Patent Document 4 describes a root-knot nematode control agent comprising an extract of an Asteraceae plant as an active ingredient. Further, Patent Document 5 describes an agent for controlling a nematode nematode or cyst nematode, which contains an extract of Ayuyusendangusa as an active ingredient. However, the control agents described in Patent Documents 4 and 5 are intended for soil harmful nematodes, and there is no description of pinewood nematode and pinewood beetle. The root-knot nematodes, the nematode nematodes, and the cyst nematodes described in Patent Documents 4 and 5 all belong to the Tylenoptera platyrenxaceae, and the pine wood nematode (Aferencoides aferencoides) that is the subject of the present invention Is a taxonomically different organism. Further, nematodes targeted by the control agents described in Patent Documents 4 and 5 are included in soil nematodes and commit the roots of vegetables. Therefore, these soil nematodes have a completely different ecology from the pine wood nematode that parasitizes the above-ground tissue of trees.

  Patent Document 6 describes a pest control agent mainly composed of an extract of a plant belonging to the genus Syndaceae. However, the insect pests targeted by the insect pest control agent described in Patent Document 6 are weevil (Lepidoptera, Noctuidae), aphids (Homoptera, Aphididae), white butterflies (Lepidoptera, Pieridae), spider mites (Acari, Acaridae) and whiteflies (Asteridae, Aphididae), which belong to a different taxon from the pine moth Beetle (Coleoptera, Cerambycidae). In addition, all of these pests are harmful to vegetables or herbaceous plants. Therefore, the pests targeted by the control agent of Patent Document 6 have a completely different ecology from the pinewood beetle that feeds on the young branches of trees and the subcutaneous tissue of trees.

JP-A-5-279223 JP 2009-268453 A Special table 2009-536937 gazette Japanese Patent No. 4528982 JP 2011-68612 A JP 2010-208994 A

Deba, F., Xuan, TD, Yasuda, M. and Tawata, S. (2008) Chemical composition and antioxidant, antibacterial and antifungal activities of the essential oil from Bidens pilosa Linn. Var. Radiata. Food Control 19, 346-352 . Masuzawa Mikio, Masuzawa Mako, Maeda Akiko, Miyata Kyoko, Katsuoka Norio (2005). Prevention effect of Kanpo tea (registered trademark) against summer ulcer of Livedo reticularis with summer ulceration. Rihka Bulletin 115 (1), 7-13. Eiji Usami, Genjiro Kusano, Takanori Katayose, Hiroshi Waichi, Yoshiyuki Seyama (2004). Evaluation of antioxidant effects of water-soluble and fat-soluble antioxidants in natural products. YAKUGAKU ZASSHI 123 (11), 847-850.

  The present invention provides a control agent and control method for pine wilt disease, which has a low environmental impact, is excellent in safety against wildlife, and has a high control effect on pinewood nematode and pinewood beetle. The purpose is to provide.

  The present invention provides a pine wilt disease control agent (hereinafter also referred to as “the pine wilt disease control agent of the present invention”) containing an extract of a plant belonging to the genus Syndaceae as an active ingredient.

  The present invention also relates to a pine wood nematode control agent (hereinafter also referred to as “the pine wood nematode control agent of the present invention”) containing an extract of the asteraceae family Sendungusa plant as an active ingredient, and an extract of the asteraceae family Sengakusa plant. The present invention provides a control agent for pine wood beetles (hereinafter, also referred to as “the pine wood beetle control agent of the present invention”) containing a product as an active ingredient.

  Furthermore, the present invention provides a pine wilt disease control agent, a pine wood nematode control agent, and a pine wood beetle control agent according to the present invention, and any one or more of a pine genus plant, a pine wood nematode, and a pine wood beetle. The present invention provides a method for controlling pine wilt disease (hereinafter also referred to as “the pine wilt control method of the present invention”).

  The pine wilt disease nematode control agent of the present invention exhibits an immobilizing, repelling and insecticidal effect on the pine wood nematode and the pine wood beetle by the components contained in the extract of the plant of the genus Sendamunga. It can prevent or reduce pine wilt disease (pine wilt) caused by seed pests.

