JP2007112759A - Insect pest feeding inhibitor and method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an insect pest feeding inhibitor for plants, and to provide a method for inhibiting insect pest feeding. <P>SOLUTION: This insect pest feeding inhibitor for the plants contains a monogalactosylglycerol as an active ingredient. This method for inhibiting the insect pest feeding includes a process for artificially imparting monogalactosylglycerol to a plant. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to pest feeding inhibitors and methods in plants.

  A filamentous fungus belonging to the genus Neotyphodium is known as a filamentous fungus (filamentous fungus endophyte) symbiotic in plant tissue (Patent Document 1). Plants infected with such filamentous fungi are known to have improved resistance to pests, resistance to pathogens, growth rate, resistance to environmental stresses such as heat and dryness compared to non-symbiotic individuals. However, the mechanism of this action is unclear.

  For example, a mechanism may be considered in which infection of a filamentous fungus endophyte with a plant causes an increase in the concentration of a substance originally present in the plant, thereby increasing the insect resistance of the plant. If such a substance can be identified, it is expected that the substance can be used as a plant feeding inhibitor for pests.

JP 2001-286277 A Japanese Patent Laid-Open No. 10-14579

  The present invention provides an insect pest feeding inhibitor and method for a plant by finding an ingredient effective for inhibiting feeding against pests among the components peculiar to plants symbiotic with the filamentous fungus endophyte belonging to the genus Neotyphodium. With the goal.

  The present inventors have used Neotyphodium sp., Which is a filamentous fungal endophyte belonging to the genus Neotyphodium. The present inventors have found that a large amount of monogalactosyldiacylglycerol is contained in the grass family Meadow Fescue in which the Po-060 strain is symbiotic. Further, it was found that monogalactosyl diacylglycerol has pest feeding inhibitory activity. And the following invention was completed.

(1) A pest feeding inhibitor for plants containing monogalactosyl diacylglycerol as an active ingredient.
(2) A method for inhibiting feeding by plant pests, comprising a step of artificially imparting monogalactosyl diacylglycerol to a plant.

  As used herein, the anti-feeding activity is, as will be apparent to those skilled in the art, substances existing on the surface of the plant or on the periphery of the plant (even if produced by the plant, exogenously. (Which may be given) means the activity of inhibiting feeding of the plant by pests.

  ADVANTAGE OF THE INVENTION According to this invention, the damage by the pest in a plant, especially a grass family plant can be reduced.

  Monogalactosyl diacylglycerol (sometimes referred to herein as “MGDG”) that can be used in the present invention is a β-galactosyl bond to any one O atom of glycerol, preferably the O atom at the 3-position. It has a bonded β-D-galactopyranosyl group and two same or different acyl groups bonded to any two O atoms of glycerol, preferably the O atom at the 1-position and 2-position. The acyl group in MGDG is usually a linear or branched saturated or unsaturated chain molecule having 2 to 30 carbon atoms, such as acetic acid, propionic acid, butyric acid, isobutyric acid, caproic acid, caprylic acid, 2 -Ethylhexanoic acid, pelargonic acid, capric acid, undecylenic acid, lauric acid, myristic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, behenic acid, palmitoleic acid, hexadeca-7,10,13-trienoic acid, olein Acyl groups derived from fatty acids such as acid, linoleic acid, linolenic acid, arachidonic acid, erucic acid, isopalmitic acid and isostearic acid. An example of the structure of MGDG that can be used in the present invention is shown below, but it is not limited to this structure as described above.

  The MGDG used in the present invention may be naturally occurring or obtained by chemical synthesis. Further, a naturally occurring MGDG (for example, MGDG represented by the above chemical formula) may be modified by performing a transesterification reaction using a microorganism or an enzyme, or using other chemical methods. Good.

  The application amount of MGDG for imparting feeding inhibition to pests to plants is appropriately selected according to the target plant and the type of pests. Typically, MGDG is applied so as to be preferably 1000 ppm or more, more preferably 1400 ppm or more in the leaves of plants. Although an upper limit is not specifically limited, Usually, it is 5000 ppm or less.

