JP2020063199A - Composition for controlling pet and method for controlling pest - Google Patents
Composition for controlling pet and method for controlling pest Download PDFInfo
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本発明は、殺虫剤の虫体への付着を阻害する虫体被覆物を有する害虫を防除するための組成物及び当該害虫を防除する方法に関する。 TECHNICAL FIELD The present invention relates to a composition for controlling a pest having an insect body coating that inhibits the attachment of an insecticide to the insect body, and a method for controlling the pest.
植物に食害を与える害虫の中には、体表がロウ状の物質を含む虫体被覆物で覆われている害虫がいる。このような虫体被覆物は殺虫剤の虫体への付着を阻害するため、これを有する害虫に対しては、殺虫剤を含む農薬を直接散布しても十分に防除することができない。そのような殺虫剤による防除が難しい害虫の１種として、例えば、コナカイガラムシ類が知られている。食害を与えるコナカイガラムシ類の雌の成虫は、ロウ状の物質で覆われており、殺虫剤を弾き、虫体に付着しにくいので、殺虫剤を虫体表面に適用しても、その効果を十分に発揮することができない。そして、コナカイガラムシ類は、病害も媒介することから、効果的な防除方法が早急に必要とされている。 Among the pests that feed on plants, there are pests whose body surface is covered with an insect body coating containing a waxy substance. Since such an insect-body coating inhibits the attachment of the insecticide to the insect body, it is not possible to sufficiently control the harmful insects having the insecticide by directly spraying the pesticide containing the insecticide. As one kind of pests that are difficult to control with such insecticides, for example, the scale insects are known. Adult female insects of the scale insect beetle, which causes feeding damage, are covered with wax-like substances and repel pesticides, making it difficult for them to attach to insect bodies. I can't show it to the full. Since the scale insects also transmit diseases, effective control methods are urgently needed.
殺虫剤の虫体への付着を阻害する虫体被覆物を有する害虫に対する防除方法としては、植物浸透移行性の殺虫剤を使用し、害虫が植物を食べることで体内に殺虫剤を取り込ませたり、天敵により防除したり、物理的に気門を塞いだり、歯ブラシなどで植物から物理的に擦り取ったりする方法が用いられている。しかしながら、植物浸透移行性の殺虫剤ではある程度の効果が期待できるものの、当該殺虫剤は植物中に残存するものであるため、使用の際に慎重な取り扱いが求められ、使用量及び使用回数などが厳しく制限されている。また、天敵防除や物理的防除は、発生初期などの個体数が少ない場合には有効であるが、増殖した際や長期的な効果は期待できない。 As a method for controlling pests that have insect body coatings that inhibit the attachment of insecticides to insect bodies, insecticides that are plant-penetrating and migrating are used, and insect pests can be incorporated into the body by the insects eating the plants. , Methods of controlling by natural enemies, physically blocking the air gate, and physically scraping from plants with a toothbrush are used. However, although some effects can be expected with a plant-penetrating migration insecticide, since the insecticide remains in the plant, careful handling is required at the time of use, and the usage amount and number of times It is strictly limited. In addition, natural enemy control and physical control are effective when the number of individuals is small, such as in the early stage of development, but cannot be expected when they multiply or in the long term.
一方、生分解性プラスチックを速やかに分解するクチナーゼ様酵素が、植物常在性の酵母や糸状菌から単離同定されている（特許文献１及び２、非特許文献１〜３）。クチナーゼ様酵素は、様々な長さの炭素鎖のエステル結合を加水分解する活性を有する、基質特異性の広いエステラーゼである（非特許文献１）。クチナーゼ様酵素を大量生産する技術も開発されており（特許文献３及び４）、近い将来、市場で、安価な酵素液が大量に入手可能になることが期待されるが、クチナーゼ様酵素の害虫に対する効果は知られていない。 On the other hand, cutinase-like enzymes that rapidly decompose biodegradable plastics have been isolated and identified from plant-resident yeasts and filamentous fungi (Patent Documents 1 and 2, Non-Patent Documents 1 to 3). A cutinase-like enzyme is an esterase with a wide substrate specificity, which has an activity of hydrolyzing ester bonds of carbon chains of various lengths (Non-Patent Document 1). Techniques for mass-producing cutinase-like enzymes have also been developed (Patent Documents 3 and 4), and it is expected that a large amount of cheap enzyme solutions will be available in the market in the near future. The effect on is unknown.
近年、環境保全型農業への人々の関心が高まり、作物中に浸透移行する殺虫剤の使用量を減らしつつ、より食の安全や環境負荷低減への配慮がなされた、新たな病害虫防除技術が求められている。また、人体に対して有毒な殺虫剤の使用量や使用回数を抑えることができる、より効率的な害虫の防除方法が求められている。したがって、本発明は、殺虫剤の虫体への付着を阻害する虫体被覆物を有する害虫を安全かつ効率的に防除することを目的としている。 In recent years, people's interest in environmentally friendly agriculture has increased, and a new pest control technology has been developed that takes into consideration food safety and environmental load reduction while reducing the amount of insecticide that penetrates into crops. It has been demanded. There is also a demand for a more efficient pest control method that can reduce the amount and number of times of use of insecticides that are toxic to the human body. Therefore, an object of the present invention is to safely and efficiently control pests having an insect-body coating that inhibits the attachment of insecticides to the insect body.
