JP4402386B2 - Plant resistance inducer, plant resistance induction method, and plant disease and food damage prevention method - Google Patents

Description

【００４４】
【化２】

【００４５】
【化３】

【００４６】
【化４】

【００４７】
【化５】

【００４８】
【化６】

【００４９】
【化７】

【００５０】
【化８】

【００５１】
【化９】

【００５２】
後述する実施例１〜３に示すごとく、シロイナズナに(E)-2-hexenalおよび(Z)-3-hexenalをそれぞれ曝露させることによって、数種の生体防御関連遺伝子が誘導された。よってこれら物質が情報伝達物質として機能し、植物内の生態防御系遺伝子を誘導させたということがいえる。また作用させる植物由来情報伝達物質の種類によって、誘導される遺伝子の種類、誘導量が異なるということがわかった。さらに実施例４〜９において示すごとくシグナル伝達経路の検討を行ったところ、一部の遺伝子はＪＡシグナル伝達経路を介して誘導されているという知見が得られた。
【００５３】
またここでいうイソプレノイドは、別名テルペン、テルペノイドともよばれるイソプレンを構成単位とする天然有機化合物のことである。上記イソプレノイドには、例えば、ステロイド、カロテノイド、ドリコール、天然ゴム等がある。また、上記イソプレノイドに分類される物質には、allo-ocimene（式（１０）参照）、β-ocimene（式（１１）参照）、limonene、myrcene、pineneなど種々の化合物が挙げられるが、その中でも、植物の抵抗性を誘導する能力が高いという理由からallo-ocimene、β-ocimeneであることが好ましい。
【００５４】
【化１０】

【００５５】
【化１１】

【００５６】
後述する実施例１〜３において、allo-ocimeneをシロイナズナに曝露させることによって、数種の生体防御関連遺伝子が誘導された。よって、allo-ocimeneが情報伝達物質として機能し、植物内の生態防御系遺伝子を誘導させたということがいえる。さらに前述と同様に、実施例４〜９において示すごとく、シグナル伝達経路の検討を行ったところ、一部の遺伝子はＪＡシグナル伝達経路を介して誘導されているという知見が得られた。
【００５７】
またここで植物ホルモンとは、外的環境の影響を受けて植物体内で合成され、微量で成長や種々の生理作用を制御する有機化合物のことである。通常は、生産部位から離れた部位で作用するが、特定の標的器官を持ったり、特定の生理機能を示すものではなく、また植物の発育に従って量的・質的に変化して異なった反応を示すことから、植物の形態形成を考える上で極めて重要な物質である。植物ホルモンの例としては、ＪＡ、サリチル酸、アブシジン酸（ＡＢＡ）、エチレン、オーキシン、ジベレニン、サイトカイニン等が挙げられる。また上記植物ホルモンのメチルエステル等のエステル化合物であってもよい。例えば、ジャスモン酸エステル、サリチル酸エステル等である。
【００５８】
また、上記の以外の植物由来情報伝達物質として、傷害によって植物全身に誘導される、ＭＡＰキナーゼ、カタラーゼ、ポリフェノールオキシダーゼ、プロシステミン、リポキシゲナーゼなどのタンパク質分解酵素群、あるいは、トマトの全身抵抗性誘導のシグナル物質として知られているシステミンなどのペプチドといったものを挙げることができる。
【００５９】
後述の実施例で示したごとく、作用させる植物由来情報伝達物質によって誘導される生体防御関連遺伝子の種類が異なることから、上述した植物由来情報伝達物質を適宜組み合わせて植物用抵抗性誘導物質とする方が、広範囲の病害・食害に対しての抵抗性が向上するという点で好ましい。
【００６０】
それゆえ、本発明の植物用抵抗性誘導剤には、上記植物由来情報伝達物質が複数含まれていてもよい。また、上記植物由来情報伝達物質のみならず、他の成分が含まれていてもよい。上記他の成分としては、例えば、ｐＨ緩衝液、安定化剤等を挙げることができる。また病害・食害予防を補助するために、適宜農薬等を添加しても良い。