  The Asteraceae plant, for example, Ayuyusendangusa, which is a raw material for the active ingredients of the pine wilt disease nematode control agent, the pinewood nematode control agent, and the pine wood beetle control agent, is widely distributed in Japan and tropical and subtropical regions. It is easy to obtain because of its vigorous growth and abundant resources. Therefore, the pine wilt disease nematode control agent, pine wood nematode control agent and pine wood beetle control agent of the present invention can be manufactured at a lower cost than conventional pest control agents using plant-derived components and the like. It is. In addition, since such plants have been treated as weeds / harmful grasses until now and are rarely used, the present invention is expected to lead to effective utilization of unused biological resources.

  In addition, Asteraceae plants such as Ayuyukisendansa are used as Kampo tea in some areas and have high safety. Furthermore, recently, there are reports that the components of the plant are effective for dermatitis and adult diseases, and it is known that there are no adverse effects on major crops and soil microorganisms. Therefore, the pine wilt disease nematode control agent, the pine wood nematode control agent, and the pine wood beetle control agent of the present invention have little influence on living organisms and the environment including humans and do not impair biodiversity.

It is a microscope picture which shows the insecticidal effect of the Ayuyukisendangususa extract with respect to a pinewood nematode (the dead nematode in the Ayuyukisendanusa extract). It is a figure which shows the repellent effect of the Ayuyukisendangususa extract with respect to a pinewood nematode. It is a figure which shows the influence of the Ayuyukisendangususa extract with respect to the diseased pine tissue nematode. Values are mean ± standard deviation, and different alphabets indicate significant differences (Tukey's multiple comparison test, p <0.05). It is a photograph which shows the insecticidal effect of the Ayuyukisendangususa extract with respect to an adult pine-nosed beetle. The left is a pinewood beetle that has been sprayed with Ayuyukisendanusa extract and the right is a pinewood beetle that has been sprayed with sterilized water (control: survival). It is a photograph which shows the repellent effect of the Ayuyukisendangususa extract with respect to an adult pine-nosed beetle. The traces of the pine beetle are shown when sprayed with Ayuyukisendanusa extract on young pine branches (left) and when sprayed with water (right). It is a photograph which shows the insecticidal effect of the Ayuyukisendangususa extract with respect to the pinewood beetle larva. The left and middle are pinewood beetle larvae (left: 10-fold diluted solution, middle: undiluted solution) that died from Ayuyukisendanusa extract, and the right is a photograph of live larvae treated with sterile water.

  The active ingredient of the pine wilt disease nematode control agent, the pinewood nematode control agent and the pine wood beetle control agent (hereinafter also referred to as “control agent of the present invention”) of the present invention is an extract obtained from a plant belonging to the genus Sendamsa It is. Examples of the Asteraceae plant that can be used as a raw material for the extract include, for example, Bendens pilosa L. and its variety Awayukisendansa (B. pilosa L. var. Radiata Scherff.), And Examples include Sendengsa (Bidens pilosa L. var. Radiata Sch. Bip) and its varieties, Hidenusu sensen ungusa (Bidens pilosa L. var. Radiata Sch. F. Decumbens Scherff.). Among these plants, it is preferable to use Ayuyukisendangusa as a raw material of the extract.

  Asteraceae plants used as the raw material of the extract may be those in the flowering stage or those in the vegetative period, and may be in a raw state or dried. It is preferable to use a dried plant body. The standard for drying is not limited, but for example, it is preferable that the water content is 0% to less than 15%. In the case of Awayukisen Dangsa, it is usually 24 hours under warm air of 40 to 80 ° C. It is sufficient to dry at 100 ° C. for about 2 hours. Further, from the viewpoint of extraction efficiency, it is preferable to use a material obtained by appropriately chopping or pulverizing a plant body, for example, a powdered material. Regarding the plant part, whole plants including flowers, leaves, stems, roots and the like can be used as an extraction raw material, but it is preferable to use the above-ground part excluding the roots.

  The extract of the Asteraceae plant in the control agent of the present invention can be obtained using a general plant component extraction method. Examples of such extraction methods include boiling extraction method, immersion extraction method, shaking extraction method, Soxhlet extraction method, steam distillation method and the like, and among these, boiling extraction method is preferable.