本発明者らは、上記課題を解決すべく鋭意検討した結果、生分解性プラスチック分解酵素として知られていたクチナーゼ様酵素が、殺虫剤の虫体への付着を阻害する虫体被覆物を溶解することを見出し、本発明を完成させた。
すなわち、本発明は、以下に示す害虫を防除するための組成物及び害虫を防除する方法を提供するものである。
〔１〕害虫を防除するための組成物であって、クチナーゼ様酵素を含み、前記害虫が、殺虫剤の虫体への付着を阻害する虫体被覆物を有することを特徴とする、組成物。
〔２〕害虫において殺虫剤の殺虫作用を増強するための組成物であって、クチナーゼ様酵素を含み、前記害虫が、前記殺虫剤の虫体への付着を阻害する虫体被覆物を有することを特徴とする、組成物。
〔３〕殺虫剤をさらに含む、前記〔１〕又は〔２〕に記載の組成物。
〔４〕前記クチナーゼ様酵素が、シュードザイマ（Ｐｓｅｕｄｏｚｙｍａ）属、モエジオマイセス属（Ｍｏｅｓｚｉｏｍｙｃｅｓ）、パラフォーマ（Ｐａｒａｐｈｏｍａ）属、クリプトコッカス（Ｃｒｙｐｔｏｃｏｃｃｕｓ）属、ムコール（Ｍｕｃｏｒ）属、フミコラ（Ｈｕｍｉｃｏｌａ）属、テルモミセス（Ｔｈｅｒｍｏｍｙｃｅｓ）属、タラロミセス（Ｔａｌａｒｏｍｙｃｅｓ）属、ケトミウム（Ｃｈａｅｔｏｍｉｕｍ）属、トルラ（Ｔｏｒｕｌａ）属、スポロトリクム（Ｓｐｏｒｏｔｒｉｃｈｕｍ）属、マルブランケア（Ｍａｌｂｒａｎｃｈｅａ）属、アルタナリア（Ａｌｔｅｒｎａｒｉａ）属、クラドスポリウム（Ｃｌａｄｏｓｐｏｒｉｕｍ）属、ぺニシリウム（Ｐｅｎｉｃｉｌｌｉｕｍ）属、ペキロマイセス（Ｐａｅｃｉｌｏｍｙｃｅｓ）属、シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属、バクテロイデス（Ｂａｃｔｅｒｏｉｄｅｓ）属、及び、アシドボラックス（Ａｃｉｄｏｖｏｒａｘ）属から成る群から選択される少なくとも１種の微生物に由来する、前記〔１〕〜〔３〕のいずれか一項に記載の組成物。
〔５〕前記クチナーゼ様酵素が、シュードザイマ・アンタクティカ（Ｐｓｅｕｄｏｚｙｍａ ａｎｔａｒｃｔｉｃａ）が産生するエステラーゼ（ＰａＥ）、パラフォーマ属類縁菌クチナーゼ様酵素（ＰＣＬＥ）、及び、クリプトコッカス・マグナス（Ｃｒｙｐｔｏｃｏｃｃｕｓ ｍａｇｎｕｓ）又はその類縁菌により産生されるクチナーゼ様酵素１（ＣｍＣｕｔ１）からなる群から選択される少なくとも１種である、前記〔１〕〜〔４〕のいずれか一項に記載の組成物。
〔６〕前記害虫が、ロウ状物質を含む虫体被覆物を有する昆虫である、前記〔１〕〜〔５〕のいずれか一項に記載の組成物。
〔７〕害虫を防除する方法であって、
クチナーゼ様酵素を含む組成物を前記害虫に接触させる工程を含み、
前記害虫が、殺虫剤の虫体への付着を阻害する虫体被覆物を有することを特徴とする、方法。
〔８〕害虫において殺虫剤の殺虫作用を増強する方法であって、
クチナーゼ様酵素を含む組成物を前記害虫に接触させる工程を含み、
前記害虫が、前記殺虫剤の虫体への付着を阻害する虫体被覆物を有することを特徴とする、方法。
〔９〕前記クチナーゼ様酵素の接触工程と同時に又はその後に、殺虫剤を含む組成物を前記害虫に接触させる工程をさらに含む、前記〔６〕又は〔７〕に記載の方法。
As a result of intensive studies to solve the above problems, the present inventors have found that a cutinase-like enzyme known as a biodegradable plastic-degrading enzyme dissolves insect body coatings that inhibit the attachment of insecticides to insect bodies. The present invention has been completed and the present invention has been completed.
That is, the present invention provides the following composition for controlling harmful insects and a method for controlling harmful insects.
[1] A composition for controlling pests, comprising a cutinase-like enzyme, wherein the pest has an insect body coating that inhibits adhesion of insecticide to insect bodies. .
[2] A composition for enhancing the insecticidal action of an insecticide in a pest, comprising a cutinase-like enzyme, wherein the pest has an insect body coating that inhibits adhesion of the insecticide to the insect body. A composition comprising:
[3] The composition according to [1] or [2], further containing an insecticide.
[4] The cutinase-like enzyme is a genus Pseudozyme, a genus Moesziomyces, a genus Paraphoma, a genus Cryptococcus, a genus Mucor, and a humicola humicola. Genus, Talaromyces genus, Chaetomium genus, Torula genus, Sporotorichum genus, Malbranchea genus, Alternaria sperium Cl genus, Cladosporium Genus (Penicillium), Paecilomyces es), Pseudomonas genus, Bacteroides genus, and Acidovorax genus, derived from at least one microorganism selected from the group consisting of the above-mentioned [1]-[3] The composition according to any one of claims.
[5] The cutinase-like enzyme is an esterase (PaE) produced by Pseudozyma antarctica, a paraforma-related cutinase-like enzyme (PCLE), and Cryptococcus magnus or its analogues. The composition according to any one of [1] to [4] above, which is at least one selected from the group consisting of cutinase-like enzyme 1 (CmCut1) produced.
[6] The composition according to any one of [1] to [5] above, wherein the pest is an insect having an insect body coating containing a waxy substance.
[7] A method for controlling pests,
Comprising the step of contacting a composition containing a cutinase-like enzyme with the pest,
The method as described above, wherein the pest has an insect body coating that inhibits adhesion of the insecticide to the insect body.