【００６１】
（ＩＩ）植物由来情報伝達物質の取得方法・植物用抵抗性誘導剤の調製方法上記植物由来情報伝達物質を取得する方法としては、植物から従来公知の方法に従って精製するという方法を用いることができる。さらに、上記植物用抵抗性誘導剤に含まれる情報伝達物質は、植物から採取したものに限らず、上記植物由来情報伝達物質と同一物質であればよい。それゆえ、情報伝達物質の取得には、微生物から従来公知の方法を用いて採取してもよいし、また従来公知の合成法に従って、植物由来情報伝達物質と同一の組成や構造を有する物質を人工的に合成するという方法などを用いてもよい。
【００６２】
例えば、(E)-2-hexenal、(Z)-3-hexenal、allo-ocimeneを植物由来情報伝達物質として植物用抵抗性誘導剤に含ませる場合に、これらの物質は、植物から単離してもよいが、既に市販されているものについては購入して使用すればよい。また市販されていない(Z)-3-hexenalなどは、後述する実施例に示すごとく酸化反応よって取得してもよいし、その他公知の方法（例えば非特許文献4参照）によって取得してもよい。
【００６３】
次に、上述の植物由来情報伝達物質、あるいは植物由来情報伝達物質と同一の物質を含む植物用抵抗性誘導剤の調製方法（製造方法）について説明する。
【００６４】
上記植物用抵抗性誘導剤は、これらの植物由来情報伝達物質を適当な濃度に希釈して調製すればよい。希釈する媒質に関して、水系、有機系などは特に限定されることはないが、有機系の場合は植物体の生育に悪影響をおよぼさない物質であることが望ましい。上記の媒質として具体的には、メタノール、エタノール等が挙げられる。メタノール、エタノール等の揮発性が高い溶媒を用いれば、当該植物用抵抗性誘導剤を使用した後に土壌等への蓄積がなく好ましい。
【００６５】
また、上記植物用抵抗性誘導剤に含まれる植物由来情報伝達物質の濃度であるが、例えば１Ｌ容器の中で植物用抵抗性誘導剤１０μｌを蒸発させることによって植物に曝露させる場合、当該植物用抵抗性誘導剤中に植物由来情報伝達物質が０．０１Ｍ以上、１Ｍ以下の濃度で含まれていることが好ましく、０．１Ｍの濃度であることがさらに好ましい。換言すれば、１Ｌの気体容積に対して、植物由来情報伝達物質が、０．１μｍｏｌ以上、１０μｍｏｌ以下の濃度で存在するような条件において植物を処理することが好ましく、１μｍｏｌの濃度で存在するような条件であることがさらに好ましい。このような濃度範囲内で植物由来情報伝達物質が含まれれば、上記条件において植物に曝露した場合に、確実に抵抗性を誘導させることができる。
【００６６】
ただし、上記植物由来情報伝達物質が(E)-2-hexenalの場合、植物用抵抗性誘導剤中に１Ｍを越えない濃度であることが好ましい。換言すれば、１Ｌの気体容積に対して、(E)-2-hexenalが、１０μｍｏｌを越えない濃度条件で、植物を処理することが好ましい。この濃度以上の(E)-2-hexenalを含む植物用抵抗性誘導剤を１０μｌ用いて、１Ｌ容器中で、植物に曝露すると、生体防御関連遺伝子の発現は顕著に見られるが、葉のネクロシスが起こり、葉の一部が枯死する場合があるため、あまり好ましくない。
【００６７】
またこれらの物質は、単独で使用してもよいが、前述のごとく適宜組み合わせることによって、より幅広く抵抗性を誘導することができるため、好ましい。
【００６８】
例えば、植物由来情報伝達物質として(E)-2-hexenalとallo-ocimeneとが含まれれば、それぞれを単独で用いた場合と比較してより多種の生体防御関連遺伝子の発現を誘導させることができる。同様のことが、(E)-2-hexenalと(Z)-3-hexenalとを組み合わせた場合、(Z)-3-hexenalとallo-ocimeneとを組み合わせた場合、あるいは、(E)-2-hexenal、(Z)-3-hexenal、およびallo-ocimeneを組み合わせた場合についても可能である。
【００６９】
なお、これらの植物由来情報伝達物質は、上記植物由来情報伝達物質が揮発性物質であれば、植物に作用させた後の余剰の植物用抵抗性誘導物剤は空気中に蒸散するため、土壌・河川等への蓄積がない等、環境へのリスクが少なく、さらに好ましい。
