JP5560499B2 - Control agent and control method for pinewood beetle - Google Patents
Control agent and control method for pinewood beetle Download PDFInfo
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- JP5560499B2 JP5560499B2 JP2012124989A JP2012124989A JP5560499B2 JP 5560499 B2 JP5560499 B2 JP 5560499B2 JP 2012124989 A JP2012124989 A JP 2012124989A JP 2012124989 A JP2012124989 A JP 2012124989A JP 5560499 B2 JP5560499 B2 JP 5560499B2
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- pine
- acid
- extract
- control agent
- beetle
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- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing carboxylic groups or thio analogues thereof, directly attached by the carbon atom to a cycloaliphatic ring; Derivatives thereof
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- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
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Description
本発明は、植物病害防除の技術分野に属し、マツノザイセンチュウおよびマツノマダラカミキリの防除剤および防除方法、ならびにこれら生物によって引き起こされるマツ材線虫病の防除剤および防除方法に関する。 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.
マツ材線虫病の原因であるマツノザイセンチュウ(線形動物門、幻器綱、アフェレンコイデス目、アフェレンコイデス科、Bursaphelenchus xylophilus(Steiner & Buhrer)Nickle)、および、その媒介虫であるマツノマダラカミキリ(節足動物門、昆虫綱、コウチュウ目、カミキリムシ科、Monochamus alternatus Hope)は、1905年に造船材料として北米から輸入された木材によって長崎に侵入し、その後、北海道、青森を除く全地域に拡大し、平成22年度のマツ材線虫病に羅病したマツの積量は約58万立方メートルに及ぶ。
日本に分布するアカマツやクロマツ、南西諸島の固有種であるヤクタネゴヨウ(絶滅危惧種)、リュウキュウマツが甚大な被害を受けており、日本各地に存在するマツが本病の脅威にさらされている。
本病による被害は日本のみならず、中国、台湾、韓国、ヨーロッパ地域においても発生しており、上記の種に加えてタイワンアカマツ、チョウセンゴヨウ、ヨーロッパアカマツ、フランスカイガンショウなどを含むマツ属(Pinus)植物も本病に対する感受性が高い。
ヨーロッパ諸国ではマツ材線虫病の感染拡大を防止するためマツ材の輸入に関して検疫を行っている。
The pine wood nematode that causes pine wilt disease (Linear phylum, Phantom, Aferenchoides, Aferenchoides, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle) and its vector, Matsunomadara Beetle (Arthropoda, Insecta, Coleoptera, Cerambycidae, Monochamus alternatus Hope) invaded Nagasaki in 1905 by timber imported from North America as a shipbuilding material, and then in all regions except Hokkaido and Aomori The volume of pine that has been expanded and affected by pine wilt disease in 2010 reaches approximately 580,000 cubic meters.
Japanese red pine and black pine, the endemic species Yakuta Neyoyo (endangered species) and Ryukyu pine are severely damaged, and pine existing in various parts of Japan are under threat. .
Damage caused by this disease has occurred not only in Japan but also in China, Taiwan, South Korea, and Europe. In addition to the above species, Pinus spp. ) plants are also highly sensitive against to the disease.
European countries have quarantined pine timber imports to prevent the spread of pine wilt disease .
マツ材線虫病は、5〜7月に枯れたマツの幹から羽化し、脱出したマツノマダラカミキリ(一般的には8千〜数万頭のマツノザイセンチュウを体内に保持)が性成熟のためにマツの若枝に飛来し、表皮を摂食する。
表皮が剥がれた部位から、マツノザイセンチュウを誘引するβミルセンやエタノールなどの化学物質が揮発することで、マツノザイセンチュウに対して正の走化性を示すと考えられているが、このようにしてマツノザイセンチュウはマツ組織内に寄生する(6〜9月)。
寄生後、早ければ2〜3週間程度で松枯れ症状を呈する。
そして、7〜8月になると、枯死したマツにマツノマダラカミキリ雌成虫が飛来し、マツの樹皮下に産卵する。
孵化したマツノマダラカミキリの幼虫は、マツの樹皮下組織を摂食しながら成長し、11〜翌5月に蛹室を形成して蛹となり、5〜6月に羽化に至る。
Pine line Mushibyo is, emerged from withered pine stem 5 to July, escape the pine sawyer (generally hold the pine wood nematode of 8 1000 to several tens of thousand animals in the body) is of sexual maturation To fly to the pine shoots and eat the epidermis.
From the site where the skin is peeled off, by chemicals such as β myrcene or ethanol to attract pine wood nematode is volatilized, it is thought to exhibit a positive chemotactic for pine wood nematodes, and thus pine wood nematode Te is you parasitic in the pine organization (6-9 may).
After parasitism, pine wilt symptoms occur as early as 2-3 weeks.
Then, at 7-8 May, flying the pine sawyer adult females in the dead pine, lay their eggs in the bark of pine.
Larvae hatched pine sawyer is, the bark under the organization of pine feeding and growth while eating, become a pupa to form a Sanagishitsu to 11 next May, leading to the emergence in May and June.
本病の防除手段は、一般的に、マツノザイセンチュウに対する予防剤の散布、媒介虫であるマツノマダラカミキリに対する殺虫剤の散布のほか、枯死木の伐倒や薫蒸処理などによって行われているが、これらの方法は有効である一方で、野生生物や環境への影響、農薬のドリフト(飛散)被害、労働および経費負担が問題視されている。
特にマツノマダラカミキリに使用される有機リン系殺虫剤は、人や他の生物にも影響することが懸念され、さらに農薬の連用は、薬剤耐性昆虫の出現を誘発することが知られている。
また抵抗性マツを利用する方法もあるが、抵抗性系統が少なく、挿木による繁殖が困難であること、外国産の系統は日本の風土に適さないなどの欠点がある。
Control means of this disease is generally spread prophylactic against pine wood nematode, other spraying insecticides against Monochamus alternatus which is mediated insect, is performed by such felling and fumigation of dead wood However, while these methods are effective, the effects on wildlife and the environment, pesticide drift damage, 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.
現在は、マツ材線虫病に対して、環境負荷が大きい防除手段が多く採られていることから、特に生物多様性保護の観点から、可能な限り農薬に依存した防除方法からの脱却が望まれている。 Currently, with respect to pine wilt disease, since it has been adopted a number environmental load is large control means, in particular break from the viewpoint of biodiversity protection, the control method depending on the pesticide as possible Nozomu It is rare.
マツノザイセンチュウまたはマツノマダラカミキリの防除方法には、網、化学物質、酵素、電流、微生物等を用いた方法があり、植物由来抽出物または成分を利用したものとしては、マツ由来の有機溶媒抽出物(特許文献1)、ヒガンバナ科球根圧搾液(特許文献2)、アフリカンマリーゴールド油、スイートバジル油およびパルマローザ油(特許文献3)などを利用する方法があるが、キク科センダングサ属植物を利用する方法は知られていない。
特許文献1〜3において利用される植物は、木材、燃料、食材、精油原料、観賞用植物、土壌改良用作物等として既に広く利用されているものであり、病害虫防除剤の原料として大量に調達すること等が容易でない場合もある。
また、これら植物を病害虫防除に用いることにより、例えば食料として有用な資源を消費してしまうことにも繋がりかねないという問題も有している。
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), an Amaryllidaceae bulb pressing solution (Patent Document 2), African Marigold oil, Sweet Basil oil, Palmarosa oil (Patent Document 3), etc. 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.
特許文献4には、キク科植物の抽出物を有効成分とするネコブセンチュウ防除剤が記載されている。また、特許文献5には、アワユキセンダングサの抽出物を有効成分とするネグサレセンチュウ又はシストセンチュウの防除剤が記載されている。 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.