  Solvents that can be used for extraction include not only water but also lower alcohols (such as methanol and ethanol), ketones (such as acetone and methyl ethyl ketone), hydrophilic organic solvents (such as acetonitrile and ethyl acetate), and ethers. (For example, ethyl ether, isopropyl ether, etc.), hydrophobic hydrocarbons such as aromatic hydrocarbons (for example, toluene, xylene, etc.), halogenated hydrocarbons (for example, methylene chloride, chloroform, etc.), and mixed solvents thereof Can also be used. The extract of the Asteraceae plant in the control agent of the present invention is preferably extracted using water as a solvent. In one preferred embodiment, the extract of the Asteraceae plant in the control agent of the present invention is an extract (water-boiled extract) obtained by a boiling extraction method using water as a solvent. Further, even when a method other than water boiling extraction is used, those skilled in the art can appropriately set conditions such as solvent and extraction time to obtain an extract of the Asteraceae plant.

  In addition, the obtained extract can be purified by a column fractionation method, a solvent fractionation method, or the like, if necessary.

  In this specification, the term “extract” includes an extract obtained by various extraction methods using a solvent, a concentrated solution obtained by concentrating the extract, and a diluted solution obtained by diluting the extract with an appropriate solvent. , Solid extracts (powder, granular, etc.) obtained by removing the solvent from the extract or its concentrated or diluted solution by various methods such as heat drying and freeze drying, and solidifying agents and gels A solid or semi-solid extract obtained by addition of an agent is also included.

  The extract of the Asteraceae plant in the control agent of the present invention is, for example, added 5 to 1000 parts by weight, preferably 5 to 100 parts by weight of water to 1 part by weight of a dried plant of the Asteraceae family Sendungusa. And boiled for 30 to 60 minutes, preferably 30 minutes. As described above, the extract (extract) obtained by such a method can be diluted or concentrated as necessary, and the solvent can be removed to obtain a solid extract.

  The extract of the Asteraceae plant that is an active ingredient of the control agent of the present invention is preferably ethyl abietic acid, isovanillin, α-caryophyllene, β-caryophyllene, coumaran, cinnamic acid, dehydroabietic acid methyl ester, syringa Aldehyde, stearic acid, vanillic acid, palmitic acid, 4-hydroxybenzoic acid, 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, butylhydroxytoluene, 2 (4H)- It contains one or more compounds selected from the group consisting of benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl and myristic acid.

  In addition to the above-mentioned compounds, Awayusendangusa has alkaloids, tannins, saponins, flavonoids, chalcone substances, caffeoyl derivatives, linole compounds, diterpenes, essential oils, phenylpropanoid glycosides, phenols, flavonoids , Flavone glycosides and polyacetylenes, ferulic acid, quercetin, caffeic acid, pyrocatetin, salicylic acid, p-vinyl guaiacol, dimesoxyphenol, eugenol, 4-ethyl-1,2-benzenediol, isovanillin, 2- It has been reported to contain hydro-6-methylbenzaldehyde, vanillin, vanillic acid, p-hydroxybenzene acid, protocatechuic acid and coumarin (coumaric) acid (Non-Patent Documents 1 to 3). Therefore, the extract of the Asteraceae plant Sendangusa plant which is an active ingredient of the control agent of the present invention may contain one or more of these substances. In addition, the control agent of the present invention may contain one or more of these substances in combination with an extract of the plant belonging to the genus Asteraceae, and further in combination with various spreading agents, osmotic transfer agents and the like. It can also be used to control pinewood nematode and pinewood beetle and pine wilt disease.

  Plants to which the control agent of the present invention can be effectively applied are Pinus plants, and more specifically, Pinus plants that are susceptible to pine wilt disease. Examples of the pine genus plant include, but are not limited to, red pine (Pinus densiflora Sieb. & Zucc.), Japanese black pine (Pinus thunbergii Parl.), Japanese white pine (Pinus amamiana Koidz.), Ryukyu pine (Pinus luchuensis Mayr), Examples include the red pine (Pinus massoniana Lambert, 1803), Korean pine (Pinus koraiensis Siebold et Zucc.), European red pine (Pinus sylvestris L.), and the French pine (Pinus pinaster Aiton).

  Hereinafter, an extract obtained by adding 5 parts by weight of water to 1 part by weight of a dry plant of Ayuyukisendansa, heating and boiling for 30 minutes (hereinafter referred to as “extraction stock solution” for convenience) is an active ingredient. The preferred concentration of the Asteraceae plant extract is described by taking the control agent of the present invention contained as an example.

  In order to obtain a certain control effect against pinewood nematodes, it is preferable to use a diluted solution up to about 20 to 100 times that of the extraction stock solution. In order to obtain a control effect of almost 100%, the extraction stock solution or its solution It is preferable to use a diluting solution up to about 10 times.