【００７０】
（ＩＩＩ）本発明の作用
続いて、本発明の植物用抵抗性誘導剤の作用について説明する。
【００７１】
上記植物用抵抗性誘導剤は、植物に曝露されることによって、植物に抵抗性を誘導させることができる。つまり、上記植物用抵抗誘導剤に暴露された植物は、病原菌や害虫などに対する抵抗性を獲得するための物質を自身の体内おいて発生させる。
【００７２】
植物が病原菌等に対する抵抗性を獲得する際に発生させる物質としては、例えば、ファイトアレキシンの合成系酵素、細胞壁強化に関与するタンパク質、ＰＲタンパク質、転写制御タンパク質、ポリガラクツロナーゼ阻害タンパク質（ＰＧＩＰ）、Ｓ−アデノシルメチオニン合成酵素（ＳＡＭＳ）、ＡＣＣ合成酵素等がある。
【００７３】
さらに、上記ファイトアレキシン合成系酵素の例としては、フェニルアラニンアンモニアリアーゼ（ＰＡＬ）、４クマリルＣｏＡリガーゼ（４ＣＬ）、カルコン合成酵素（ＣＨＳ）、３−ヒドロキシ−３−メチルグルタリルＣｏＡ還元酵素（ＨＭＧＲ）等がある。また細胞壁強化に関与するタンパク質の例としては、ハイドロキシプロリンリッチ糖タンパク質（ＨＲＧＰ）、グリシンリッチタンパク質（ＧＲＰ）、ペルオキシダーゼ（ＰＯＸ）等がある。
【００７４】
そのほか、後述する実施例１〜９において検討した、フェニルプロパノイド合成経路の鍵酵素であるカファイン酸−Ｏ−メチルトランスフェラーゼ（ＣＯＭＴ）、低温ストレス誘導性のジアシルグリセロールキナーゼ（ＤＧＫ１）、ＪＡやＣ₆−アルデヒドが合成されるオキシリピン経路の鍵酵素であるリポキシゲナーゼ（ＬＯＸ２）、β−１，３−グルカナーゼ（ＰＲ２）、ＰＤＦ１．２遺伝子によってコードされている抗菌タンパク質、ＶＳＰ２遺伝子遺伝子にコードされている栄養貯蔵タンパク質、グルタチオン−Ｓ−トランスフェラーゼ（ＧＳＴ１）等がある。
【００７５】
これらの物質が植物の体内で発生することによって、植物は病害虫に対する抵抗性を獲得し、病原菌の感染を防止したり、害虫による食害を防止したりすることができる。
【００７６】
（ＩＶ）本発明の利用
本発明にかかる植物の抵抗性誘導方法は、前述の植物用抵抗性誘導剤を対象となる植物に曝露させることによって達成される。
【００７７】
対象となる植物は特に限定されるものではなく、例えば、食用作物、果実や野菜、花・木その他の有効樹木を含む園芸作物、工芸作物、さらには飼肥料作物等が挙げられる。なお、上述した食用作物園芸作物、工芸作物、飼肥料作物にそれぞれ含まれる具体的な作物については、例えば、農学大辞典改訂第４版（養賢堂・１９９７年４月３０日発行）の目次８〜１６頁にも詳細に記載されている。
【００７８】
また、後述の実施例では、アブラナ科の植物であるシロイナズナ（Arabidopsis thaliana）においては、有効に抵抗性が向上していた。それゆえ、本発明の植物用抵抗性誘導剤は、アブラナ科植物に対して使用されることが好ましく、それに伴って、本発明の植物の抵抗性誘導方法も、アブラナ科植物に対する抵抗性の誘導に使用されることが好ましい。なお、アブラナ科植物として具体的には、シロイヌナズナ、アブラナ、キャベツ、カブ、ハクサイ、ダイコン、ワサビ、カラシ、ナズナなどを挙げることができる。
【００７９】
本発明にかかる植物用抵抗性誘導剤の曝露方法は、特に限定されるものではないが、例えば、霧吹き、スプレー容器等に当該植物用抵抗性誘導剤を充填し、対象植物に直接噴霧する方法などが挙げられる。また、上記植物由来情報伝達物質が揮発性物質の場合には、当該植物用抵抗性誘導剤を浸透させた脱脂綿・スポンジ等を対象植物の近隣に設置し、それらから蒸発することで植物に曝露させる方法を採用することが好ましい。また、当該植物用抵抗性誘導剤とゲル化剤とを混和してゲル化させ、そのゲルを対象植物の近隣に設置し、それらから蒸発することで植物に曝露させる方法を採用してもよい。ビニルハウス・温室等の密閉系（半密閉系）で対象植物を栽培している場合では、空調から当該植物用抵抗性誘導剤を曝露させる方法を採用することが好ましい。
【００８０】