[8] A method for enhancing the insecticidal action of an insecticide in a pest, comprising:
Comprising the step of contacting a composition containing a cutinase-like enzyme with the pest,
The method as described above, wherein the pest has an insect body coating that inhibits adhesion of the insecticide to the insect body.
[9] The method according to [6] or [7] above, which further comprises a step of bringing a composition containing an insecticide into contact with the harmful insects, simultaneously with or after the step of contacting the cutinase-like enzyme.
本発明に従えば、クチナーゼ様酵素を害虫防除のための有効成分として使用することにより、従来の殺虫剤では防除が難しいとされていた害虫を防除することができる。また、クチナーゼ様酵素と従来の殺虫剤とを併用すれば、相乗的な防除作用が奏される。したがって、人体に対して有毒な殺虫剤の使用量を低減し、殺虫剤の虫体への付着を阻害する虫体被覆物を有する害虫でも安全かつ効率的に防除することが可能となる。 According to the present invention, by using a cutinase-like enzyme as an active ingredient for controlling pests, it is possible to control pests which have been difficult to control with conventional pesticides. In addition, when a cutinase-like enzyme and a conventional insecticide are used in combination, a synergistic control action is exhibited. Therefore, it becomes possible to reduce the amount of the insecticide toxic to the human body and safely and efficiently control even the pests having the insect body coating that inhibits the attachment of the insecticide to the insect body.
以下、本発明をさらに詳細に説明する。
本発明は、害虫を防除するための組成物又は害虫において殺虫剤の殺虫作用を増強するための組成物に関しており、当該組成物は、クチナーゼ様酵素を有効成分として含み、前記害虫が、殺虫剤の虫体への付着を阻害する虫体被覆物を有することを特徴としている。前記虫体被覆物は、ロウ状物質を含み得るものであり、殺虫剤が虫体に付着しにくいため、そのような虫体被覆物を有している害虫（特に昆虫）を従来の殺虫剤単独で防除することは難しい。前記ロウ状物質は、高級脂肪酸及び／又は高級アルコールなどの比較的鎖長の長い構成部分を有するエステルを含み得る。前記虫体被覆物を有する害虫は、特に限定されないが、例えば、ミカンコナカイガラムシ、ミカンヒメコナカイガラムシ、及びフジコナカイガラムシなどのコナカイガラムシ類、並びに、カラマツカサアブラムシ、ヒメカサアブラムシなどのカサアブラムシ類などであってもよい。
Hereinafter, the present invention will be described in more detail.
The present invention relates to a composition for controlling a pest or a composition for enhancing the insecticidal action of an insecticide in a pest, wherein the composition contains a cutinase-like enzyme as an active ingredient, and the pest is an insecticide. It is characterized by having a worm body coating that inhibits adhesion of the worm body to the worm body. Since the insect-body coating may contain a waxy substance, and the insecticide does not easily adhere to the insect body, a pest (especially an insect) having such an insect-body coating is used as a conventional insecticide. It is difficult to control alone. The waxy material may include esters having relatively long chain moieties such as higher fatty acids and / or higher alcohols. The insect pest having the worm body coating is not particularly limited, for example, beetle scales such as citrus scale insects, citrus scale bugs, and aphid insects such as Fujita beetle, and aphids such as larch moss aphid and Aphididae. And so on.
本明細書に記載の「クチナーゼ様酵素」とは、生分解性プラスチックを分解する酵素として植物常在性の酵母や糸状菌から単離同定された酵素であって、そのアミノ酸配列中にクチナーゼと共通するリパーゼボックスを有しているが、当該クチナーゼとはアミノ酸配列同一性が低く、代表的なクチナーゼであるフザリウム・ソラニ（Ｆｕｓａｒｉｕｍ ｓｏｌａｎｉ）由来クチナーゼを比較対象としてマキシマムマッチングにより解析したときのアミノ酸配列同一性が、約６０％以下好ましくは約３０％以下の酵素のことをいう。なお、前記クチナーゼ様酵素は、必ずしもクチナーゼのようなクチン分解活性を有しているわけではないし、タンパク質全体のアミノ酸配列に関してクチナーゼとの相同性が高いわけでもない。基質特異性に関しても、クチナーゼでは、短鎖脂肪酸エステルの分解活性が高く、長鎖脂肪酸エステルの分解活性は低いのに対して、前記クチナーゼ様酵素は、短鎖脂肪酸エステル及び長鎖脂肪酸エステルのどちらもよく分解する。このように、クチナーゼとクチナーゼ様酵素とは、それぞれ特性が異なる別種の酵素であると認識されている。 The "cutinase-like enzyme" described in the present specification is an enzyme isolated and identified from plant-resident yeast or filamentous fungus as an enzyme that decomposes biodegradable plastics, and cutinase in the amino acid sequence thereof. Although it has a common lipase box, it has low amino acid sequence identity with the cutinase, and the cutinase from Fusarium solani, which is a typical cutinase, is analyzed by maximum matching as a comparison target. An enzyme having an identity of about 60% or less, preferably about 30% or less. The cutinase-like enzyme does not necessarily have a cutin-degrading activity like cutinase, and the amino acid sequence of the whole protein does not have high homology with cutinase. Regarding the substrate specificity, cutinase has a high activity of decomposing short-chain fatty acid esters and a low activity of degrading long-chain fatty acid esters, whereas the cutinase-like enzyme has a high activity of decomposing short-chain fatty acids and long-chain fatty acids. Also decomposes well. As described above, cutinase and cutinase-like enzyme are recognized as different types of enzymes having different characteristics.