  In addition, when aiming at an immediate effect on pinewood nematode, that is, an insecticidal effect, it is preferable to use an extraction stock solution or a diluted solution of up to about 100 times, and by immobilizing or repelling pinewood nematode, For the purpose of preventing invasion of adult pine beetle winged adults, it is preferable to use an undiluted extract or a diluted solution up to about 200 times, and the pine of the pinewood nematode at the end of the meal When it is intended to prevent entry into the genus plant tissue, it is preferable to use an extraction stock solution or a diluted solution up to about 20 times.

  On the other hand, in order to exert an insecticidal effect and repellency against adult pine beetle, it is preferable to use an extraction stock solution.

  Moreover, in order to exhibit an immediate effect, that is, an insecticidal effect, on the pinewood beetle larvae, it is preferable to use an extraction stock solution or a diluted solution up to about 20 times.

  The extract of the Asteraceae plant, which is an active ingredient of the control agent of the present invention, exhibits immobilization and insecticidal action against pinewood nematodes and pinewood beetle larvae and adults. Therefore, it is possible to control pine wilt disease by applying the control agent of the present invention to any one or more of the pine genus plants, the pinewood nematode and the pinewood beetle.

  For example, if the control agent of the present invention is applied to a pine genus plant suffering from pine wilt disease, (1) killing pine wood nematode and pine wood beetle larvae present in the tissue of the pine plant, 2) It is possible to block the migration of the pine wood nematode, the pine wood beetle, and (3) to prevent the emergence of the vector worm, thereby preventing pine wilt disease And control becomes possible.

  In addition, since the extract of the Asteraceae genus Sengungusa exhibits repellent properties against pinewood nematodes and pinewood beetles, by applying the control agent of the present invention to healthy pine plants, a postprandial meal of pinewood beetles (Action for maturity: feeding on the bark of shoots) and pine wood nematode invasion into pine plant tissues can be prevented or reduced.

  Here, “application” of the control agent of the present invention refers to bringing the control agent into contact with an object to be applied (for example, a pine genus plant, a pinewood nematode, or a pinewood beetle). Application methods of the control agent include, but are not limited to, spraying, coating, dipping, in-tree injection, soil irrigation (absorption from roots), and the like.

  The control agent of the present invention can be formulated into various dosage forms that can be employed in agricultural chemicals and the like according to a desired application method. Examples of dosage forms include, but are not limited to, powders (including general powders, DL powders, flow dust (FD) agents), granules, pastes, wettable powders, water solvents, liquids, emulsions, flowables , Microemulsions, microcapsules and the like. In addition, excipients, media, carriers, additives, and the like that are usually used in the technical field can be appropriately selected and used for formulation of the control agent. For example, extenders, media for dissolving or dispersing active ingredients (solvents, dispersion media, etc.), dispersants, emulsifiers, solidifying or gelling agents for solidification or semi-solidification, imparting sustained release A matrix or the like can be used.

  For application to pine plants, liquid control agents can be injected into the living or dead trees of the pine plant, and liquid or solid (e.g. powders) on the surface of the living or dead trees of the pine plant. (Form) control agent can also be applied. The application in this case is intended to fix the extract of the plant belonging to the genus Asteraceae on the surface of the pine plant, or to permeate it into the tissue of the pine plant. Therefore, the extract can be applied alone, but various spreading agents and osmotic transfer agents can be used to improve the fixation on the surface of the pine plant and the penetration into the tissue of the pine plant. Can also be used together. Spreading agents and osmotic transfer agents are generally liquids mainly composed of surfactants, such as general spreading agents (spreaders), functional spreading agents (adjuvants), adhesive spreading agents (stickers), etc. Can be used in combination.

  For example, as the nonionic surfactant, polyoxyethylene alkyl ether, polyoxyethylene alkylalkyl phenyl ether, polyoxyethylene fatty acid ester, polyoxyethylene hexitan fatty acid ester, polyoxyalkylene alkyl ether, polyoxyalkylene fatty acid ester N-octylpyrrolidone, acetylene surfactants, and the like. Examples of the anionic surfactant include alkylbenzene sulfonate, dialkyl sulfosuccinate, lignin sulfonate, and formalin condensate of naphthalene sulfonate. For improving adhesion, anionic surfactants include lignin sulfonate, condensate of naphthalene sulfonate, paraffin, wax, motan wax, polyoxyethylene resin acid ester, polyvinyl alcohol, methyl vinyl ether / maleic anhydride There are polymers and the like. Examples of the material that imparts permeability include polyoxyethylene hexitan fatty acid ester and polyoxyethylene dodecyl ether.