後述する実施例では、当該植物用抵抗性誘導剤を浸透させた脱脂綿を用いて密閉容器中でシロイナズナに曝露させたが、複数の生体防御関連遺伝子の誘導が確認されており、本発明にかかる植物の抵抗性誘導方法が有効であるということがわかった。また、実施例１０〜１２に示すごとく当該植物用抵抗性誘導剤を曝露した植物の灰色カビ病の原因菌であるBotrytis cinerea(以下B.cinereaと称す)の抗菌活性を検討したが、明らかに抗菌活性が向上しており、抗菌性物質の発現が確認できた。この結果から、本発明の植物抵抗性誘導剤は、ボトリチス（Botrytis）属の菌に対する抗菌剤として使用されることが好ましい。また、本発明の植物の抵抗性誘導方法は、ボトリチス（Botrytis）属の菌の感染を防止するために利用されることが好ましい。
【００８１】
また実施例１３〜１５において示すごとく、葉租汁液を分子量分画した画分の抗菌性の検討においては、明らかに性質の異なる抗菌物質の存在が認められ、複数の抗菌性物質の発現が確認された。
【００８２】
以上のように、本発明の植物の抵抗性誘導方法によれば、上記植物用抵抗性誘導剤を植物に曝露することによって、植物自身の体内での作用によって抗菌物質を生成させることができ、病原菌に対する抵抗性を身につけることができる。それゆえ、例えば、食用の作物に対して本発明の植物の抵抗性誘導方法を用いれば、病害を防止するための農薬の散布量を減少させることができると考えられ、人体に対して安全性の高い作物を収穫することができる。また、環境に対しても悪影響の少ない農業を実現できる可能性も有している。
【００８３】
本発明の植物の病害・食害予防方法は、上述の植物由来情報伝達物質と同一物質を含む植物用抵抗性誘導剤を用いて、植物の抵抗性を誘導することによって、病原菌や害虫などからの被害を防止するというものである。
【００８４】
本発明が適用可能な病害としては、特に限定されるものではないが、例えば、B.cinereaをはじめとするボトリチス（Botrytis）属の菌を原因とする病害、Fusarium oxysporumをはじめとするフサリウム（Fusarium）属の菌を原因とする病害、うどんこ病、べと病、さび病、黒穂病、モモ縮葉病、イネいもち病、イネごま葉枯れ病、黒斑病、コムギ斑点病等が挙げられる。
【００８５】
上述植物の抵抗性誘導方法を用いることによって、後述の実施例１〜１５に説示したごとく、本発明にかかる植物用抵抗性誘導剤、および植物の抵抗性誘導方法を用いることによって、生体防御関連遺伝子の発現のみならず、抗菌性物質の誘導発現が植物体において確認され、このことによって植物の病害等を有効に予防できるといえる。
【００８６】
特に、後述の実施例では、(E)-2-hexenalを植物用抵抗性誘導剤として用いることによって、ボトリチス（Botrytis）属の菌であるB.cinereaに対する抗菌作用が確認されている。そのため、本発明の植物の病害および／または食害の予防方法は、ボトリチス（Botrytis）属の菌の感染による病害の予防に使用されることが好ましい。
【００８７】
【実施例】
以下実施例および比較例により、本発明をさらに詳細に説明するが、本発明はこれに限定されるものではない。各実施例について説明する前に、各実施例において用いられた試験材料および実験方法について以下に説明する。
【００８８】
〔供試植物の前処理〕
シロイナズナ（Arabidopsis thaliana）の種子の表面を滅菌し、０．１％寒天中で４℃、４８時間放置後、２％スクロース入りのＭＳプレート上にまいた。植物は、明期２３℃、１６時間および暗期２１℃、８時間の条件で生育させた。前処理までに、この条件下で２１日間生育させた。前処理するためにシャーレのフタを取り、１Ｌ容の密閉式のジャーに移し、供試揮発性成分の投入前２４時間インキュベートした。
【００８９】
〔揮発性成分の曝露条件〕
植物用抵抗性誘導剤として、揮発性を有する(E)-2-hexenal（和光純薬工業株式会社製）、(Z)-3-hexenal、およびallo-ocimene（東京化成工業株式会社製）を準備した。
【００９０】