前記クチナーゼ様酵素としては、当技術分野で通常用いられている種々の酵素を、特に制限されることなく採用することができるが、例えば、微生物により産生されるクチナーゼ様酵素を、当該微生物の培養液（培養ろ液）の形態で使用してもいいし、そこから単離精製して使用してもよい。前記クチナーゼ様酵素を産生する微生物は、特に限定されるものではないが、例えば、シュードザイマ（Ｐｓｅｕｄｏｚｙｍａ）属、モエジオマイセス属（Ｍｏｅｓｚｉｏｍｙｃｅｓ）、パラフォーマ（Ｐａｒａｐｈｏｍａ）属、クリプトコッカス（Ｃｒｙｐｔｏｃｏｃｃｕｓ）属、ムコール（Ｍｕｃｏｒ）属、フミコラ（Ｈｕｍｉｃｏｌａ）属、テルモミセス（Ｔｈｅｒｍｏｍｙｃｅｓ）属、タラロミセス（Ｔａｌａｒｏｍｙｃｅｓ）属、ケトミウム（Ｃｈａｅｔｏｍｉｕｍ）属、トルラ（Ｔｏｒｕｌａ）属、スポロトリクム（Ｓｐｏｒｏｔｒｉｃｈｕｍ）属、マルブランケア（Ｍａｌｂｒａｎｃｈｅａ）属、アルタナリア（Ａｌｔｅｒｎａｒｉａ）属、クラドスポリウム（Ｃｌａｄｏｓｐｏｒｉｕｍ）属、ぺニシリウム（Ｐｅｎｉｃｉｌｌｉｕｍ）属、ペキロマイセス（Ｐａｅｃｉｌｏｍｙｃｅｓ）属、シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属、バクテロイデス（Ｂａｃｔｅｒｏｉｄｅｓ）属、及び、アシドボラックス（Ａｃｉｄｏｖｏｒａｘ）属から成る群から選択される微生物であってもよい。 As the cutinase-like enzyme, various enzymes commonly used in the art can be adopted without particular limitation. For example, a cutinase-like enzyme produced by a microorganism may be cultured in the microorganism. It may be used in the form of a liquid (culture filtrate) or may be used after being isolated and purified from it. Microorganisms that produce the cutinase-like enzyme are not particularly limited, and include, for example, Pseudozyme genus, Moesziomyces genus, Paraforma genus, Cryptococcus genus, and Mucor. Genus, Humicola, Thermomyces, Talaromyces, Chaetomium, Torulaa, Sporotrichumal, Sporotrichum M, Marblanca. Genus Cladosporium, Penicillium ium) genus Pekiromaisesu (Paecilomyces) genus Pseudomonas (Pseudomonas) genus Bacteroides (Bacteroides) genus, and it may be a microorganism selected from the group consisting of Acidovorax (Acidovorax) genus.
前記クチナーゼ様酵素は、好ましくは、シュードザイマ・アンタクティカ（Ｐｓｅｕｄｏｚｙｍａ ａｎｔａｒｃｔｉｃａ）が産生するエステラーゼ（ＰａＥ）、パラフォーマ属類縁菌クチナーゼ様酵素（ＰＣＬＥ）、又は、クチナーゼ様酵素１（ＣｍＣｕｔ１）である。ＰａＥは、例えば、ＧＢ−４（１）Ｗ株、ＧＢ−４（０）−ＨＰＭ７株（独立行政法人製品評価技術基盤機構特許微生物寄託センターに寄託された酵母；受託番号ＮＩＴＥ Ｐ−０２２３８）及びＯＭＭ６２−２株（独立行政法人製品評価技術基盤機構特許微生物寄託センターに寄託された酵母；受託番号ＮＩＴＥ Ｐ−０２２３９）などのシュードザイマ・アンタクティカ（最近分類が変更され、モエジオマイセス・アンタクティクス（Ｍｏｅｓｚｉｏｍｙｃｅｓ ａｎｔａｒｃｔｉｃｕｓ）と呼ばれることもある。）により産生される酵素である。ＰＣＬＥは、パラフォーマ属類縁菌Ｂ４７−９株（独立行政法人製品評価技術基盤機構特許微生物寄託センターに寄託された糸状菌；受託番号ＮＩＴＥ Ｐ−５７３；要すれば特許第５０８２１２５号参照）などのパラフォーマ属類縁菌により産生される酵素であり、ＣｍＣｕｔ１は、クリプトコッカス・マグナス（Ｃｒｙｐｔｏｃｏｃｃｕｓ ｍａｇｎｕｓ）類縁菌ＢＰＤ１Ａ株（独立行政法人製品評価技術基盤機構特許微生物寄託センターに寄託された酵母；受託番号ＮＩＴＥ Ｐ−０２１３４）などのクリプトコッカス・マグナス又はその類縁菌により産生される酵素である。Ｆ．ソラニ由来クチナーゼを比較対象としてマキシマムマッチングにより解析したときのアミノ酸配列同一性は、ＰａＥでは２０．０８％、ＰＣＬＥでは５７．２６％、そしてＣｍＣｕｔ１では２６．０１％である。前記クチナーゼ様酵素は、遺伝子組換え技術によって生産された酵素であってもよい。 The cutinase-like enzyme is preferably an esterase (PaE) produced by Pseudozyma antarctica, a paraforma-related fungus cutinase-like enzyme (PCLE), or a cutinase-like enzyme 1 (CmCut1). PaE includes, for example, GB-4 (1) W strain, GB-4 (0) -HPM7 strain (yeast deposited at the Patent Microorganism Depositary Center, National Institute for Product Evaluation Technology, National Institute for Product Evaluation Technology; accession number NITE P-02238) and OMM62-2 strain (Yeast deposited at the Patent Microorganism Depositary Center, National Institute of Product Evaluation Technology, Japan; Accession No. NITE P-02239) and other Pseudozyma antactica (recently changed classification, Moesziomyces antactics) It is also called.) The enzyme produced by. PCLE includes Paraforma related strain B47-9 strain (filamentous fungi deposited at the Patent Microorganism Depositary Center, National Institute of Technology for Product Evaluation and Technology; Deposit No. NITE P-573; see Patent No. 5082125 if necessary). CmCut1, which is an enzyme produced by Paraforma-related fungi, is Cryptococcus magnus-related BPD1A strain (yeast deposited at the Patent Microorganism Depositary Center, National Institute for Product Evaluation Technology, National Institute for Product Evaluation Technology; Accession No. NITE P). -02134) and other enzymes produced by Cryptococcus magnus or related fungi. F. The amino acid sequence identities when the Solani-derived cutinase was analyzed by maximum matching as a comparison were 20.08% for PaE, 57.26% for PCLE, and 26.01% for CmCut1. The cutinase-like enzyme may be an enzyme produced by a gene recombination technique.