  Hereinafter, the present invention will be described in more detail with reference to examples.

1. Test Nematodes The pinewood nematodes used in the experiments described herein were obtained according to the following procedure.
(1) Wood chips were collected from diseased Ryukyu pine on the campus of Ryukyu University using a manual 15 mm diameter wood drill.
(2) Using the obtained wood waste, the Bellman method (Nematode Experimental Method, 2004, p87-88, Editor: Nematode Experimental Method Editorial Committee, Publisher: Shinji Shingu, Publisher: Japanese Nematode 10 ml of the nematode in the tissue was separated into a sterilized centrifuge tube by an academic society) and centrifuged at 3,000 rpm (1 minute) with a centrifuge.
(3) After centrifugation, remove 9 ml of the supernatant with a Pasteur pipette 5 times, add 9 ml of lactic acid diluted to 10% using a Pasteur pipette, centrifuge, remove 9 ml of the supernatant, and again After adding 9 ml of sterilized water and centrifuging, 9 ml of the supernatant was removed to wash the nematodes.
(4) The surface sterilized nematode is preliminarily stored on a PDA medium (potato: 200 to 300 g, glucose: 10 to 20 g, agar: 10 to 20 g, water: 1,000 ml) on-campus stored bacteria Botrytis cinerea Persoon After being grown on a pre-cultured medium for 7 days, about 10 ml of sterilized water was added dropwise using a Pasteur pipette, and the suspension was recovered and used for the experiment.

2. Test insects 1) Adult pine beetle Adults from the Okinawa Prefectural Forest Resource Research Center that emerged and escaped from the diseased Ryukyu pine in the breeding gauge, and were distributed after 1 to 2 weeks of adult pine beetle adults It was used for the experiment.

2) Matsunomadori Beetle Larva We collected larvae of the Japanese pine beetle using a wood-splitting machine and tweezers from a cocoon in the log of the diseased Ryukyu pine that was distributed from the Okinawa Prefectural Forest Resource Research Center. Thereafter, the sample was allowed to stand in the dark at 25 ° C. for 1 day and selected one having a good state was subjected to the following experiment.

3. Identification of pinewood nematode The pinewood nematode was identified by observing morphological features such as oral cavity, esophagus and vulva with a stereomicroscope. The genus was identified using a search table (William et al., 1996). Next, the species was identified by PCR-RFLP method (Iwahori et al., 1998) using one individual morphologically identified as the genus Bursaphelenchus, and the sequence of the spacer region between the 18S and 26S rRNA genes of the nematode was further determined. Decoding (the primers used for PCR amplification and sequencing were 18S primer (SEQ ID NO: 1 5'-TTGATTACGTCCCTGCCCTTT-3 ') and 26S primer (SEQ ID NO 2: 5'-GGAATCATTGCCGCTCACTTT-3')) BLAST searches were performed on the DDBJ (DNA Data Bank of Japan) website to identify the species. As a result, in any of the above methods, the nematode used in the following experiment was identified as the pine wood nematode (Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle).

[Test Example 1]
Preparation of Ayuyukisendangusa extract The ground part of the flowering Ayuyusendangusa that grows naturally in the field center of the University of the Ryukyus is cut off, cut with a dry heat sterilizer at 100 ° C for 2 hours, and then the mixer is cut. 10 g of the finely pulverized product was put into a beaker and 50 ml of water was added. This was covered with aluminum foil, boiled for 30 minutes, and filtered with filter paper to obtain an extract (hereinafter also referred to as “extract of test example 1”).