また、(Z)-3-hexenalは市販されていないため、(Z)-3-hexen-1-olをデスマーチン酸化によって酸化して製造し、試験に用いた。
【００９１】
上記の方法で入手した(E)-2-hexenal、(Z)-3-hexenal、およびallo-ocimeneを、それぞれ０．１Ｍとなるようにメタノールで希釈し、４℃に冷却した溶液１０μｌを植物の上で滅菌綿棒にたらした後、容器を密閉した。コントロールは、各種薬剤を含まないメタノールのみで同様の処理を行った。
【００９２】
〔試験に使用する植物、化学物質、ＤＮＡプローブの入手〕
シロイナズナＪＡ非感受性変異株（Staswick PE ,Su W,Howell SH .、Proc Natl Acad Sci USA (1992.8.1) Vol.89 no.15、6837-6340 頁参照）およびシロイナズナエチレン非感受性変異株（Chang C ,Kwok SF ,Bleecker AB, Meyerowitz EM .、Science (1993.10.22) Vol.262 no.5133、539-544 頁参照）は、オハイオ州立大学 The Arabidopsis Biological Resource Center (ABRC)から入手した。
【００９３】
ＮＡＤはベーリンガーマンハイムより購入した。その他の化学物質は、９５％純度以上のものを使用し、それらは、シグマアルドリッチから購入した。
【００９４】
ＤＧＫ１は、ジアシルグリセロールキナーゼの遺伝子である。
【００９５】
ＬＯＸ２およびＶＳＰ２は、それぞれリポキシゲナーゼ遺伝子、vegetative strange proteinをコードする遺伝子でである。
【００９６】
ＣＨＳは、カリクローンシンターゼ遺伝子である。
【００９７】
ＧＳＴ１は、グルタチオン−Ｓ−トランスフェラーゼ遺伝子である。
【００９８】
ＣＯＭＴは、カフェイン酸−Ｏ−メチルトランスフェラーゼ遺伝子である。
【００９９】
ＰＤＦ１．２は、抗菌性タンパク質をコードする遺伝子である。
【０１００】
ＰＲ２は、β−１、３−グルカナーゼ遺伝子である。
【０１０１】
なお上記遺伝子に関する情報は、National Center for Biological Information のデータベース Gen Bank から検索することによって入手した。またこれらの遺伝子は、その公開されている遺伝子配列情報をもとに独自にプライマーを設計し、ＰＣＲによって取得した。
【０１０２】
〔半定量的ＲＴ−ＰＣＲ〕
遺伝子発現量の定量原理は、反応液中のサイバーグリーンの蛍光を指標にしており、ＰＣＲ反応が進むと、転写産物の量に応じてサイバーグリーンの蛍光が増加し、Gene Amp が輝度を測定するというものである。
【０１０３】
具体的には、植物用抵抗性誘導剤で処理した葉から、TRIzol (Invitrogen 社製)を用いてＲＮＡを抽出した。次に、このＲＮＡを、THERMOSCRIPT RT-PCR kit (Invitrogen 社製)を用いてｃＤＮＡに変換した。このｃＤＮＡを、Cyber Green PCR Core Reagent Kit (Applied Biosystem 社製)を用いて増幅した。この際にGene Amp 5700 (Applied Biosystem 社製)を用いてＰＣＲとＰＣＲ産物の定量を同時に行なった。なお、プライマーは、Primer Express (Applied Biosystem 社製)を用いてデザインした。そのほかのＲＴ−ＰＣＲの詳細な条件については、添付のマニュアルに従って試験を行なった。
【０１０４】
〔実施例１〕
(E)-2-hexenalを上述の条件で、シロイナズナに曝露させて４時間後、１２時間後、２４時間後の、８種類の生体防御関連遺伝子（ＣＯＭＴ遺伝子、ＣＨＳ遺伝子、ＤＧＫ１遺伝子、ＬＯＸ２遺伝子、ＰＲ２遺伝子、ＰＤＦ１．２遺伝子、ＶＳＰ２遺伝子、ＧＳＴ１遺伝子）の発現量をActinの転写量との相対値で評価した。その結果を、図１，２に示す。なお、図１，２の各グラフは、上記８種類の生体防御関連遺伝子発現量の相対値について、各種揮発成分（(E)-2-hexenal、(Z)-3-hexenal、allo-ocimene）曝露後の経時変化を示したものである。本実施例１の(E)-2-hexenalを曝露した場合の遺伝子の発現量は、各グラフ中の実線で示す。