前記クチナーゼ様酵素の力価は、生分解性プラスチックであるポリブチレンサクシネート−ｃｏ−アジペート（ＰＢＳＡ）エマルジョン（昭和電工株式会社、ＥＭ−３０１）の濁度の減少量として規定することができる。本明細書においては、ＯＤ６６０ｎｍの値を１分間に１低下させるときの力価を１Ｕと定義する。酵素活性測定時の緩衝液としては、例えば、トリス塩酸緩衝液（２０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ９．０、塩化カルシウム（２ｍＭ）なし又はあり）を使用してもよい。 The titer of the cutinase-like enzyme can be defined as the amount of turbidity reduction of polybutylene succinate-co-adipate (PBSA) emulsion (Showa Denko KK, EM-301), which is a biodegradable plastic. In the present specification, the titer when the value of OD660nm is decreased by 1 per minute is defined as 1U. As the buffer solution for measuring the enzyme activity, for example, a Tris-hydrochloric acid buffer solution (20 mM Tris-HCl, pH 9.0, without or with calcium chloride (2 mM)) may be used.
特定の理論に拘束されるものではないが、前記クチナーゼ様酵素は、害虫の体表において殺虫剤の虫体への付着を阻害している虫体被覆物を溶解し脱離させることができ、溶出した虫体被覆物は、虫体の周囲で乾燥して再度固化することによって、その虫体の運動を阻害し、さらには当該虫体の気門を塞いで窒息死させることができると考えられる。また、前記虫体被覆物を溶解し脱離させると、前記害虫の体表が露出するため、殺虫剤が容易に付着して虫体内に浸潤し、その防除活性を有効に発揮することができると考えられる。 Without being bound to a particular theory, the cutinase-like enzyme is capable of dissolving and releasing the worm body coating that inhibits the attachment of the insecticide to the worm body on the body surface of the pest, It is considered that the eluted worm body coating can be dried around the worm body and solidified again to inhibit the motility of the worm body, and further to block the air gates of the worm body to cause suffocation. To be Further, when the insect body coating is dissolved and released, the body surface of the pest is exposed, so that the insecticide easily attaches and infiltrates into the insect body, and its control activity can be effectively exerted. it is conceivable that.
本発明の組成物は、殺虫剤をさらに含んでもよい。前記殺虫剤としては、当技術分野で通常使用されるものを、特に制限されることなく採用することができるが、例えば、フェニトロチオン（ＭＥＰ）、ジメチルジカルベトキシエチルジチオホスフェート、ヘキシチアゾクス、ジクロルボス（ＤＤＶＰ）、及び／又は、酸化フェンブタスズなどを有効成分として含む殺虫剤を使用してもよい。なお、上述したように、前記クチナーゼ様酵素を作用させると前記害虫の体表が露出するため、害虫の体表に付着し虫体内に浸潤して作用するタイプの殺虫剤を前記クチナーゼ様酵素と併用すると、顕著な併用効果が観察されやすい。 The composition of the present invention may further include an insecticide. As the insecticide, those commonly used in the art can be employed without particular limitation, and examples thereof include fenitrothion (MEP), dimethyldicarbetoxyethyldithiophosphate, hexithiazox, dichlorvos (DDVP). ), And / or an insecticide containing fenbutatin oxide or the like as an active ingredient may be used. As described above, since the body surface of the pest is exposed when the cutinase-like enzyme is acted on, the insecticide of a type that acts on the body surface of the pest by invading the body of the pest and acting as the cutinase-like enzyme. When used in combination, a remarkable combination effect is easily observed.
また、本発明の組成物は、害虫の防除活性を妨げない限り、任意の添加剤をさらに含んでもよい。前記添加剤としては、特に限定されないが、例えば、直鎖アルキルベンゼンスルホン酸塩、ポリオキシエチレンノニフィルフェニルエーテル、及び、ポリオキシエチレンドデシルエーテルなどの界面活性剤などを採用してもよい。 In addition, the composition of the present invention may further contain any additive as long as it does not interfere with the pest control activity. The additive is not particularly limited, but for example, a linear alkylbenzene sulfonate, a surfactant such as polyoxyethylene noniphenyl phenyl ether, and polyoxyethylene dodecyl ether may be adopted.
別の態様では、本発明は、害虫を防除する方法又は害虫において殺虫剤の殺虫作用を増強する方法にも関しており、当該方法は、クチナーゼ様酵素を含む組成物を前記害虫に接触させる工程を含み、前記害虫が、殺虫剤の虫体への付着を阻害する虫体被覆物を有することを特徴としている。本発明の方法は、前記クチナーゼ様酵素の接触工程と同時に又はその後に、殺虫剤を含む組成物を前記害虫に接触させる工程をさらに含んでもよい。 In another aspect, the invention also relates to a method of controlling a pest or a method of enhancing the insecticidal action of an insecticide in a pest, the method comprising contacting a composition comprising a cutinase-like enzyme with said pest. And the pest has a worm body coating that inhibits adhesion of the insecticide to the worm body. The method of the present invention may further comprise a step of contacting a composition containing an insecticide with the pest, simultaneously with or after the step of contacting the cutinase-like enzyme.
以下、実施例により本発明を具体的に説明するが、本発明の範囲はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to examples, but the scope of the present invention is not limited to these examples.