  Examples of plants subject to the inhibition of pest feeding according to the present invention include gramineous plants. The pest feeding inhibitor or method of the present invention includes Agropyron, Agrostis, Alopecurus, Anthoxyanthum, Arrhenathiatum, Asprella, Brachylystom, Brachypodium, Brachyposum, Bromusgostom, Bromusgostom, Festuca, Glyceria, Hierochloe, Holcus, Hystrix, Koeleria, Lolium, Leymus, Melica, Millium, Phalaroides, Phleum, Poa, Puccinellia, Secale, Secale, Site olis, Stipa, Triticum, Andropogon, Axonopus, Cenchrus, Cymbopogon, Cynodon, Cyrtococcum, Eremochloa, Hymenachne, Ichnanthus, Lasiacis, Oplismenus, Panicum, Paspalum, Saccharum, Sacciolepis, Setaria, Sorghum, Aristida, Bouteloua, Calamovilfa, Chasmanthium, Chloris, Ctenuim, Eragrostis, Eragrostria, Gymnopogon, Muhlenbergia, Sporobulus, Thraysa, Tridens, Uniola, Bam usa, Chusquea, Guadua, Olya, Orthoclada, Pariana, Leersia, Luziola, Oryza, Danthonia, Zea, a plant belonging to any genus of Zoysia, among which agrostis, Festucal, FestucalP, In particular, it is particularly effective for tall fescue, meadow fescue, Italian ryegrass, perennial ryegrass, and labrador glass.

  The pests that can inhibit the feeding of plants by the present invention include lepidoptera, aphids, leafhoppers, stink bugs, crickets, potato beetles, weevil, weevil, trash beetles, scarab beetles, crabs, planthoppers And insects belonging to grasshoppers, locusts and the like. Inhibition of feeding according to the present invention is particularly effective against insects belonging to the order Lepidoptera, in particular, larvae thereof, and specifically, the moth family (for example, Tensujitsutoga, Tsutoga, Shibata Toga, Lobno Meiga, Wamon noiga, Awanogaiga, Akafutsu Riga) For example, Sedokukuga), Higgariidae (for example, Obihitori, Kiharagomadarahitori, Shilohitori) or Yagai (for example, Ezochairoyotou, Shiroshitayotou, Futaobikiyotou, Tampokiyotou, Kashishirokiyotou, Inagiyotou, Insect-together) It is valid.

  When MGDG is applied to a plant, MGDG can be used as it is, but it is usually used as a composition formulated in various forms such as a liquid such as lotion or aerosol, or a cream. Examples of liquids include lotions, aerosols, oils, and the like. Carriers used in these liquids include, for example, water, alcohols such as methanol, ethanol, isopropyl alcohol, and cetyl alcohol, and aliphatics such as petroleum benzine. Examples thereof include hydrocarbons, esters such as isopropyl myristate and cetyl acetate. Depending on the type of the liquid, add an emulsifier, dispersant, spreading agent, wetting agent, suspending agent, preservative, preservative, auxiliary agent for formulation, coating film forming agent, etc. to obtain the desired formulation. Can do.

  Examples of emulsifiers and dispersants include soaps, polyoxyethylene fatty acid alcohol ethers such as polyoxyethylene oleyl ether, polyoxyethylene alkyl aryl ethers such as polyoxyethylene nonylphenyl ether, polyoxyethylene fatty acid esters, fatty acid glycerides, Examples include sorbitan fatty acid esters such as polyoxyethylene sorbitan monostearate, sulfates of higher alcohols, alkylaryl sulfonates such as sodium dodecylbenzene sulfonate, and spreading agents or wetting agents include, for example, glycerin, propylene glycol And polyethylene glycol. Examples of the suspending agent include casein, gelatin, alginic acid, carboxymethylcellulose, gum arabic, hydroxypropylcellulose, and bentonite. Examples of the preservative include salicylic acid, ethyl paraoxybenzoate, propyl paraoxybenzoate, An example is butyl paraoxybenzoate. Examples of the propellant include dimethyl ether, chlorofluorocarbon, carbon dioxide gas, and LPG. Examples of the film forming agent include cellulose derivatives such as nitrocellulose, acetylcellulose, acetylbutylcellulose, and methylcellulose, and vinyl acetate resin. Vinyl resin, polyvinyl alcohol, methylpolysiloxane, octylmethylcyclotetrasiloxane, decamethylcyclopentasiloxane, dimethylpolysiloxane, methylphenylpolysiloxanemethylpolycyclopolysiloxane, dimethylsiloxane-methyl (polyoxyethylene) siloxane copolymer , Dimethylsiloxane methyl (polyoxyethylene polyoxypropylene) siloxane copolymer, trimethylsiloxysilicic acid, octamethylcyclotetrasi Silicones such as hexane silicone polyether polymers.

  Carriers used in creams include, for example, hydrocarbons such as paraffin, liquid paraffin, petrolatum, silicon compounds such as dimethylsiloxane, colloidal silica, bentonite, ethanol, stearyl alcohol, lauryl alcohol, ethylene glycol, polyethylene glycol, Examples thereof include alcohols such as glycerin, carboxylic acids such as lauric acid and stearic acid, and esters such as beeswax and lanolin. Furthermore, it can be set as the target formulation by adding suitably the preparation adjuvant similar to what is used in the case of a liquid formulation. The content of MGDG in these preparations is usually 1 to 70% by weight, preferably 3 to 40% by weight.