【０１０５】
〔実施例２〕
(Z)-3-hexenalを上述の条件で、シロイナズナに曝露させて４時間後、１２時間後、２４時間後の、８種類の生体防御関連遺伝子（ＣＯＭＴ遺伝子、ＣＨＳ遺伝子、ＤＧＫ１遺伝子、ＬＯＸ２遺伝子、ＰＲ２遺伝子、ＰＤＦ１．２遺伝子、ＶＳＰ２遺伝子、ＧＳＴ１遺伝子）の発現量をActinの転写量との相対値で評価した。その結果を、図１，２に示す。なお、本実施例２の(Z)-3-hexenalを曝露した場合の遺伝子の発現量は、図１，２に示す各グラフ中の破線で示すものである。
【０１０６】
〔実施例３〕
allo-ocimeneを上述の条件で、シロイナズナに曝露させて４時間後、１２時間後、２４時間後の、８種類の生体防御関連遺伝子（ＣＯＭＴ遺伝子、ＣＨＳ遺伝子、ＤＧＫ１遺伝子、ＬＯＸ２遺伝子、ＰＲ２遺伝子、ＰＤＦ１．２遺伝子、ＶＳＰ２遺伝子、ＧＳＴ１遺伝子）の発現量をActinの転写量との相対値で評価した。その結果を、図１，２に示す。なお、本実施例３のallo-ocimeneを曝露した場合の遺伝子の発現量は、図１，２に示す各グラフ中の一点鎖線で示すものである。
【０１０７】
実施例１〜３の結果を、以下に説明する。
【０１０８】
図１では、フェニルプロパノイド合成経路の鍵酵素である、カファイン酸−Ｏ−メチルトランスフェラーゼ（ＣＯＭＴ）、その派生物からフラボノイドを合成するカルコンシンターゼ（ＣＨＳ）、低温ストレス誘導性のジアシルグリセロールキナーゼ（ＤＧＫ１）、ＪＡやＣ６−アルデヒドが合成されるオキシピン経路の鍵酵素であるリポキシゲナーゼ（ＬＯＸ２）の誘導を示している。その結果、(E)-2-hexenal、allo-ocimeneは、上記タンパク質をコードする各遺伝子発現をいずれも顕著に誘導した。一方、(Z)-3-hexenalは、ＣＨＳ遺伝子、ＤＧＫ１遺伝子の発現をほとんど誘導しなかった。
【０１０９】
図２では、β−１，３−グルカナーゼ（ＰＲ２）、抗菌タンパク質をコードするＰＤＦ１．２遺伝子、栄養貯蔵タンパク質をコードするＶＳＰ２遺伝子、グルタチオン−Ｓ−トランスフェラーゼ（ＧＳＴ１）遺伝子の発現量をみた。これらの遺伝子はいずれも、病原菌の感染によって遺伝子発現が誘導されることが知られている。しかし、今回のケースではＰＲ２遺伝子、ＶＳＰ２遺伝子の発現誘導は認められなかった。一方、ＰＤＦ１．２遺伝子、ＧＳＴ１遺伝子は、いずれの実施例においても顕著な誘導がみられた。
【０１１０】
〔実施例４〕
(E)-2-hexenalを上述の条件で、ＪＡ非感受性シロイナズナ変異体（ｊａｒ１変異体）に曝露させて、４時間後、１２時間後、２４時間後の、８種類の生体防御関連遺伝子（ＣＯＭＴ遺伝子、ＣＨＳ遺伝子、ＤＧＫ１遺伝子、ＬＯＸ２遺伝子、ＰＲ２遺伝子、ＰＤＦ１．２遺伝子、ＶＳＰ２遺伝子、ＧＳＴ１遺伝子）の発現量をActinの転写量との相対値で評価した。ＣＨＳ遺伝子およびＧＳＴ１遺伝子についての結果を、図３（ｂ）、（ｅ）に示す。本実施例４の(E)-2-hexenalを曝露した場合の遺伝子の発現量は、図３（ｂ）、（ｅ）に示す各グラフ中の実線で示すものである。
【０１１１】
〔実施例５〕
(E)-2-hexenalを上述の条件で、エチレン非感受性シロイナズナ変異体（ｅｔｒ１変異体）に曝露させて、４時間後、１２時間後、２４時間後の、８種類の生体防御関連遺伝子（ＣＯＭＴ遺伝子、ＣＨＳ遺伝子、ＤＧＫ１遺伝子、ＬＯＸ２遺伝子、ＰＲ２遺伝子、ＰＤＦ１．２遺伝子、ＶＳＰ２遺伝子、ＧＳＴ１遺伝子）の発現量をActinの転写量との相対値で評価した。ＣＨＳ遺伝子およびＧＳＴ１遺伝子についての結果を、図３（ｃ）、（ｆ）に示す。本実施例５の(E)-2-hexenalを曝露した場合の遺伝子の発現量は、図３（ｃ）、（ｆ）に示す各グラフ中の実線で示すものである。
【０１１２】
〔実施例６〕