[Test Example 2]
Analysis of components containing Ayuyukisendansa (1) The collected Ayuyunsendansa is dried (100 ° C., 2 hours) and chopped, and 50 ml of water is added per 10 g of shredded material in the same manner as in Test Example 1 (aluminum Boiled for 30 minutes (covered with foil). After boiling, centrifugation and filtration were performed to obtain about 25 ml of a stock solution.
(2) 25 ml of the stock solution was transferred to a 100 ml beaker containing a rotating stone, sodium hydroxide (4N) was added at a ratio of 1: 1, and the mixture was stirred at 50 ° C. for 4 hours using a water bath. After stirring, hydrochloric acid (12N) was added to adjust the solution to pH1. The density was measured using litmus paper.
(3) After adjusting the pH, a half amount (25 ml) of the obtained solution and 100 ml of ethyl acetate were added to a separatory funnel and stirred, and allowed to stand for a while. After confirming the separation of the ethyl acetate layer and the aqueous layer, the lower aqueous layer was recovered in a 50 ml capacity beaker, and the remaining ethyl acetate fraction was recovered in a 1,000 ml capacity Erlenmeyer flask. The same operation was repeated twice for the recovered aqueous layer. The same operation was performed for the remaining half of the solution to obtain a total of 600 ml of ethyl acetate fractions. In addition, in order to remove water contained in a trace amount in the collected ethyl acetate fraction, about 1 tablespoon of sodium sulfate was added and stirred sufficiently, and stored in a cold room (2 ° C.).
(4) The ethyl acetate fraction was filtered, and 100 ml was divided in half, put into a 100 ml volume eggplant type flask, and ethyl acetate was volatilized using a rotary evaporator to obtain a solid substance. The obtained substance was dissolved in acetone to adjust to 10,000 ppm, filtered, and about 1.5 ml in a 2 ml vial was used as a sample.
(5) Component analysis was performed on the sample using GC-MS (GCMS-QP2010, column: DB-SMS, column length: 30 m, inner diameter φ0.25 mm).

  As a result, ethyl abietic acid, isovanillin, α-caryophyllene, β-caryophyllene, coumaran, cinnamic acid, dehydroabietic acid methyl ester, syringaldehyde, stearic acid, vanillic acid, palmitic acid, 4-hydroxybenzoic acid, 4H-pyran -4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, butylhydroxytoluene, 2 (4H) -benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl and Myristic acid was detected.

[Example 1]
Insecticide and immobilization experiment against pinewood nematode (1) Using a pipette (1 ml), extract 1 ml of the test example 1 (stock solution) and its 10-, 20-, and 100-fold dilutions into a sterile small test tube After the addition, add nematode suspension of pine wood nematode adjusted to 100 / 0.1 ml using a micropipette (20-200 μl), cover with parafilm and incubator (at 25 ° C.) The nematodes that were allowed to stand and immobilized after 1, 4 and 7 days were counted under a light microscope. A sterile water section was provided as a control. In addition, dead nematodes were counted after 8 days. The experiment was repeated 7 times, and immobilization and mortality were calculated as percentages (%).
(2) On the 7th day after the start of the experiment, the number of moving nematodes and immobilized nematodes were counted, and the immobilization rate was determined.
(3) One day after counting the number of immobilized nematodes, the supernatant was removed, and the number of live and dead nematodes was counted with a microscope (40 times) to determine the proportion of lethal nematodes among all nematodes (lethal rate). It was.

  As a result, high lethality and immobilization were observed at all concentrations (Table 1 and FIG. 1). Specifically, the immobilization rate showed a high lethality of 86.5 to 100% in all 100-fold dilutions from the stock solution (Table 1). Moreover, regarding the lethality, the value of the control solution of the stock solution to 20 times dilution was 76.4 to 100%, and the value of the 100 times dilution solution was reduced to 12.6% (Table 1).

[Example 2]
Repellent experiment against pinewood nematode (1) A circle with a diameter of 2 cm was previously drawn with black magic on the center of the bottom of a petri dish that had been sterilized by dry heat.
(2) After 10 ml of an agar medium (agar: 10 g, distilled water: 1,000 ml) was dispensed and coagulated in the petri dish, extraction of Test Example 1 was performed using a micropipette (20-200 μl) in a circle. 3.5 μl of the solution (stock solution) and its 10, 20, 100 and 200-fold dilutions were dropped and allowed to air dry in a clean bench for 10 minutes.
(3) Next, a nematode suspension of pine wood nematode adjusted to 100 / 3.5 μl using a micropipette (20 to 200 μl) was added to the circle in each section and allowed to stand at 25 ° C. . Thereafter, pinewood nematodes remaining in the circle were counted under an optical microscope from 30 minutes to 24 hours later. Sterile water was provided as a control, and the experiment was repeated 7 times.