  The method for inhibiting plant feeding by pests according to the present invention includes the step of artificially imparting MGDG to a plant. Here, “manually assigning MGDG to a plant” means applying MGDG or a formulated composition to the plant from the outside, using genetic engineering techniques to the plant body. A method for increasing the amount of MGDG in the plant body by increasing the expression of the contained MGDG synthase, a method for increasing the amount of MGDG in the plant body by introducing an exogenous MGDG synthase gene into the plant body, A method for increasing the amount of MGDG in the plant body by increasing the expression level or activity of other enzymes involved in the MGDG synthesis pathway in the body, or a symbiotic bacterium that has the ability to increase the amount of MGDG in the plant body in symbiosis It includes a method for increasing the amount of MGDG by artificially symbiotic with a plant. MGDG synthase is disclosed in, for example, Japanese Patent Application Laid-Open No. 10-14579. Examples of symbiotic bacteria having the ability to symbiosis with plants and increase the amount of MGDG in the plant body include, for example, Neotyphodium sp. Po-060 stock (this stock was deposited on March 31, 2005 as a deposit number NITE P-94 at the Patent Microorganisms Depositary Center for Product Evaluation Technology (2-5-8, Kazusa-Kamashita, Kisarazu-shi, Chiba) ).

  Preferred examples of the present invention are shown below, but the scope of the present invention is not limited to these examples.

  The following experiment was conducted for the purpose of extracting and identifying a component exhibiting an antifeeding activity to Sugikiryoto from a gramineous plant (meadow fescue) infected with endophyte.

Materials and methods
1) Ligro which is a cultivar of a plant Meadow Fescue (Festuka platenosis) was used. Meadow fescue was produced by growing a neotyphodium endophyte (Neotyphodium sp. Po-060 strain, which may be abbreviated as “Po-060” hereinafter) infected and non-infected sections.

2) Use insect As a lepidopterous insect for evaluating the feeding inhibition action, the main insect pest of turfgrass (Spodoptera depravata Butler) was used. Feeding inhibition test was performed using larvae of 2-3 years old.

3) Selective feeding test First, a selective feeding test for Po-060 infected medusa fescue and endophyte non-infected medusa fescue was carried out for alternative larvae.

4) Extraction of feeding inhibitory active substance The extraction of the feeding inhibitory active substance for Spodoptera was performed using the stem and leaf part of Po-060 strain-infected medo fescue (Ei: endophyte infected). Further, as a control test, extraction was similarly carried out from the stem and leaf portion of endophyte-free infected meadow fescue (Ef: endophyte free).
The stems and leaves were each extracted with 100 ml of 90% acetone, filtered and dehydrated, and concentrated to dryness.

5) Isolation and purification of feeding-inhibiting active substance The acetone-extracted sample of the above-mentioned Meadowesque was fractionated by silica gel column chromatography and C-18 column chromatography. The fractionation and elution classification are shown in FIG. In addition, bioassay using swordfish was carried out at each stage, and the fraction containing an anti-feeding substance was confirmed. Samples used in the bioassay were adjusted to a constant concentration with ethyl acetate or methanol and dissolved.

6) Feeding inhibition test (bioassay)
For the evaluation of each fraction, a test method using cut leaves was used. Apply each fraction to both sides of cut leaves (length: 2cm, weight: about 3mg) of non-infected medope fescue, air-dry well and volatilize the solvent, then leave it in a petri dish, and 20-40 heads of 2-3 years old swordfish , And fed for 6 to 8 hours at 25 ° C., and the degree of feeding was measured by image analysis in pixel units.
The evaluation of feeding was performed by calculating according to the following formula proposed by Alkofahi based on the feeding rate calculated in pixel units.
Antifeedant index (AFI)
= 100xT / (T + C)
In the formula, T: feeding rate of the cut leaves treated with the Po-060 infected individual extract, C: feeding rate of the cut leaves treated with the non-infected individual extract (control).
When this index is 30 or less, it has feeding inhibitory activity, and when the index is higher than that, it is considered that feeding inhibitory activity is inactive. When the feeding rates of the Po-060 infected individual extract-treated cut leaves and the non-infected individual extract-treated cut leaves are equal, the feeding inhibition index indicates 50.