(Z)-3-hexenalを上述の条件で、ｊａｒ１変異体に曝露させて４時間後、１２時間後、２４時間後の、８種類の生体防御関連遺伝子（ＣＯＭＴ遺伝子、ＣＨＳ遺伝子、ＤＧＫ１遺伝子、ＬＯＸ２遺伝子、ＰＲ２遺伝子、ＰＤＦ１．２遺伝子、ＶＳＰ２遺伝子、ＧＳＴ１遺伝子）の発現量をActinの転写量との相対値で評価した。ＣＨＳ遺伝子およびＧＳＴ１遺伝子についての結果を、図３（ｂ）、（ｅ）に示す。本実施例６の(Z)-3-hexenalを曝露した場合の遺伝子の発現量は、図３（ｂ）、（ｅ）に示す各グラフ中の破線で示すものである。
【０１１３】
〔実施例７〕
(Z)-3-hexenalを上述の条件で、ｅｔｒ１変異体に曝露させて４時間後、１２時間後、２４時間後の、８種類の生体防御関連遺伝子（ＣＯＭＴ遺伝子、ＣＨＳ遺伝子、ＤＧＫ１遺伝子、ＬＯＸ２遺伝子、ＰＲ２遺伝子、ＰＤＦ１．２遺伝子、ＶＳＰ２遺伝子、ＧＳＴ１遺伝子）の発現量をActinの転写量との相対値で評価した。ＣＨＳ遺伝子およびＧＳＴ１遺伝子についての結果を、図３（ｃ）、（ｆ）に示す。本実施例７の(Z)-3-hexenalを曝露した場合の遺伝子の発現量は、図３（ｃ）、（ｆ）に示す各グラフ中の破線で示すものである。
【０１１４】
〔実施例８〕
allo-ocimeneを上述の条件で、ｊａｒ１変異体に曝露させて４時間後、１２時間後、２４時間後の、８種類の生体防御関連遺伝子（ＣＯＭＴ遺伝子、ＣＨＳ遺伝子、ＤＧＫ１遺伝子、ＬＯＸ２遺伝子、ＰＲ２遺伝子、ＰＤＦ１．２遺伝子、ＶＳＰ２遺伝子、ＧＳＴ１遺伝子）の発現量をActinの転写量との相対値で評価した。ＣＨＳ遺伝子およびＧＳＴ１遺伝子についての結果を、図３（ｂ）、（ｅ）に示す。本実施例８のallo-ocimeneを曝露した場合の遺伝子の発現量は、図３（ｂ）、（ｅ）に示す各グラフ中の一点鎖線で示すものである。
【０１１５】
〔実施例９〕
allo-ocimeneを上述の条件で、ｅｔｒ１変異体に曝露させて４時間後、１２時間後、２４時間後の、８種類の生体防御関連遺伝子（ＣＯＭＴ遺伝子、ＣＨＳ遺伝子、ＤＧＫ１遺伝子、ＬＯＸ２遺伝子、ＰＲ２遺伝子、ＰＤＦ１．２遺伝子、ＶＳＰ２遺伝子、ＧＳＴ１遺伝子）の発現量をActinの転写量との相対値で評価した。ＣＨＳ遺伝子およびＧＳＴ１遺伝子についての結果を、図３（ｃ）、（ｆ）に示す。本実施例９のallo-ocimeneを曝露した場合の遺伝子の発現量は、図３（ｃ）、（ｆ）に示す各グラフ中の一点鎖線で示すものである。
【０１１６】
実施例４〜９の結果を、図３を参照して以下に説明する。なお、図３（ａ）および（ｄ）のには、実施例１〜３におけるＣＨＳ遺伝子およびＧＳＴ１遺伝子についての結果を比較として示している。
【０１１７】
図３（ｂ）に示すように、ｊａｒ１変異体では、ＣＨＳ遺伝子の(E)-2-hexenalによる誘導が抑制されていた。図には示していないが、同様の挙動をみせた遺伝子は、その他にはＤＧＫ１遺伝子があった。また、図３（ｅ）に示すように、ＧＳＴ１遺伝子は、ｊａｒ１変異体において全ての揮発成分による遺伝子発現量が低下した。図には示していないが、ＬＯＸ２遺伝子、ＣＯＭＴ遺伝子、ＰＤＦ１．２遺伝子の発現誘導は、ｊａｒ１変異体において抑制されなかった。一方、図３（ｃ）および（ｆ）に示すように、いずれの遺伝子においても、ｅｔｒ１変異体では、遺伝子の誘導が抑制されなかった。
【０１１８】
これまでの実施例１から９の結果をまとめると、(E)-2-hexenal、(Z)-3-hexenal、およびallo-ocimeneによって、種々の生体防御関連遺伝子の発現が誘導されることが確認された。また、同じ遺伝子でも、揮発成分の種類によって発現量が異なる場合があることがわかった。さらに、ｊａｒ１変異体およびｅｔｒ１変異体を用いた解析から、各揮発成分によるいくつかの遺伝子発現誘導にはＪＡシグナル伝達経路の関与が示唆された。
【０１１９】
〔実施例１０〕
(E)-2-hexenalを曝露した２４時間後のシロイヌナズナに、２．５％（ｗ/ｖ）グルコースを含んだ１×１０⁵個／ｍｌB.cinereaの分生胞子懸濁液を接種して二日後の病徴を観察した。その結果を図４（ａ）に示す。
【０１２０】
また、その病徴の程度を、Berrocal-Loboらの方法（Berrocal-Lobo, M., Molina, A.,Solano, R.,Plant J. (2002) Vol.29 no.1,23-32 頁参照）でグラフ化したもの図５（ａ）に示す。さらに、このときの葉の様子を図６（ａ）に示す。