しかし、特許文献4および5に記載された防除剤は土壌有害線虫を対象とするものであり、マツノザイセンチュウおよびマツノマダラカミキリについては記載が無い。特許文献4および5に記載のネコブセンチュウ類、ネグサレセンチュウ類およびシストセンチュウ類は、いずれもティレンクス目に属し(それぞれメロイドギネ科、プラティレンクス科、ヘテロデラ科)、本発明で対象となるマツノザイセンチュウ(アフェレンコイデス目アフェレンコイデス科)とは属する目が異なることから、両者は分類学上はもちろんのこと遺伝的観点からも全く別の生物である。 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, nepless nematodes, and cyst nematodes described in Patent Documents 4 and 5 all belong to the Tylenoptera ( Meloid gynecidae, Platyrenxaceae , Heterodera family , respectively ) , and the pinewood nematode targeted in the present invention (Aferenchoides eyes Aferenchoides family) because they belong to different eyes, they are completely different organisms from a genetic point of view as well as taxonomic.
また、特許文献4および5に記載された防除剤が対象とする線虫類は、土壌線虫類に含まれ、野菜類の根を犯すものである。つまり、土壌線虫類は、単独で、土壌中の野菜類の根に侵入する。
これに対し、本発明で対象となるマツノザイセンチュウは、マツノマダラカミキリという寄生宿主の存在があって初めてマツ属植物の地上部に到達し、マツ属植物の組織内に侵入して増殖することができる。
そして、この寄生宿主は、マツノマダラカミキリのみである。
このように、マツノザイセンチュウと土壌線虫類とは、生態的特徴からしても全く異なる。
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. In other words, soil nematodes enter the roots of vegetables in the soil alone.
In contrast, pine wood nematode ing and the target in the present invention, reaches the ground portion of the first Pinus plants there exists parasitic host of Monochamus alternatus, proliferate invade into the tissue of Pinus plants be able to.
And this parasitic host is only the pinewood beetle.
Thus, pine wood nematode and soil nematode are completely different from each other even in ecological characteristics .
特許文献6には、キク科センダングサ属植物の抽出物を主成分とする害虫防除剤が記載されている。
しかし、特許文献6に記載の害虫防除剤が対象とする害虫は、ヨトウムシ類(チョウ目、ヤガ科)、アブラムシ類(カメムシ目、アブラムシ科)、シロチョウ類(チョウ目、シロチョウ科)、ハダニ類(ダニ目、ハダニ科)およびコナジラミ類(カメムシ目、コナジラミ科)であり、マツノマダラカミキリ(コウチュウ目、カミキリムシ科)とは異なる分類群に属するものである。
また、これらの害虫類はいずれも、野菜類または草本植物を食害し、それ単体で病害虫として認められるのに対し、マツノマダラカミキリは、単体では単にマツ属植物の若枝と樹皮下組織を摂食するのみで病害虫として認められるほどの甚大な被害を発生させないという点で違いがある。
さらに、マツノマダラカミキリは、マツノザイセンチュウを体内に宿し、マツノザイセンチュウをマツ属植物に伝搬する役割を果たすことで、マツ材線虫病を媒介する。
つまり、マツ材線虫病の発症原因という点からみると、マツノマダラカミキリのみが、マツ材線虫病の原因であるマツノザイセンチュウを体内に宿してマツ属植物へ伝搬する役割を果たすのであり、特許文献6の防除剤が対象とする害虫類がマツ材線虫病の発症原因に関与することはなく、マツ材線虫病を媒介しないという点で、両者は全く異なる。
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 feed on vegetables or herbaceous plants and are recognized as pests by themselves, whereas the pine beetle simply feeds on the shoots and subcutaneous tissues of the pine genus plants. However, there is a difference in that it does not cause enormous damage that is recognized as a pest.
Further, the pine wood beetle mediates pine wilt disease by carrying pine wood nematode in the body and transmitting pine wood nematode to pine plants.
In other words, in terms of the cause of the development of pine wilt disease, only the pine wood nematode is responsible for transmitting the pine wood nematode, which is the cause of pine wilt disease, to the pine plant. Yes, they are completely different in that the pests targeted by the control agent of Patent Document 6 are not involved in the onset of pine wilt disease and do not mediate pine wilt disease.
本発明は、環境に対して低負荷であり、野生生物に対する安全性に優れた、マツノザイセンチュウおよびマツノマダラカミキリの防除剤及び防除方法、ならびにこれら生物によって引き起こされるマツ材線虫病の防除剤および防除方法を提供することを目的とする。 The present invention is a low load on the environment, it is excellent in safety to wildlife, control agent and a method for controlling pine wood nematode and Monochamus alternatus and controlling the induced Rumatsu wilt disease by these organisms, An object is to provide an agent and a control method.
本発明は、キク科センダングサ属植物の抽出物を有効成分として含む、マツ材線虫病の防除剤および防除方法を提供する。 The present invention provides a control agent and method for controlling pine wilt disease comprising an extract of an Asteraceae plant as an active ingredient.
また、本発明は、キク科センダングサ属植物の抽出物を有効成分として含む、マツノザイセンチュウの防除剤及び防除方法を提供する。 Moreover, this invention provides the control agent and control method of a pinewood nematode which contain the extract of the Asteraceae plant Sendangusa plant as an active ingredient .
さらに、本発明は、キク科センダングサ属植物の抽出物を有効成分として含む、マツノマダラカミキリの防除剤及び防除方法を提供する。 Furthermore, the present invention provides a control agent and control method for pinewood beetle, which contains an extract of a plant belonging to the genus Syringaaceae as an active ingredient.
本発明のマツ材線虫病防除剤は、キク科センダングサ属植物の抽出物に含まれる成分がマツノザイセンチュウ及びマツノマダラカミキリに対して不動化、忌避及び殺虫効果を発揮することにより、マツノザイセンチュウによって生じるマツ材線虫病を予防、軽減、駆除することができるものである。 Bursaphelenchus xylophilus control agent of the present invention, the components contained in extracts of Asteraceae Bidens plant immobilized against pine wood nematode and Monochamus alternatus, by exerting a repelling and insecticidal effects, Matsunozai It can prevent, reduce or eliminate pine wilt disease caused by nematodes .
本発明のマツ材線虫病防除剤、マツノザイセンチュウ防除剤及びマツノマダラカミキリ防除剤の有効成分の原料となるキク科センダングサ属植物、特にアワユキセンダングサは、日本及び熱帯・亜熱帯地域に広く分布しており、かつ、生育が旺盛で資源量が豊富であるため、入手が容易である。
したがって、本発明のマツ材線虫病防除剤、マツノザイセンチュウ防除剤及びマツノマダラカミキリ防除剤は、従来の植物由来成分等を利用した病害虫防除剤と比較して、安価に製造することが可能である。
加えて、かかる植物はこれまで雑草・害草として扱われ、ほとんど利用されていないのが現状であるため、本発明は未利用生物資源の有効活用にもつながるものと期待される。
The Asteraceae plant, which is a raw material for the active ingredients of the pine wilt disease nematode control agent, the pine wood nematode control agent, and the pine wood beetle control agent, in particular Ayuyusendangusa, 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, the pine wood nematode control agent and the 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.
また、アワユキセンダングサ等のキク科センダングサ属植物は、一部地域においては漢方茶等として利用されており、高い安全性を有している。
さらに、最近ではキク科センダングサ属植物の成分が皮膚炎や成人病に有効とする報告もあるほか、主要な農作物や土壌微生物に対しても悪影響がないことが知られている。
したがって、本発明のマツ材線虫病防除剤、マツノザイセンチュウ防除剤及びマツノマダラカミキリ防除剤は、ヒトを含む生物や環境への影響が小さく、生物多様性を損なうことがないものである。
In addition, Asteraceae plants such as Ayuyukisendansa are used as Kampo tea in some areas and have high safety.