〔実施例１〕
Ｐ．アンタクティカを常法により培養し、その培養ろ液から５０％飽和硫安沈殿物、７０％エタノール可溶性成分、そして９０％エタノール沈殿物を順に回収することによって、クチナーゼ様酵素であるＰａＥを粗精製した（要すれば特願２０１８−０５２９５２号を参照）。このＰａＥを１０Ｕ、５０Ｕ、又は１００Ｕ含む酵素液（２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ９．０））を、それぞれ１ｍＬずつ調製した。シャーレ内にミカンコナカイガラムシ（雌）約１５匹を入れ、ここへスプレーを用いて各酵素液を噴霧した。対照区では、緩衝液のみを１ｍＬ噴霧処理した。シャーレの蓋を閉めて室温で２４時間置いた後、仰天などの状態で動かない又は動けなくなった虫体の割合をノックダウン（ＫＤ）活性として評価した。結果を表１に示す。
[Example 1]
P. The tactica was cultured by a conventional method, and 50% saturated ammonium sulfate precipitate, 70% ethanol-soluble component, and 90% ethanol precipitate were sequentially recovered from the culture filtrate to roughly purify the cutinase-like enzyme PaE ( See Japanese Patent Application No. 2018-052952 if necessary). 1 mL of each enzyme solution (20 mM Tris-HCl (pH 9.0)) containing 10 U, 50 U, or 100 U of this PaE was prepared. Approximately 15 citrus scale insects (female) were placed in a petri dish, and each enzyme solution was sprayed thereto using a spray. In the control group, only 1 mL of the buffer solution was spray-treated. After the lid of the petri dish was closed and left at room temperature for 24 hours, the proportion of worms that did not move or could not move due to astonishment was evaluated as knockdown (KD) activity. The results are shown in Table 1.
表１に示されているように、ＰａＥ濃度が高くなるにつれてＫＤ活性が上昇した。したがって、ＰａＥは、ミカンコナカイガラムシの防除に使用することができることがわかった。ＫＤ活性を評価した時点のミカンコナカイガラムシの様子を実体顕微鏡で観察すると、ミカンコナカイガラムシへの酵素液の噴霧によって虫体被覆物が溶けて虫体の周りに流れ出し、そこで再度固化した様子が見て取れる（図１）。図１の左図では、虫体の背側がシャーレ表面に固定されており、図１の右図では、虫体の腹側がシャーレ表面に固定されている。このことから、ＰａＥを含む酵素液を噴霧すると、虫体被覆物が溶出し、噴霧した酵素液がシャーレ内で蒸発するにつれて溶出した虫体被覆物が虫体の周囲で再度固化することによって、その運動を阻害し、さらには当該虫体の気門を塞いで窒息死させたと考えられる。 As shown in Table 1, the KD activity increased with increasing PaE concentration. Therefore, it was found that PaE can be used for controlling citrus scale insects. Observing the state of the citrus scale bug at the time of evaluating the KD activity with a stereomicroscope, it can be seen that the worm body coating melts and flows out around the worm body by spraying the enzyme solution on the citrus scale bug, which is then solidified again. (Figure 1). In the left diagram of FIG. 1, the dorsal side of the insect body is fixed to the petri dish surface, and in the right diagram of FIG. 1, the ventral side of the insect body is fixed to the petri dish surface. From this, when the enzyme solution containing PaE is sprayed, the insect body coating is eluted, and as the sprayed enzyme solution evaporates in the petri dish, the eluted insect body coating solidifies again around the insect body, It is considered that the movement was inhibited, and further, the air gate of the insect body was blocked to cause suffocation.
〔実施例２〕
（１）ミカンコナカイガラムシの虫体被覆物への酵素液処理の影響
実施例１に記載のように粗精製したＰａＥ（粗精製酵素）を１０Ｕの濃度で含む酵素液（２０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ９．０）又はＰａＥを含む培養ろ液（４Ｕ）を各２ｍＬずつ用意し、その中にミカンコナカイガラムシ（雌）を室温で浸漬した。対照区は酵素液を調製する際に使用した緩衝液（２０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ９．０）を用いた。浸漬２４時間後にミカンコナカイガラムシの状態を実体顕微鏡で観察した。結果を図２に示す。
[Example 2]
(1) Effect of Enzyme Solution Treatment on Insect Body Coating of Citrus scales, Enzyme Solution (20 mM Tris-HCl, pH 9) containing PaU (crude purified enzyme) crudely purified as described in Example 1 at a concentration of 10 U .0) or PaE-containing culture filtrate (4 U) was prepared in an amount of 2 mL each, and the citrus scale bug (female) was immersed therein at room temperature. As a control, the buffer solution (20 mM Tris-HCl, pH 9.0) used when preparing the enzyme solution was used. Twenty-four hours after the immersion, the state of the scale insecticide, Citrus algae, was observed with a stereoscopic microscope. The results are shown in Figure 2.
使用した酵素の濃度は、実施例１で十分なＫＤ活性が示された濃度よりも低いものであったが、上記酵素液又は培養ろ液の処理によって、ミカンコナカイガラムシの体表を覆うロウ状の物質が剥がれ、反応液中に分散するとともに、ミカンコナカイガラムシの体表が露わとなった（図２）。対照区では体表を覆うロウ状の物質は失われなかった。したがって、ＰａＥは、ミカンコナカイガラムシの体表において、ロウ状の物質からなる虫体被覆物を分解できることがわかった。 The concentration of the enzyme used was lower than the concentration at which sufficient KD activity was shown in Example 1, but the wax-like shape covering the body surface of the citrus scale insect was treated by the treatment with the enzyme solution or the culture filtrate. The substance was peeled off and dispersed in the reaction solution, and the body surface of the citrate scale insect was exposed (Fig. 2). In the control group, the wax-like substance covering the body surface was not lost. Therefore, it was found that PaE can decompose the worm body coating consisting of a wax-like substance on the body surface of the citrus scale insect.