  As a result, the number of endocytic nematode heads tended to decrease with time in all concentrations of the extract, and after 24 hours of treatment, it was 39.3 in sterilized water, whereas the stock solution was diluted 10 and 20 times. In the liquid section, the number of in-circle nematode heads decreased to 10.1, 7.1, and 18.7, respectively (FIG. 2).

[Example 3]
Insecticidal and immobilization experiments on diseased pine tissue nematodes (1) A rubber tube of about 10 cm was attached to a funnel on which a sieve of 3 mm mesh was placed, and a pinch clamp was attached at a location 2 to 3 cm from the bottom.
(2) Next, 20 g of wood scrap collected from a diseased Ryukyu pine in the Ryukyu University with a manual drill with a diameter of 15 mm was wrapped with Kimwipe, which was stacked two times, placed on a sieve, and the extract of Test Example 1 The (stock solution) and its 10, 20, 50, 100 and 200-fold dilutions were poured and allowed to stand.
(3) After 1, 3 and 5 days, slowly open the pinch clamp, take 10 ml of the liquid containing nematodes in a sterilized small test tube, centrifuge at 3,000 rpm (1 minute), and then remove 9 ml of the supernatant. Removed with a Pasteur pipette and counted the nematode in the remaining 1 ml. Sterile water was provided as a control, and the experiment was repeated 7 times.

  As a result, 458.8 heads were separated in sterilized water, whereas the number of separated nematodes in the stock solution, 10, 20, 50, 100 and 200-fold diluted solutions was 8, 32.8, 103.4, It decreased to 213.6, 214.4, 285.8. Further, the number of separated nematode heads tended to decrease as the concentration increased in the extract section (FIG. 3).

[Example 4]
(1) Insecticidal and immobilization experiment against adult pine beetle, 2 ml of the extract (stock solution) of Test Example 1 was directly sprayed onto one adult pine beetle using a nebulizer. The adult was placed in a cylindrical plastic container (diameter 6 cm, height 9 cm) together with healthy Ryukyu pine shoots (length 5 cm, width 2 cm) collected from Ryukyu University and allowed to stand at 25 ° C. Thereafter, immobile and dead adults in plastic containers were counted after 1, 2 and 3 days, respectively. As a control, sterile water was provided and the experiment was repeated 6 times.

  As a result, in the stock solution treatment group, all test insects were immobilized or lethal after one day, and all individuals were lethal after three days (Table 2 and FIG. 4).

(2) Effects on the repellent properties of the pinewood beetle, 2 ml of the extract (stock solution) of Test Example 1 was directly sprayed using a nebulizer on three shoots (length 5 cm, width cm) of Ryukyu pine. Then, it was placed in a plastic container together with a healthy adult pine beetle, and allowed to stand at 25 ° C. Thereafter, the size of the feeding traces of the pine shoots was measured after 1, 2 and 3 days. Judgment criteria are strong repellency when the pine shoots treated have no eating traces (++), and those with a total area of 1 cm ² or less per edible trace (+) More than that, no repellency (−). A sterilized water section was provided as a control, and the experiment was repeated 6 times.

  As a result, all the individuals showed strong repellency one day after the treatment (Table 3 and FIG. 5). Also, depending on the individual, the effect may decrease as the number of days elapses (Table 3).

(3) Insecticidal effect experiment against pine moth beetle larvae On a sterilized petri dish, the extract of the test example 1 (stock solution) and its 10, 20 and 100 samples using a mespipet (2 ml) on one pine moth beetle larvae After 2 ml of the double diluted solution was dropped, the solution was allowed to stand under dark conditions at 25 ° C. Thereafter, lethal larvae were counted after 1, 3 and 7 days. As a control, a sterilized water section and an untreated section were provided, and the experiment was repeated five times.

  As a result, the insecticidal action was not observed in the untreated and sterile water, whereas the insecticide action was observed at all concentrations when the Ayuyu-Dengusa extract was treated, and 100% in the stock solution and 80% in the 10-fold dilution. %, 20% dilution was 60%, and 100% dilution was 20% (Table 4 and FIG. 6).

  From the above results, the control agent of the present invention comprising an extract of the Asteraceae plant family as an active ingredient can exhibit a high control effect against pinewood nematode and pinewood beetle by setting its concentration appropriately. Proven.

  For the pinewood nematode, when the control agent of the present invention comes into contact with the nematode, it exhibits insecticidal properties in the tissues of the genus Pinus and in the body of the pine moth Beetle.