Furthermore, recently, there have been reports that components of the Asteraceae plant species are effective against 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 effect on living organisms and the environment including humans and do not impair biodiversity.
本発明のマツ材線虫病防除剤、マツノザイセンチュウ防除剤及びマツノマダラカミキリ防除剤の有効成分は、キク科センダングサ属植物から得られる抽出物である。
キク科センダングサ属植物は、アワユキセンダングサ(B. pilosa L. var. radiata Scherff.)、タチアワユキセンダングサ(Bidens pilosa L. var. radiata Sch. Bip)、ハイアワユキセンダングサ(Bidens pilosa L. var. radiata Sch. f. decumbens Scherff.)などが挙げられる。これらの植物の中でも、アワユキセンダングサを抽出物の原料として用いることが好ましい。
The active ingredients of the pine wilt disease nematode control agent, the pinewood nematode control agent, and the pine wood beetle control agent of the present invention are extracts obtained from the plant of the family Asteraceae.
The Asteraceae plants include the genus Ayuna, B. pilosa L. var. Radiata Scherff., Bidens pilosa L. var. Radiata Sch. Bip, and 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.
キク科センダングサ属植物は、開花期のものであっても栄養生長期のものであってもよく、生の状態であっても乾燥させたものであってもよいが、乾燥させた植物体を用いることが好ましい。
乾燥の目安としては、限定されるものではないが、例えば水分含量が0%〜15%未満であることが好ましく、アワユキセンダングサであれば、通常、40〜80℃の温風下で24時間または100℃で2時間程度の条件で乾燥させれば十分である。また、抽出効率の点から、植物体を適宜細断ないし粉砕等したもの、例えば粉末状のものを抽出原料として用いることが好ましい。
植物体の部位に関しては、花、葉、茎、根などを含む全草を抽出原料として用いることが可能であるが、根を除いた地上部を用いることが好ましい。
Asteraceae plants may be in the flowering period or in the vegetative period, and may be in the raw state or dried, but the dried plant body It is preferable to use it.
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.
キク科センダングサ属植物の抽出物は、一般的な植物成分抽出方法を用いて得ることができる。かかる抽出方法としては、例えば、煮沸抽出法、浸漬抽出法、振とう抽出法、ソックスレー抽出法、水蒸気蒸留法などが挙げられ、これらの中でも煮沸抽出法が好ましい。 An extract of Asteraceae plants 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.
また、抽出に用い得る溶媒としては、水だけでなく低級アルコール類(例えばメタノール、エタノール等)、ケトン類(例えばアセトン、メチルエチルケトン等)、親水性有機溶媒(例えばアセトニトリル、酢酸エチル等)、エーテル類(例えばエチルエーテル、イソプロピルエーテル等)、芳香族炭化水素類(例えばトルエン、キシレン等)、ハロゲン化炭化水素類(例えば塩化メチレン、クロロホルム等)などの疎水性有機溶媒が挙げられ、これらの混合溶媒も利用できる。一つの好ましいキク科センダングサ属植物の抽出物は、溶媒として水を用い、煮沸抽出法によって得られる抽出物(水煮沸抽出物)である。また、水煮沸抽出以外の方法を用いる場合であっても、適宜、溶媒や抽出時間等の条件を設定してキク科センダングサ属植物の抽出物を得ることが可能である。 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. One preferable extract of the Asteraceae plant is an extract (boiled extract) obtained by boiling extraction using water as a solvent. In addition, even when a method other than water boiling extraction is used, it is possible to appropriately set conditions such as a solvent and an extraction time to obtain an extract of the Asteraceae plant.
また、得られた抽出物を、必要によりカラム分画法や溶媒分画法などにより精製することも可能である。 In addition, the obtained extract can be purified by a column fractionation method, a solvent fractionation method, or the like, if necessary.
本発明における「抽出物」とは、溶媒を用いる各種抽出方法によって得られる抽出液のほかに、抽出液を濃縮した濃縮液、抽出液を適当な溶媒で希釈した希釈液、抽出液またはその濃縮液もしくは希釈液から加熱乾燥や凍結乾燥などの種々の方法によって溶媒を除くことにより得られる固形の抽出物(粉末状や粒状のもの等)、ならびに固形化剤やゲル化剤の添加等によって得られる固形もしくは半固形の抽出物も含まれるものとする。 In the present invention, the “extract” means , in addition to the extract obtained by various extraction methods using a solvent, a concentrate obtained by concentrating the extract, a diluted solution obtained by diluting the extract with an appropriate solvent, an extract or a concentration thereof. Obtained by adding a solid extract (powdered or granular, etc.) obtained by removing the solvent from the solution or dilution by various methods such as heat drying or freeze drying, and addition of a solidifying agent or gelling agent Also included are solid or semi-solid extracts.
本発明の防除剤におけるキク科センダングサ属植物の抽出物は、例えば、キク科センダングサ属植物の乾燥植物体1重量部に対して5〜1000重量部、好ましくは5〜100重量部の水を加えて加熱し、30〜60分間、好ましくは30分間沸騰させることによって得ることができる。かかる方法によって得られる抽出物(抽出液)は、上述の通り、必要に応じて希釈もしくは濃縮することができ、溶媒を除去して固形の抽出物とすることもできる。 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.
本発明の防除剤におけるキク科センダングサ属植物の抽出物は、好ましくは、アビエチン酸エチル、イソバニリン、α-カリオフィレン、β-カリオフィレン、クマラン、桂皮酸、デヒドロアビエチン酸メチルエステル、シリンガアルデヒド、ステアリン酸、バニリン酸、パルミチン酸、4-ヒドロキシ安息香酸、4H-ピラン-4-オン,2,3-ジヒドロ-3,5-ジヒドロキシ-6-メチル、ブチルヒドロキシトルエン、2(4H)-ベンゾフラノン,5,6,7,7a-テトラヒドロ-4,4,7a-トリメチル及びミリスチン酸からなる群より選択される1種以上の化合物を含むものである。 The extract of the Asteraceae plant in the control agent of the present invention is preferably 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, It contains one or more compounds selected from the group consisting of 6,7,7a-tetrahydro-4,4,7a-trimethyl and myristic acid.