（２）染色液の浸潤
ＰａＥ処理によって体表が露わになったミカンコナカイガラムシについて、外部から添加した溶液の浸潤のしやすさを検討するために、上記（１）と同様に各処理を行ったミカンコナカイガラムシに対し、水溶性染色試薬であるメチレンブルー（０．１％水溶液）を添加した。ロウ状の物質に覆われた対照区のミカンコナカイガラムシは、メチレンブルー水溶液を弾き、青色には呈色しなかった。一方、粗精製酵素を含む酵素液又は培養ろ液を用いた酵素処理によって体表の露わとなったミカンコナカイガラムシは、メチレンブルーによって青色に呈色した（図３）。したがって、ミカンコナカイガラムシの虫体被覆物をＰａＥ処理によって分解すると、外部から添加された溶液が虫体内に浸潤しやすくなることがわかった。
(2) Infiltration of dyeing solution In order to study the ease of infiltration of the solution added from the outside, the same treatment as in (1) above should be performed on the scale insecticide Beetle, Pseudomonas aeruginosa, whose surface is exposed by PaE treatment. Methylene blue (0.1% aqueous solution), which is a water-soluble staining reagent, was added to the citrus scale insects that had been tested. The control citrus scale insects covered with wax-like substances repelled the methylene blue aqueous solution and did not turn blue. On the other hand, the scale insects of the citrus scale, whose surface was exposed by the enzyme treatment using an enzyme solution containing a crudely purified enzyme or a culture filtrate, turned blue with methylene blue (FIG. 3). Therefore, it was found that when the coat of insects of Citrus scales was decomposed by the PaE treatment, the solution added from the outside was easily infiltrated into the insect body.
（３）殺虫剤と酵素の併用によるＫＤ活性
上記（２）の結果から、ミカンコナカイガラムシのＰａＥ処理により、殺虫剤も虫体に付着して虫体内に浸潤しやすくなる効果が期待された。そこで、緩衝液又は粗精製ＰａＥを含む酵素液と住化スミチオン乳剤（住友化学株式会社製）（一般名フェニトロチオン、ＭＥＰ）とを混合して、その混合液によるＫＤ活性を調べた。ＰａＥについては、終濃度４Ｕ／ｍＬとなるように、緩衝液（２０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ９．０）で調製した。住化スミチオン乳剤については、使用時に１０００倍希釈液となるような通常使用される濃度で調製した処理区と、そこから段階的にさらに希釈した処理区とを用意した。このようにして調製した混合液１ｍＬを、シャーレに採集したミカンコナカイガラムシ（雌）１０匹に対して噴霧処理し、その２４時間後に、ミカンコナカイガラムシのＫＤ活性を測定した。４回試行した結果の平均値を図４に示す。
(3) KD activity due to combined use of insecticide and enzyme From the results of (2) above, it was expected that the insecticide would be attached to the insect body and easily infiltrate into the insect body by PaE treatment of citrus scale insects. Therefore, a buffer solution or an enzyme solution containing crudely purified PaE and a Sumika Sumithion emulsion (manufactured by Sumitomo Chemical Co., Ltd.) (generic name: fenitrothion, MEP) were mixed, and the KD activity of the mixed solution was examined. PaE was prepared with a buffer solution (20 mM Tris-HCl, pH 9.0) so that the final concentration was 4 U / mL. As for the Sumika Sumition emulsion, a treatment group prepared at a concentration usually used so as to be a 1000-fold diluted solution at the time of use, and a treatment group further diluted stepwise therefrom were prepared. 1 mL of the thus-prepared mixed solution was spray-treated on 10 citrus scale insects (female) collected in a petri dish, and 24 hours after that, the KD activity of citrus scale insects was measured. The average value of the results of four trials is shown in FIG.
住化スミチオン乳剤の１０００倍希釈液単独のＫＤ活性は５４．５％程度にすぎなかった。このように殺虫剤が効きにくいミカンコナカイガラムシのような害虫は、難防除害虫として認識されているものである。ＰａＥ単独のＫＤ活性も、ＰａＥの終濃度が４Ｕ／ｍＬだと、たったの１４．６％にしかならなかった。しかしながら、ＰａＥと住化スミチオン乳剤との混合液でミカンコナカイガラムシを処理すると、ＫＤ活性が９０．４％にも達し、相乗的な殺虫作用が示された。そして、この相乗的な殺虫作用は、住化スミチオン乳剤の濃度をさらに薄くしても示された。したがって、ＰａＥと殺虫剤を併用すると、相乗的な殺虫作用が示されること、そして、人体にとっても有毒な殺虫剤の使用量を低減しても、通常の使用量と同等の殺虫作用を示すことが可能であることがわかった。 The KD activity of the 1000-fold diluted Sumika Sumithion emulsion alone was only about 54.5%. Thus, pests such as citrus scale insects, for which insecticides are difficult to act, are recognized as difficult-to-control pests. The KD activity of PaE alone was only 14.6% when the final concentration of PaE was 4 U / mL. However, when citrus scale insects were treated with a mixed solution of PaE and Sumika Sumition emulsion, the KD activity reached 90.4%, showing a synergistic insecticidal action. And, this synergistic insecticidal action was shown even when the concentration of the Sumika Sumithione emulsion was further reduced. Therefore, when PaE and an insecticide are used in combination, a synergistic insecticidal action is shown, and even if the amount of the insecticide that is toxic to the human body is reduced, it shows an insecticidal action equivalent to the usual amount used. It turns out that is possible.