7) High Performance Liquid Chromatography (HPLC) Analysis The HPLC analysis conditions are as follows.
Model: TOSOH HPLC system DP-8020
Detector: TOSOH HPLC PDA PD-8020
Detection wavelength: PDA multi-channel (UV 205nm)
Column used: TOSOH TSk-gel ODS-120A (7.8 mm ID x 300 mm, 10 μm)
Column temperature: room temperature Mobile phase composition: mobile phase A (water 100%), mobile phase B (acetonitrile 100%)
Elution ratio: 0-5 minutes (A100%), 5-20 minutes (A100% -0%, linear concentration gradient), 20-30 minutes (B100%)
Flow rate: 2.0 ml / min.
Injection volume: 20 μl

8) Structural analysis Structural analysis was performed using NMR (JNMGX500 NMR Spectrometer).

result
1) Selective feeding test In order to confirm the feeding-inhibiting activity of Po-060 endophyte-infected Meadow fescue against Japanese horse chestnut, an endophyte-infected / non-infected Meadowfesk feeding comparative test was conducted. FIG. 2 shows a photograph of the leaves at the end of the test. Moreover, the data of a feeding rate are shown in FIG.
As shown in FIG. 2 and FIG. 3, it was confirmed that infection of Meadowfesc with Po-060 strain showed a very strong feeding inhibitory activity against Scots biloba.

2) Isolation / purification of feeding-inhibiting active substance Fractionation was carried out using the feeding-inhibiting activity against Aspergillus oryzae (FIG. 1). A bio-assay of a crude extract of Po-060 infected Meadowfesc foliage, that is, 90% acetone extract was performed to confirm the activity.
Next, the 90% acetone extract was fractionated by silica gel column chromatography. The active ingredient was eluted with 100% ethyl acetate. These were again fractionated by silica gel column chromatography. Similarly, the feeding-inhibiting active ingredient was eluted with 100% ethyl acetate.
This ethyl acetate 100% elution fraction was further fractionated using C-18 column chromatography. The sample was applied to a C-18 column and eluted sequentially with methanol 30%, 60%, and 100%, and the feeding inhibitory activity against swordfish was measured for each. As a result, feeding inhibition activity was observed in the methanol 100% elution section.

3) HPLC analysis HPLC analysis was performed on C-18 column chromatography methanol 100% elution section where feeding inhibitory activity against Shirokiyoto was observed. FIG. 4a shows the HPLC analysis results of the same section of Po-060 infected medoufesc, and FIG. 4b shows the HPLC analysis results of the same section of Po-060 non-infected medoufesc.
From the results of FIG. 4, the retention time (Rt) of 27.3 min. In the Po-060 infected Meadow Fesc test group was compared with that in the non-infected Meadow fesc test group. It was observed that there was a very large peak at.

4) Isolation and identification of an antifeedant substance Rt 27.3 min. The material was isolated. The substance was isolated by HPLC (ODS), and the purity was confirmed again by HPLC analysis.
As a result of structural analysis of each substance by NMR, Rt 27.3 min. This substance was identified as Monogalactosyl diacylglycerol (MGDG) having the following structure.

  It was confirmed that MGDG was present at 1400 ppm in the Po-060 infected Meadowfesc live leaves. On the other hand, MGDG was present at 180 ppm in the uninfected medoufesc raw leaves. That is, it was found that the amount of MGDG accumulated in the plant in vivo increased by 7.7 times by infection with MYK-006 endophyte.

5) Activity evaluation Using the isolated MGDG, the feeding inhibitory activity was evaluated for the pearl millet. The results are shown in Table 1. The results of Table 1 are shown in FIG.

  From this result, the activity was not observed at the same application amount (180 ppm) as the content of MGDG in the endophyte-infected medope fescue, whereas the same application amount (1400 ppm) as that of the MGDG in the endophyte-infected medope fescue. The activity was confirmed at the above application amount.

It is a figure which shows the fractionation method of an eating inhibitory substance. It is a photograph which shows the result of a feeding selectivity test. It is a figure which shows the feeding rate in a feeding selectivity test. It is a figure which shows the HPLC analysis result of the C-18 column chromatography methanol 100% elution group of Po-060 infection Meadow fescue. It is a figure which shows the HPLC analysis result of the C-18 column chromatography methanol 100% elution group of an endophyte non-infected meadow fescue. It is a figure which shows the feeding-inhibiting effect with respect to a swordfish of MGDG.

Claims (2)

  A plant pest feeding inhibitor containing monogalactosyldiacylglycerol as an active ingredient.   A method for inhibiting feeding by plant pests comprising the step of artificially imparting monogalactosyldiacylglycerol to a plant.

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