また、キク科センダングサ属植物の抽出物は、上記の化合物以外にも、アワユキセンダングサは、アルカロイド、タンニン、サポニン、フラボノイド、カルコン系物質、カフェオイル誘導体、リノール系化合物、ジテルペン、精油、フェニルプロパノイド配糖体、フェノール類、フラボノイド類、フラボン配糖体及びポリアセチレン類、フェルラ酸、ケルセチン、カフェ酸、ピロカテチン、サリチル酸、p-ビニルグワヤコール、ジメソキシフェノール、オイゲノール、4-エチル-1,2-ベンゼネジオール、イソバニリン、2-ハイドロ-6-メチルベンザルデハイド、バニリン、バニリン酸、p-ハイドロキシベンゼン酸、プロトカテチュイック酸及びクマリン(クマリック)酸を含有することが報告されている(非特許文献1〜3)。したがって、本発明の防除剤におけるキク科センダングサ属植物の抽出物は、これらの物質のうち1種以上を含むものであってもよい。また、本発明の防除剤は、これらの物質のうち1種以上とキク科センダングサ属植物の抽出物とを組み合わせて含むものであってもよく、さらに各種展着剤及び浸透移行剤等と組み合わせることもできる。 In addition to the above-mentioned compounds , the extract of the plant belonging to the genus Asteraceae is Acacia genus, which is an alkaloid, tannin, saponin, flavonoid, chalcone substance, caffeoyl derivative, linol compound, diterpene, essential oil, phenylpropaline. Noid glycosides, phenols, flavonoids, flavone glycosides and polyacetylenes, ferulic acid, quercetin, caffeic acid, pyrocatechin, salicylic acid, p-vinyl guaiacol, dimesoxyphenol, eugenol, 4-ethyl-1, Contains 2-benzenediol, isovanillin, 2-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 in 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 is further combined with various spreading agents, osmotic transfer agents and the like. You can also
本発明の防除剤が有効な植物はマツ属(Pinus)植物であり、より具体的には、マツ材線虫病に対して感受性が高い植物、例えば、アカマツ(Pinus densiflora Sieb. & Zucc.)、クロマツ(Pinus thunbergii Parl.)、ヤクタネゴヨウ(Pinus amamiana Koidz.)、リュウキュウマツ(Pinus luchuensis Mayr)、タイワンアカマツ(Pinus massoniana Lambert, 1803)、チョウセンゴヨウ(Pinus koraiensis Siebold et Zucc.)、ヨーロッパアカマツ(Pinus sylvestris L.)、フランスカイガンショウ(Pinus pinaster Aiton)等が挙げられる。 Plants in which the control agent of the present invention is effective are Pinus plants, and more specifically plants that are highly susceptible to pine wilt disease, such as Pinus densiflora Sieb. & Zucc. , Pinus thunbergii Parl., Pinus amamiana Koidz., Pinus luchuensis Mayr, Pinus massoniana Lambert, 1803, Pinus koraiensis Siebold et Zucc. sylvestris L.), French pinaster Aiton, and the like.
以下、アワユキセンダングサの乾燥植物体1重量部に対して5重量部の水を加えて加熱し、30分間沸騰させることによって得られる抽出液(以下、「抽出原液」と称する)を有効成分として含む本発明の防除剤における抽出原液の好適濃度について記載する。 Hereinafter, an extract obtained by adding 5 parts by weight of water to 1 part by weight of dried plant of Ayuyukisendansa, heating and boiling for 30 minutes (hereinafter referred to as “extraction stock solution”) as an active ingredient It describes preferred concentration of definitive to control agent extracted stock solution of the present invention including.
マツノザイセンチュウに対して、100%不動化させる効果を得るためには、抽出原液もしくはその10倍程度までの希釈液を用いることが好ましく、100%ではないが高い確率で不動化させる効果を得るためには、抽出原液の20〜100倍程度までの希釈液を用いることが好ましい。 Against pine wood nematodes, in order to obtain the effect of 100% immobilisation, extracted undiluted or rather preferable be used dilutions of up to ten times, but not 100% is immobilized with high probability effect In order to obtain the above, it is preferable to use a diluted solution up to about 20 to 100 times the extraction stock solution.
つまり、マツノザイセンチュウに対する不動化、忌避、殺虫効果を目的とする場合は、抽出原液もしくはその100倍程度までの希釈液を用いることが好ましい。
また、マツ属植物の表皮にできたマツノマダラカミキリによる摂食痕から、マツノザイセンチュウがマツ属植物の皮下組織内へ侵入することを阻止するには、抽出原液またはその200倍程度までの希釈液を用いることが好ましい。
That is, immobilization against pine wood nematode, repellent, for the purpose of insecticidal effect, extracted stock solution or it is not preferable to use a dilution of up to that 100 times.
In addition, in order to prevent pine wood nematode from entering into the subcutaneous tissue of the pine genus plant from the eating traces by the pine pine beetle on the epidermis of the pine genus plant, it is diluted to about 200 times the extraction stock solution. it preferably is used liquid.
一方、マツノマダラカミキリの成虫に対して、忌避、不動化、殺虫効果を発揮するためには、抽出原液を用いることが好ましい。 On the other hand , in order to exert repellent, immobilization, and insecticidal effects against adult pine beetle, it is preferable to use an extraction stock solution.
また、マツノマダラカミキリの幼虫に対して、不動化、殺虫効果を発揮するためには、抽出原液もしくはその20倍程度までの希釈液を用いることが好ましい。 Further, with respect to larvae of Monochamus alternatus, immobilization, in order to exhibit the insecticidal effect, extracted stock solution also properly it is preferable to use a dilute solution up to the 20 times.
本発明の防除剤の有効成分であるキク科センダングサ属植物の抽出物は、マツノザイセンチュウならびにマツノマダラカミキリ(幼虫及び成虫)に対して、忌避、不動化または殺虫効果を有する。したがって、本発明の防除剤を、マツ属植物、マツノザイセンチュウまたはマツノマダラカミキリのいずれか1種以上に施用することにより、マツ材線虫病を防除することが可能である。 Extract of Asteraceae Bidens plant as an active ingredient of the control agent of the present invention, the chromatic against pine wood nematode and pine sawyer (larvae and adults), repellent, the immobilized or insecticidal effect. Therefore, the control agent of the present invention, Pinus plants, by for facilities to any one or more nematode or Monochamus alternatus, it is possible to control the pine wilt disease.
例えば、マツ材線虫病に罹病したマツ属植物に本発明の防除剤を適用すれば、(1)マツ属植物の組織内に存在するマツノザイセンチュウおよびマツノマダラカミキリ幼虫を殺虫すること、(2)マツノザイセンチュウの媒介虫であるマツノマダラカミキリ体内への移行を阻止すること、および(3)該媒介虫の羽化を阻止することが可能であり、その結果、マツ材線虫病の予防および防除が可能となる。
また、キク科センダングサ属植物の抽出物はマツノザイセンチュウおよびマツノマダラカミキリに対して忌避性を示すため、健全なマツ属植物に本発明の防除剤を適用することにより、マツノマダラカミキリの後食(成熟のための行動:若枝の樹皮を摂食する)およびマツノザイセンチュウのマツ属植物組織内への侵入を阻止または軽減することができる。
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.
本発明における防除剤の「施用」とは、防除剤を施用対象物(例えば、マツ属植物、マツノザイセンチュウまたはマツノマダラカミキリ)に接触させることを意味する。摂食させる手段は、噴霧、塗布、浸漬、樹幹内注入、土壌灌注(根からの吸収)等が挙げられるが、いかなる手段を用いることもできる。 The "for facilities" of control agent in the present invention, facilities for object a control agent (e.g., Pinus plants, pine wood nematode or Monochamus alternatus) means contacting the. Examples of the means for feeding include spraying, application, dipping, injection into the trunk, soil irrigation (absorption from the roots) , and any means can be used.
本発明の防除剤は、所望の施用方法に応じ、農薬等において採用され得る各種の剤形に製剤化することが可能である。剤形の例としては、これらに限定されないが、粉剤(一般粉剤、DL粉剤、フローダスト(FD)剤を含む)、粒剤、ペースト剤、水和剤、水溶剤、液剤、乳剤、フロアブル剤、マイクロエマルション剤、マイクロカプセル剤等が挙げられる。また、防除剤の製剤化のために、当該技術分野において通常用いられる賦形剤、媒体、担体、添加剤等を適宜選択して用いることが可能である。例えば、増量剤、有効成分を溶解または分散させるための媒体(溶媒や分散媒等)、分散剤、乳化剤、固形化もしくは半固形化のための固形化剤やゲル化剤、徐放性付与のためのマトリックス等を用いることができる。 Control agent of the present invention can be formulated into desired depending on the method for facilities, various dosage forms may be employed in agricultural chemicals. 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.