〔実施例３〕
クチナーゼ様酵素であるＰａＥ、ＰＣＬＥ（パラフォーマ属類縁菌由来）、及びＣｍＣｕｔ１（クリプトコッカス・マグナス類縁菌由来）について、ミカンコナカイガラムシの体表を覆うロウ状の物質に対する影響を評価した。対照としては、クチナーゼ様酵素ではないリパーゼであるＣａｌＢ（シュードザイマ・アンタクティカ由来）を使用した。
[Example 3]
The effects of cutinase-like enzymes PaE, PCLE (derived from paraforma-related fungi), and CmCut1 (derived from cryptococcus magnus-related fungi) on wax-like substances that cover the body surface of the citrus scale insect were evaluated. As a control, CalB (from Pseudozyma antactica), which is a lipase that is not a cutinase-like enzyme, was used.
クチナーゼ様酵素は生分解性プラスチック分解酵素としても知られており、ＰＢＳＡエマルジョン分解活性によって規格化することができる。常法によってＰＣＬＥ及びＣｍＣｕｔ１を調製し、緩衝液（１ｍＭ Ｃａ2+含有２０ｍＭ ＨＥＰＥＳ緩衝液、ｐＨ７．３）中でのこれらのＰＢＳＡ分解活性が４ＵのＰａＥ（３１μｇタンパク質／ｍＬ）と等しくなるように各酵素の濃度を調整したところ、ＰＣＬＥの濃度及びＣｍＣｕｔ１の濃度は、どちらも３７μｇタンパク質／ｍＬになった。また、ＣａｌＢは、ＰａＥと同じタンパク質濃度（３１μｇタンパク質／ｍＬ）で使用した。ＨＥＰＥＳ緩衝液又は各酵素液１ｍＬに対し、ミカンコナカイガラムシ（雌）３匹を浸漬し、室温で２４時間静置した後、ミカンコナカイガラムシの状態を実体顕微鏡で観察した。結果を図５に示す。 Cutinase-like enzymes are also known as biodegradable plastic degrading enzymes and can be normalized by their PBSA emulsion degrading activity. PCLE and CmCut1 were prepared by a conventional method so that their PBSA degrading activity in a buffer (20 mM HEPES buffer containing 1 mM Ca 2+ , pH 7.3) was equal to 4 U PaE (31 μg protein / mL). When the concentration of each enzyme was adjusted, the concentration of PCLE and the concentration of CmCut1 were both 37 μg protein / mL. CalB was also used at the same protein concentration as PaE (31 μg protein / mL). Three citrus scale insects (female) were dipped in 1 mL of the HEPES buffer solution or each enzyme solution, allowed to stand at room temperature for 24 hours, and then the state of the citrus scale insects was observed with a stereomicroscope. Results are shown in FIG.
ＨＥＰＥＳ緩衝液又はＣａｌＢ溶液に浸漬したミカンコナカイガラムシでは、虫体被覆物の脱落又は溶解は観察されなかった。一方、ＰａＥ溶液、ＰＣＬＥ溶液、又はＣｍＣｕｔ１溶液に浸漬したミカンコナカイガラムシでは、酵素液に触れていた部分（図５中の矢印部分）の虫体被覆物が溶けて脱離し、虫体が露出していた。特に、ＰａＥ溶液を使用した場合には、ＰＣＬＥ溶液又はＣｍＣｕｔ１溶液を使用した場合と比べて、虫体被覆物の溶解の程度が顕著であった。したがって、クチナーゼ様酵素により奏される虫体被覆物の脱離又は溶解作用は、リパーゼなどの他のエステラーゼでは奏されない特異な作用であることがわかった。 No detachment or dissolution of the worm body coating was observed in the scale insects of the citrus scale insects dipped in the HEPES buffer solution or the CalB solution. On the other hand, in citrus scale insects dipped in PaE solution, PCLE solution, or CmCut1 solution, the insect body coating on the part (the arrow part in FIG. 5) that had been in contact with the enzyme solution was dissolved and released, exposing the insect body. Was there. In particular, when the PaE solution was used, the degree of dissolution of the worm body coating was remarkable as compared with the case where the PCLE solution or the CmCut1 solution was used. Therefore, it was found that the detachment or dissolution action of the insect body coating by the cutinase-like enzyme is a unique action not exhibited by other esterases such as lipase.
以上より、前記クチナーゼ様酵素は、殺虫剤の虫体への付着を阻害する虫体被覆物を有する害虫に対して単独で防除作用を奏すること、そして、殺虫剤と併用した場合には相乗的な防除作用を奏することがわかった。前記クチナーゼ酵素自体は、人体に対する毒性が低いので、人体に対して有毒な殺虫剤の使用量を低減し、殺虫剤の虫体への付着を阻害する虫体被覆物を有する害虫でも安全かつ効率的に防除することが可能となる。 From the above, the cutinase-like enzyme exerts a control action alone against a pest having an insect body coating that inhibits the attachment of the insecticide to the insect body, and, when used in combination with the insecticide, is synergistic. It was found to have a good control effect. Since the cutinase enzyme itself has low toxicity to the human body, it reduces the amount of insecticide toxic to the human body, and is safe and effective even for pests having insect body coatings that inhibit the attachment of insecticide to insect bodies. Can be controlled.
Claims (9)
クチナーゼ様酵素を含む組成物を前記害虫に接触させる工程を含み、
前記害虫が、殺虫剤の虫体への付着を阻害する虫体被覆物を有することを特徴とする、方法。 A method of controlling pests,
Comprising the step of contacting a composition containing a cutinase-like enzyme with the pest,
The method as described above, wherein the pest has an insect body coating that inhibits adhesion of the insecticide to the insect body.
クチナーゼ様酵素を含む組成物を前記害虫に接触させる工程を含み、
前記害虫が、前記殺虫剤の虫体への付着を阻害する虫体被覆物を有することを特徴とする、方法。 A method of enhancing the insecticidal action of an insecticide in a pest,
Comprising the step of contacting a composition containing a cutinase-like enzyme with the pest,
The method as described above, wherein the pest has an insect body coating that inhibits adhesion of the insecticide to the insect body.
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