  Moreover, since both the pinewood beetle and the pinewood nematode have shown repellent properties against the extracts of the genus Syndaceae, the control agent of the present invention is applied to the surface of the pine genus plant. It was confirmed that the invasion of pine wood nematode into the tissues of the plant can be prevented.

  Furthermore, since the extract of the Asteraceae family Sendungusa plant showed insecticidal activity against adult pine moths and larvae, the insecticides of the present invention were directly contacted with adult larvae, It was confirmed that the insecticidal effect can be exhibited by applying the control agent of the present invention to the tissues of the genus Pinus.

[References]
William, FM and Peter, GM with Howard, HL and Kent L. (1996) Plant-parasitic nematodes a pictorial key to genera, 5 th edition, pp.1-277, Comstock publishing associates a division of cornel university press, Ithaca and London.
Iwahori, H. Tsuda, K. Kanzaki, N. Izui, K. & Futai, K (1998) PCR-RFLP and sequencing analysis of ribosomal DNA of Bursaphelenchus nematodes related to pine wilt disease. Fundam. Appl. Nematol., 21 ( 6) .655-666

  The pine wood nematode control agent of the present invention promotes environment-friendly control for preserving the environment and biodiversity attracting attention recently, and is a pine material while being a single control agent. Since it has high anti-insect activity (insecticidal and repellent effect) against both pinewood nematode, which is the pathogen of nematode disease, and its vector insect, pinewood beetle, it is an effective control agent for pinewood nematode disease. It can be effectively used as a control method.

Claims (11)

  An agent for controlling pine wilt disease containing an extract of a plant belonging to the genus Syndungaceae as an active ingredient.   The Asteraceae plants are genus Bendens (Bidens pilosa L.), Ayuyusendangusa (Bidens pilosa L. var. Radiata Scherff.), Batiensa dungusa (Bidens pilosa L. var. Radiata Sch. Bip) and Hawai'i The control agent according to claim 1, which is one or more kinds of plants selected from the group consisting of Sendensa (Bidens pilosa L. var. Radiata Sch. F. Decumbens Scherff.).   The control agent according to claim 2, wherein the plant belonging to the genus Sendamusa is Awayusendangusa.   The extract is obtained by an extraction method selected from the group consisting of boiling extraction method, immersion extraction method, shaking extraction method, Soxhlet extraction method, and steam distillation method. Control agent.   The control agent according to claim 4, wherein the extract is obtained by a boiling extraction method using water.   Extract is ethyl abietic acid, isovanillin, α-caryophyllene, β-caryophyllene, coumaran, cinnamic acid, dehydroabietic acid methyl ester, syringaldehyde, stearic acid, vanillic acid, palmitic acid, 4-hydroxybenzoic acid, 4H- Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, butylhydroxytoluene, 2 (4H) -benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl The control agent in any one of Claims 1-5 containing 1 or more types of compounds selected from the group which consists of and myristic acid.   Alkaloids, tannins, saponins, flavonoids, chalcone substances, caffeoyl derivatives, linole compounds, diterpenes, essential oils, phenylpropanoid glycosides, phenols, flavonoids, flavone glycosides and polyacetylenes, ferulic acid, quercetin, Caffeic acid, pyrocatechin, salicylic acid, p-vinyl guaiacol, dimethyloxyphenol, eugenol, 4-ethyl-1,2-benzenediol, isovanillin, 2-hydro-6-methylbenzaldehyde, vanillin, vanillic acid, The control agent according to any one of claims 1 to 6, further comprising one or more substances selected from the group consisting of p-hydroxybenzene acid, protocatechuic acid and coumaric acid.   Any of the Claims 1-7 which are the dosage forms selected from the group which consists of a powder agent, a granule, a paste agent, a wettable powder, a water solvent, a liquid agent, an emulsion, a flowable agent, a microemulsion agent, and a microcapsule agent. Crab control agent.   An agent for controlling pine wood nematode (Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle) containing an extract of a plant belonging to the genus Syndaceae as an active ingredient.   A control agent for Monochamus alternatus Hope, which contains an extract of the plant belonging to the genus Sendamsa as an active ingredient.   A method for controlling a pine wilt disease comprising applying the control agent according to any one of claims 1 to 10 to at least one of a pine genus plant, a pinewood nematode, and a pinewood beetle.