マツ属植物への施用に関しては、マツ属植物の生木または枯死木内に液状の防除剤を注入することができ、また、マツ属植物の生木または枯死木の表面に液状または固形(例えば粉状)の防除剤を施用することもできる。この場合の施用は、キク科センダングサ属植物の抽出物をマツ属植物の表面に定着させるかあるいはマツ属植物の組織内に浸透移行させることを目的としている。したがって、キク科センダングサ属植物の抽出物を単独で施用することもできるが、マツ属植物の表面への定着性やマツ属植物の組織内への浸透性を良くするために、各種の展着剤や浸透移行剤を併用することもできる。展着剤や浸透移行剤とは、一般に界面活性剤を主成分とする液体であり、例えば一般展着剤(スプレッダー)、機能性展着剤(アジュバント)、固着性展着剤(スチッカー)などを併用することができる。 Regarding for facilities to Pinus plants, can be injected control agent liquid in rough wood or dead Kiuchi Pinus plants, also liquid or solid on the surface of the rough wood or dead wood of pine plants (e.g. a control agent of powder) can also be for the facilities. For facilities in this case it is intended to be infiltrated migrated into the tissue of the Asteraceae Bidens or Pinus plants a plant extract to be fixed on the surface of the Pinus plants. Therefore, although it is also possible to use facilities an extract of Asteraceae Bidens plant alone, in order to improve the penetration into the tissue of the fixing property and Pinus plants to the surface of the pine plants, various exhibitions Adhesives and permeation transfer agents can also be used in combination. 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.
例えば、非イオン性界面活性剤としては、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンアリキルフェニルエーテル、ポリオキシエチレン脂肪酸エステル、ポリオキシエチレンヘキシタン脂肪酸エステル、ポリオキシアルキレンアルキルエーテル、ポリオキシアルキレン脂肪酸エステル、N―オクチルピロリドン、アセチレン系界面活性剤などがある。アニオン系界面活性剤としては、アルキルベンゼンスルホネート、ジアルキルスルホサクシネート、リグニンスルホネート、ナフタレンスルホネートのホルマリン縮合物などがある。固着性を良くするものには、アニオン性界面活性剤として、リグニンスルホンジェート、ナフタレンスルホネートの縮合物、パラフィン、ワックス、モタンワックス、ポリオキシエチレン樹脂酸エステル、ポリビニルアルコール、メチルビニルエーテル/無水マレイン酸共重合体などがある。浸透性を付与するものにはポリオキシエチレンヘキシタン脂肪酸エステルやポリオキシエチレンドデシルエーテルなどがある。 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.
本発明における「防除」とは、本剤を施用することでマツノザイセンチュウ、マツノマダラカミキリを忌避、不動化、殺虫すること、または、これによってマツ材線虫病を予防、症状の軽減、駆除することを意味し、「防除剤」とは、本剤を施用することでマツノザイセンチュウ、マツノマダラカミキリを忌避、不動化、殺虫する効果を有する薬剤、または、これによってマツ材線虫病を予防、症状の軽減、駆除する効果を有する薬剤を意味する。In the present invention, “control” refers to the use of this agent to repel, immobilize, and kill pinewood nematodes and pinewood beetles, or to prevent, reduce, and eliminate pine wilt disease. `` Control agent '' means a drug that has the effect of repelling, immobilizing, and killing pinewood nematode and pinewood beetle by applying this agent, or pine wilt disease by this It means drugs that have preventive, symptomatic, and extermination effects.
以下、実施例によって本発明をより詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to examples.
1.供試線虫
本明細書に記載される実験に供試したマツノザイセンチュウは、以下の手順に従って得られたものである。
(1)手動の直径15mm木工用ドリルを用いて、琉球大学校構内のマツ材線虫病に罹病したリュウキュウマツから木くずを採取した。
(2)得られた木くずの組織内から、ベルマン法(線虫学実験法、2004、p87〜88、編集者:線虫学実験法編集委員会、発行者:真宮靖治、発行所:日本線虫学会)によりマツノザイセンチュウを滅菌済み遠沈管に10ml分離し、遠心機で3,000rpm(1分間)遠心した。
(3)遠心後の上澄みの9mlをパスツールピペットで取り除いて、沈殿したマツノザイセンチュウに新たに滅菌水を加えて再度遠心分離を行う工程を計5回繰り返した後、上澄みの9mlを取り除いて残った沈殿したマツノザイセンチュウに、10%に希釈した乳酸をパスツールピペットで9ml加えて遠心分離を行い、さらに上澄みの9mlを取り除いた後に、再度滅菌水を9ml加えて遠心し、上澄みの9mlを取り除くことで、マツノザイセンチュウの洗浄を行った。
(4)得られた表面殺菌済みのマツノザイセンチュウを、あらかじめPDA培地(ジャガイモ:200〜300g、ブドウ糖:10〜20g、寒天:10〜20g、水:1,000ml)上で学内保存菌Botrytis cinerea Persoonを前培養した培地上で7日間増殖させた後、パスツールピペットを用いて滅菌水を約10ml滴下し、良く懸濁して回収したものを以下の実験に供試した。
1. Test Nematodes The pinewood nematodes used in the experiments described herein were obtained according to the following procedure.
(1) Using a manual 15 mm diameter woodworking drill, wood chips were collected from Ryukyu pine that had pine wilt disease in the Ryukyu University campus.
(2) From the obtained wood waste organization, the Bellman method (Nematologic Experimental Method, 2004, p87-88, Editor: Nematocidal Experimental Method Editorial Committee, Publisher: Shinji Mamiya, Publisher: Japan Line) 10 ml of pine wood nematode was separated into a sterilized centrifuge tube by the Entomological Society, and centrifuged at 3,000 rpm (1 minute) with a centrifuge.
(3) the 9ml supernatant after centrifugation with the exception taken with a Pasteur pipette, after the step of performing centrifugal separation again by adding new sterile water was repeated 5 times in total precipitated pine wood nematode, removed 9ml supernatant Add 9 ml of lactic acid diluted to 10% to the remaining pinewood nematode remaining by centrifugation using a Pasteur pipette, centrifuge, remove 9 ml of the supernatant, and then add 9 ml of sterile water again and centrifuge. The pine wood nematode was washed by removing 9 ml.
(4) The surface-sterilized pine wood nematode obtained 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). after growth for 7 days on a pre-cultured medium of Persoon, about 10ml dropwise sterile water using a pasteur pipette, it was tested with those recovered by well suspended in the following experiments.
2.供試昆虫
(1)マツノマダラカミキリ成虫
マツ材線虫病に罹病したリュウキュウマツから羽化、脱出し、1〜2週間経過したマツノマダラカミキリ成虫を、沖縄県森林資源研究センターから譲り受け、以下の実験に供試した。
2. Test insects (1) Japanese pine beetle adult
Emergence from Ryukyu pine that was diseased in pine wilt disease, to escape, 1 to 2 weeks elapsed pine sawyer adult, took over from Okinawa Prefecture forest resources Research Center, tried subjected to the following experiment.
(2)マツノマダラカミキリ幼虫
沖縄県森林資源研究センターから譲り受けたマツ材線虫病に罹病したリュウキュウマツの丸太内の蛹室から、薪割り機及びピンセットを用いてマツノマダラカミキリの幼虫を採取した。その後、25℃暗黒条件下に1日静置し状態の良いものを選抜して以下の実験に供試した。
(2) from the pupal chamber of the Japanese pine sawyer larvae in the log of the Ryukyu pine that was diseased from Okinawa Prefecture forest resources Research Center in Yuzuru Ri received pine wilt disease, the larvae of the pine sawyer using a wood-splitting machine and tweezers Collected. 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.マツノザイセンチュウの同定
マツノザイセンチュウの同定は、ベルマン法により分離された線虫を実体顕微鏡で口腔、食道及び陰門部などの形態的特徴を観察し同定を行った。属の同定は、検索表(Williamら、1996)を用いて行った。次に、形態的にBursaphelenchus属と同定した個体1頭を用いてPCR−RFLP法(Iwahoriら、1998)により種の同定を行い、さらに線虫の18S及び26SrRNA遺伝子間スペーサー領域の配列を解読し(PCR増幅及び配列決定のために使用したプライマーは、18Sプライマー(配列番号1:5'-TTGATTACGTCCCTGCCCTTT-3')及び26Sプライマー(配列番号2:5'-GGAATCATTGCCGCTCACTTT-3'))、得られた配列についてDDBJ(DNA Data Bank of Japan)のWebサイトでBLAST検索を行なって種を特定した。その結果、上記いずれの手法においても、以下の実験に供試したセンチュウは、マツノザイセンチュウ(Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle)であると同定された。
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, species were identified by PCR-RFLP method (Iwahori et al., 1998) using one individual morphologically identified as the genus Bursaphelenchus, and further, the sequence of the 18S and 26S rRNA intergenic spacer regions of the nematode was decoded. (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')). It was to identify the species by performing BLAST search on the Web site of the DDBJ (DNA Data Bank of Japan) for the sequence. 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).
[試験例1]
アワユキセンダングサ抽出液の作製
琉球大学農学部フィールドセンター内に自生する開花期のアワユキセンダングサの地上部を刈り取り、乾熱滅菌機で、100℃、2時間乾燥させたアワユキセンダングサを切断し、ミキサーでさらに細かく粉砕したものをビーカーに10g入れ、50mlの水を加えた。これにアルミ箔で蓋をして、30分間煮沸し、濾紙で濾過することにより抽出液を得た(以下、「試験例1の抽出液」とも称する)。
[Test Example 1]
Manufacture of Ayuyukisendangusa extract The upper part of the flowering Ayuyusendangusa that grows naturally in the field center of the University of the Ryukyus is cut off and cut with a dry heat sterilizer at 100 ° C for 2 hours. 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”).
[試験例2]
アワユキセンダングサ含有成分の分析
(1)試験例1の抽出液25mlを、回転石を入れた100mlビーカーに移し、水酸化ナトリウム(4N)を1:1の割合で加え、ウォーターバスを用いて50℃、4時間撹拌した。撹拌後、塩酸(12N)を加え、溶液をpH1に調整した。濃度の測定はリトマス紙を用いた。
(2)pHの調整後、得られた溶液の半分量(25ml)と酢酸エチル100mlを分液漏斗に入れて撹拌し、しばらく静置したのち、酢酸エチル層と水層とが分離したら、水層を50mlの容量ビーカーに回収し、残った酢酸エチル分画を1,000ml容量の三角フラスコに回収した。
(3)回収した(2)の水層に対しても、さらに(2)と同じ工程で酢酸エチルと抽出液の二層分配を計2回行い、酢酸エチル分画を回収した。
(4)pHの調整後の、(2)の残りの溶液(25ml)についても、(2)と同じ工程で酢酸エチルと抽出液の二層分配を複数回行い、計600mlの酢酸エチル分画を得た。また、回収した酢酸エチル分画中に微量に混入している水を除去するため、硫酸ナトリウムを0.5g程度加え、十分に撹拌し、冷室(2℃)に保存した。
(5)(4)の酢酸エチル分画を濾過して得られたうちの100mlをフラスコに入れて、ロータリーエバポレーターを用いて酢酸エチルを揮発させ、固形の物質を得た。得られた物質をアセトンで溶解して10,000ppmに調製し、濾過した後に2ml容量のバイアルに約1.5ml入れたものをサンプルとした。
(6)サンプルについて、GC-MS(GCMS-QP2010、カラム:DB-SMS、カラム長:30m、内径φ0.25mm)を用いて成分分析を行った。
[Test Example 2]
Analysis of components containing Ayuyukisendansa (1) Transfer 25 ml of the extract of Test Example 1 to a 100 ml beaker containing a rotating stone, add sodium hydroxide (4N) at a ratio of 1: 1, and use a water bath to add 50 ml. The mixture was stirred at 4 ° C. for 4 hours. After stirring, addition of hydrochloric acid (12N), the solution was pH1 two adjust the. The density was measured using litmus paper.
(2) After adjusting the pH, half the amount of the resulting solution (25 ml) and 100 ml of ethyl acetate are placed in a separatory funnel and stirred, and after standing for a while, when the ethyl acetate layer and the aqueous layer are separated , the layers were collected to a volume beaker 50 ml, was collected and the remaining ethyl acetate fraction Erlenmeyer flask 1,000ml capacity.
(3) For the recovered aqueous layer of (2), two-layer distribution of ethyl acetate and the extract was further performed twice in the same step as in (2) to recover the ethyl acetate fraction.
(4) After the pH adjustment, the remaining solution (25 ml) of (2) was also subjected to two-layer partitioning of ethyl acetate and the extract several times in the same process as (2) , for a total of 600 ml of ethyl acetate fractions. Got. Moreover, in order to remove the trace amount of water contained in the collected ethyl acetate fraction, about 0.5 g of sodium sulfate was added, stirred sufficiently, and stored in a cold room (2 ° C.).
(5) 100 ml of the ethyl acetate fraction obtained by filtering the ethyl acetate fraction of (4) was put into a flask, and the 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.
(6) Component analysis was performed on the sample using GC-MS (GCMS-QP2010, column: DB-SMS, column length: 30 m, inner diameter φ0.25 mm).
その結果、サンプルからは、アビエチン酸エチル、イソバニリン、α-カリオフィレン、β-カリオフィレン、クマラン、桂皮酸、デヒドロアビエチン酸メチルエステル、シリンガアルデヒド、ステアリン酸、バニリン酸、パルミチン酸、4-ヒドロキシ安息香酸、4H-ピラン-4-オン,2,3-ジヒドロ-3,5-ジヒドロキシ-6-メチル、ブチルヒドロキシトルエン、2(4H)-ベンゾフラノン,5,6,7,7a-テトラヒドロ-4,4,7a-トリメチル及びミリスチン酸が検出された。 As a result , from the sample , 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 were detected.
[実施例1]
マツノザイセンチュウに対する殺虫及び不動化実験
(1)試験例1の抽出液の、原液、10、20、100倍希釈液を、それぞれ滅菌済み小型試験管にメスピペット(1ml)を用いて1ml加えた後、マイクロピペット(20〜200μl)を用いて100頭/0.1mlに調整したマツノザイセンチュウの線虫懸濁液を添加し、パラフィルムで蓋をしてインキュベーター(25℃条件下)に静置し、1日後、4日後、7日後(計3回)に不動化したマツノザイセンチュウを、8日後に致死したマツノザイセンチュウを、それぞれ光学顕微鏡下で計数した。対照として滅菌水区を設け、実験は7反復行った。
(2)7日後の動いているマツノザイセンチュウと不動化しているマツノザイセンチュウをそれぞれ計数し、不動化率を求めた。
(3)8日後の生存しているマツノザイセンチュウと致死しているマツノザイセンチュウをそれぞれ計数し、致死率を求めた。攪拌機で十分な刺激を与えても運動性がみられないものは、致死と判断した。
[Example 1]
Insecticide and immobilization experiment against pinewood nematode (1) Add 1 ml of the stock solution of the extract of Test Example 1 to each sterilized small test tube using a measuring pipette (1 ml). After that, a nematode suspension of pine wood nematode adjusted to 100 / 0.1 ml using a micropipette (20-200 μl) was added, and the lid was covered with parafilm and placed in an incubator (under 25 ° C.). The pine wood nematodes that were allowed to stand and immobilized after 1 day, 4 days, and 7 days (total 3 times) and the pine wood nematodes that died after 8 days were counted under an optical microscope. Sterile water-ku, provided as a control, the experiment was Tsu 7 repetitive line.
(2) a moving pine wood nematode and immobilized to have pine wood nematode was counting each points after 7 days was determined immobility rate.
(3) Eight days later, the surviving pine wood nematode and the dead pine wood nematode were counted to determine the mortality rate. Those that showed no motility even when given sufficient stimulation with a stirrer were judged to be lethal.
実験の結果、全ての実験区において、86.5%以上の高い不動化率を示し、原液〜20倍希釈液の実験区において、76.4%以上の致死率を示したことからも明らかなとおり(表1)、アワユキセンダングサ抽出液のマツノザイセンチュウに対する高い殺虫効果及び不動化作用が認められた(表1及び図1)。 As a result of the experiment , all the experimental plots showed a high immobilization rate of 86.5% or higher , and the experimental plot of the stock solution to 20-fold diluted solution showed a fatality of 76.4% or higher. As shown (Table 1), the high insecticidal effect and the immobilization action of the Ayuyukisendanusa extract on pinewood nematode were observed (Table 1 and FIG. 1).
[実施例2]
マツノザイセンチュウに対する忌避実験
(1)乾熱滅菌したシャーレ底面の中央部にあらかじめ黒マジックで直径2cmの円を描いた。
(2)そのシャーレに素寒天培地(寒天:10g、蒸留水:1,000ml)を10ml分注し、凝固させた後、試験例1の抽出液の、原液、10、20、100、200倍希釈液を、それぞれマイクロピペット(20〜200μl)を用いて円内に3.5μl滴下し、クリーンベンチ内で10分間風乾させた。
(3)次に各実験区の円内に、マイクロピペット(20〜200μl)を用いて100頭/3.5μlに調整したマツノザイセンチュウの線虫懸濁液を添加し、25℃条件下に静置した。その後、30分から24時間後まで、円内に残存したマツノザイセンチュウの頭数を光学顕微鏡下で計数した。対照として滅菌水区を設け、実験は7反復行った。
[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) 10 ml of an agar medium (agar: 10 g, distilled water: 1,000 ml) was dispensed into the petri dish and allowed to solidify, and then the stock solution of the extract of Test Example 1, 10, 20, 100, 200 times 3.5 μl of the diluted solution was dropped into a circle using a micropipette (20 to 200 μl) , respectively , and air-dried 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 into the circle of each experimental group, and the condition was kept at 25 ° C. Left to stand. Thereafter, from 30 minutes to 24 hours, the number of pine wood nematodes remaining in the circle was counted under an optical microscope . Sterile water was provided as a control, and the experiment was repeated 7 times.
アワユキセンダングサ抽出液を滴下後、24時間経過した時点で、円内に残存したマツノザイセンチュウは、対照区では39.3頭であったのに対し、原液、10、20倍希釈液の各実験区では、それぞれ10.1頭、7.1頭、18.7頭と減少していることからも明らかなとおり(図2)、時間の経過と共に円内に残存するマツノザイセンチュウの頭数は減少傾向にあり、アワユキセンダングサ抽出液のマツノザイセンチュウに対する忌避効果が認められた。At the time when 24 hours had passed after the Ayuyukisendansa extract was added dropwise, the number of pinewood nematodes remaining in the circle was 39.3 in the control group, whereas the stock solution, 10-fold and 20-fold diluted solutions were used. In the experimental plot, the number of pine wood nematodes remaining in the circle over time is clear, as is evident from the decrease of 10.1, 7.1, and 18.7, respectively (Fig. 2). There was a tendency to decrease, and the repellent effect of Ayuyukisendanusa extract on pinewood nematode was recognized.
[実施例3]
マツ材線虫病に罹病したマツ組織内のマツノザイセンチュウに対する殺虫及び不動化実験
(1)3mmメッシュの篩を乗せた漏斗に約10cmのゴム管を付け、そこから2〜3cm下をピンチクランプで止めた。
(2)次に、琉球大学校内のマツ材線虫病に罹病したリュウキュウマツから手動の直径15mm木工用ドリルで木くず20gを採取し、これを2枚重ねにしたキムワイプで包み、篩の上に置いて、試験例1の抽出液の、原液、10、20、50、100、200倍希釈液をそれぞれ注ぎ静置した。
(3)1日後、3日後、5日後のマツノザイセンチュウを、次の手順で計数した。
ピンチクランプをゆっくり開けてマツノザイセンチュウを含む10mlの液体を滅菌済み小型試験管に取り、遠心機で3,000rpm(1分間)遠心した後、上澄みの9mlをパスツールピペットで取り除き、残り1ml中のマツノザイセンチュウの頭数を計数した。
なお、対照として、滅菌水区を設けた。また、実験は7反復行った。
[Example 3]
With the insecticidal and immobilization experiments (1) rubber tubes of approximately 10cm funnel topped with 3mm mesh sieve for pine wood nematode in pine tissue diseased pine wilt disease, its From there pinch under 2~3cm Stopped with a clamp.
(2) Next, 20g of wood waste was collected from a Ryukyu pine afflicted with pine wilt disease at the University of the Ryukyus with a manual 15mm diameter woodworking drill, wrapped in Kimwipe, which was stacked in two pieces, and placed on the sieve. Then , a stock solution, a 10, 20, 50, 100, and 200-fold diluted solution of the extract of Test Example 1 were poured and allowed to stand.
(3) After 1 day, 3 days, and 5 days later , pine wood nematode was counted according to the following procedure.
Slowly open the pinch clamp and take 10 ml of liquid containing pine wood nematode in a sterilized small test tube, centrifuge at 3,000 rpm (1 minute), remove 9 ml of the supernatant with a Pasteur pipette, and in the remaining 1 ml The number of pine wood nematodes was counted.
As a control, a sterilized water section was provided . The experiment was repeated 7 times.
実験の結果、対照区では、平均458.8頭のマツノザイセンチュウを確認できたのに対し、原液、10、20、50、100及び200倍希釈液の各実験区では、それぞれ平均、8頭、32.8頭、103.4頭、213.6頭、214.4頭、285.8頭のマツノザイセンチュウしか確認できなかった。また、実験区間では、アワユキセンダングサ抽出液の濃度が高くなるにつれて、分離されたマツノザイセンチュウの頭数が減少する傾向が認められた(図3)。
これは、不動化、致死したマツノザイセンチュウは運動性が失われていることから、キムワイプの隙間を通り抜けることができないため分離できなかったことによるものであるが、アワユキセンダングサ抽出液がマツノザイセンチュウに対して殺虫及び不動化作用を有することを示すものである。
なお、図3のグラフの値は、平均±標準偏差によるものであり、図3中のアルファベットは有意差を示す(Tukeyの多重比較検定、p<0.05)。
The results of the experiment, the control group, whereas it was confirmed pine wood nematode average 458.8 head, stock, in each experimental zone of 10, 20, 50, 100, and 200-fold dilution, the average respectively, 8 animals , 32.8 cows, 103.4 head, 213.6 head, 214.4 head, was only able to confirm the pine wood nematode of dogs 285.8. Further, in the experiment section, the hand As the concentration of bubbles snow Bidens extract increases, tend number of horses is reduced in isolated nematode was observed (Fig. 3).
This is because immobilized and deadly pinewood nematodes have lost their motility and cannot be separated because they cannot pass through the gaps in the Kimwipes. It shows an insecticidal and immobilizing action against nematodes.
In addition, the value of the graph of FIG. 3 is based on an average +/- standard deviation, and the alphabet in FIG. 3 shows a significant difference (Tukey's multiple comparison test, p <0.05).
[実施例4]
(1)マツノマダラカミキリ成虫に対する殺虫及び不動化実験
マツノマダラカミキリ成虫1頭に対し、試験例1の抽出液の原液2mlを噴霧器を用いて直接噴霧した。その成虫を、琉球大学校内から採取した、アワユキセンダングサ抽出液を施用していないリュウキュウマツの若枝(長さ5cm、幅2cm)と共に、円柱型プラスチック容器(直径6cm、高9cm)に入れ、25℃条件下に静置した。その後、1日後、2日後、3日後に、プラスチック容器内の不動化及び致死した成虫をそれぞれ計数した。対照として滅菌水区を設け、実験は6反復行った。
[Example 4]
(1) Insecticidal and immobilization experiments for adult pine sawworms 2 ml of the stock solution of the extract of Test Example 1 was directly sprayed on a single adult pine spoter using a sprayer. The adult is collected in a cylindrical plastic container (6cm in diameter and 9cm in height ) together with the young branch of Ryukyu pine (5cm in length and 2cm in width) that has not been applied to the Ayuyukisendansa extract extracted from Ryukyu University. It left still on 25 degreeC conditions. Thereafter , after 1 day, 2 days, and 3 days, immobile and dead adults in the plastic container were counted. As a control, sterile water was provided and the experiment was repeated 6 times.
実験の結果、全ての実験区(原液)で、1日後には不動化あるいは致死、3日後には致死の状態に至っており(表2及び図4)、アワユキセンダングサ抽出液がマツノマダラカミキリ成虫に対して殺虫及び不動化作用を有することが認められた。 As a result of the experiment , all the experimental plots (stock solution) were immobilized or lethal after 1 day, and reached lethal after 3 days (Table 2 and Fig. 4). Has been found to have an insecticidal and immobilizing action.
(2)マツノマダラカミキリ成虫の忌避性に及ぼす影響
試験例1の抽出液の原液2mlを噴霧器を用いて直接噴霧したリュウキュウマツの若枝(長さ5cm、幅1.5〜2cm)3本を、アワユキセンダングサ抽出液を施用していないマツノマダラカミキリ成虫1頭(体長約2〜3cm)と共にプラスチック容器に入れ、25℃条件下に静置した。その後、1日後、2日後、3日後に、それぞれマツの若枝に残ったマツノマダラカミキリの摂食痕の大きさを測定した。
判定基準は、処理したマツの若枝に摂食痕が全くみられないものを「強い忌避性有り(++)」、1本あたりの摂食痕の総面積が1cm2以下のものを「忌避性有り(+)」、それ以上のものを「忌避性無し(−)」とした。対照として滅菌水区を設け、実験は6反復行った。
(2) Effects on the repellent properties of adult pine beetles
Three pine branches (5 cm in length and 1.5-2 cm in width ) of Ryukyu pine that were directly sprayed with 2 ml of the stock solution of the extract of Test Example 1 using a nebulizer , were not applied with Ayuyukisendansa extract. It was placed in a plastic container together with one adult beetle (length: about 2 to 3 cm) and allowed to stand at 25 ° C. Thereafter , after 1 day, 2 days, and 3 days, the size of the eating traces of the pinewood beetle remaining on the pine shoots was measured.
Judgment criteria are "strong repellent (++)" if the treated pine shoots show no eating marks at all, and if the total area of eating marks per 1 cm 2 or less is "repellent" Existence (+) ", more than that was" no repellent (-) ". As a control, sterile water was provided and the experiment was repeated 6 times.
実験の結果、全ての実験区において、1日後に、アワユキセンダングサ抽出液のマツノマダラカミキリに対する強い忌避性が認められた(表3及び図5)。 As a result of the experiment, a strong repellency against the pinewood beetle was observed in the all experimental sections after 1 day (Table 3 and FIG. 5).
(3)マツノマダラカミキリ幼虫に対する殺虫効果実験
滅菌済みシャーレに、マツノマダラカミキリ幼虫各1頭を入れたうえで、試験例1の抽出液の、原液、10、20、100倍希釈液をそれぞれメスピペット(2ml)を用いて2ml滴下した後、25℃暗黒条件下に静置した。その後、1日後、3日後、7日後にそれぞれ致死幼虫を計数した。対照として滅菌水区及び無処理区を設け、実験は5反復行った。
(3) Insecticidal effect on pine moth beetle larvae After placing one each of pine moth beetle larvae in a sterilized petri dish , the stock solution of the extract of Test Example 1, 10, 20, and 100-fold diluted solution, respectively After dropping 2 ml with a pipette (2 ml), the mixture was allowed to stand at 25 ° C. under dark conditions. Thereafter, lethal larvae were counted after 1 day, 3 days, and 7 days, respectively . As a control, a sterilized water section and an untreated section were provided, and the experiment was repeated five times.
実験の結果、対照区(無処理、滅菌水)は致死した幼虫が0頭であったのに対し、実験区は全ての濃度で致死した幼虫が見られ、原液では100%、10倍希釈液では80%、20倍希釈液では60%、100倍希釈液では20%の割合で致死した(表4及び図6)。 As a result of the experiment , the control group (untreated, sterilized water) had 0 dead larvae, whereas the experimental group showed dead larvae at all concentrations . The stock solution was 100%, 10-fold diluted solution. The fatalities were 80%, 60% in the 20-fold diluted solution , and 20% in the 100-fold diluted solution (Table 4 and FIG. 6).
以上の結果から、キク科センダングサ属植物の抽出物を有効成分として含む防除剤は、その濃度を適切に設定することにより、マツノザイセンチュウ及びマツノマダラカミキリ(幼虫、成虫のいずれも)に対する高い防除効果(忌避、不動化、殺虫)を示すことが認められた。 From the above results, control agent comprising by an extract of Asteraceae Bidens plant as an active ingredient, by setting the concentration appropriately, for pine wood nematode and pine sawyer (larvae either adults) It was confirmed that it exhibited a high control effect (repellency, immobilization, insecticidal).
マツノザイセンチュウに対しては、本発明の防除剤が該線虫に対して接触すれば、マツ属植物の組織内および媒介虫であるマツノマダラカミキリの体内において殺虫性を発揮する。
また、マツノマダラカミキリおよびマツノザイセンチュウが共にキク科センダングサ属植物の抽出物に対して忌避性を示したことから、本発明の防除剤をマツ属植物の表面に適用することにより、マツ属植物の組織内へのマツノザイセンチュウの侵入を防止できることが確認された。
さらに、キク科センダングサ属植物の抽出物がマツノマダラカミキリ成虫および幼虫に対して殺虫性を示したことから、成虫に対しては本発明の防除剤を直接接触させることにより、また、幼虫に対しては本発明の防除剤をマツ属植物の組織内に適用することにより、殺虫効果を発揮できることが確認された。
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.
[参考文献]
William, F. M. and Peter, G. M. with Howard, H. L. 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
[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
本発明のマツ材線虫病防除剤は、昨今注目されている環境及び生物多様性を保全するための環境配慮型防除を促進するものであり、また単一の防除剤でありながら、マツ材線虫病の病原であるマツノザイセンチュウとその媒介虫であるマツノマダラカミキリの双方に対して高い抗虫活性(殺虫及び忌避効果)を有することから、効率的なマツ材線虫病の防除剤及び防除方法として有効に利用可能である。 The pine wood nematode control agent of the present invention promotes environmentally conscious 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 and its vector insect, pinewood beetle, it is an effective control agent for pinewood nematode disease. And it can be effectively used as a control method.
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