【０１２１】
〔実施例１１〕
(Z)-3-hexenalを曝露した２４時間後のシロイヌナズナに、２．５％（ｗ／ｖ）グルコースを含んだ１×１０⁵個／ｍｌB.cinereaの分生胞子懸濁液を接種して二日後の病徴を観察した。その結果を図４（ｂ）に示す。
【０１２２】
また、その病徴の程度を、Berrocal-Loboらの方法でグラフ化したもの図５（ｂ）に示す。さらに、このときの葉の様子を図６（ｂ）に示す。
【０１２３】
〔実施例１２〕
allo-ocimeneを曝露した２４時間後のシロイヌナズナに、２．５％（ｗ／ｖ）グルコースを含んだ１×１０⁵個／ｍｌB.cinereaの分生胞子懸濁液を接種して二日後の病徴を観察した。その結果を図４（ｃ）に示す。
【０１２４】
また、その病徴の程度を、Berrocal-Loboらの方法でグラフ化したもの図５（ｃ）に示す。さらに、このときの葉の様子を図６（ｃ）に示す。
【０１２５】
〔比較例１〕
メタノールのみで処理したシロイヌナズナに、２．５％（ｗ／ｖ）グルコースを含んだ１×１０⁵個／ｍｌB.cinereaの分生胞子懸濁液を接種して二日後の病徴を観察した。その結果を図４（ｄ）に示す。
【０１２６】
また、その病徴の程度をグラフ化したもの図５（ｄ）に示す。さらに、このときの葉の様子を図６（ｄ）に示す。
【０１２７】
図４に示すように、実施例１０,１１および１２では、比較例１に比べて明らかに壊死病斑が少ないことが確認された。特に、実施例１０（(E)-2-hexenal）では高い病徴抑制効果が見られた。
【０１２８】
また、図５では、実施例１０〜１２、および比較例１における１２０株中の病徴の割合を円グラフで示している。各円グラフにおいて、白色のエリアは無病徴株を、斜線を付したのエリアは１〜４枚の葉に壊死斑が見られた軽度の病徴株を、網掛けのエリアは５枚以上の葉に壊死斑が見られた重度の病徴株、黒色のエリアは完全に枯死した株の割合を示している。
【０１２９】
この結果、実施例１０、１１、１２では、比較例１に比して枯死した株の割合が、３倍程度高いことがわかった。特に、図５（ａ）に示す実施例１０（(E)-2-hexenal）の場合が、重度の病徴株の割合が他の実施例よりも少ないという結果であった。
【０１３０】
また図６に示すように、比較例１では、B.cinereaの葉上で菌糸が繁茂していた他、侵入菌糸の旺盛な生育が認められた。一方、実施例１０、１１、１２では、菌糸生育や菌糸の侵入が抑制されていた。
【０１３１】
以上の結果より、実施例における(E)-2-hexenal、(Z)-3-hexenal、およびallo-ocimeneをシロイナズナに曝露することによって、何らかの抗菌性物質が生産され、菌糸生育を抑制しているということがわかった。
【０１３２】
〔実施例１３〕
(E)-2-hexenalを曝露して２４時間後のシロイヌナズナの葉租汁液を透析し、分子量１２，０００以下の低分子画分と１２，０００以上の高分子画分を取得し、これらのB.cinerea分生胞子に対する抗菌活性を調査した。
【０１３３】
図７（ａ）には高分子画分における結果を、図８（ａ）には低分子画分における結果を示す。また、低分子画分および高分子画分をそれぞれ７０℃、３０分間加熱処理を行ったときの抗菌活性についても検討した。このうち高分子画分の結果を図９（ａ）に示す。
【０１３４】
〔実施例１４〕
(Z)-3-hexenalを曝露して２４時間後のシロイヌナズナの葉租汁液を透析し、分子量１２，０００以下の低分子画分と１２，０００以上の高分子画分を取得し、これらのB.cinerea分生胞子に対する抗菌活性を調査した。
【０１３５】
図７（ｂ）には高分子画分における結果を、図８（ｂ）には低分子画分における結果を示す。また、低分子画分および高分子画分をそれぞれ７０℃、３０分間加熱処理を行ったときの抗菌活性についても検討した。このうち高分子画分の結果を図９（ｂ）に示す。
【０１３６】
〔実施例１５〕
allo-ocimeneを曝露して２４時間後のシロイヌナズナの葉租汁液を透析し、分子量１２，０００以下の低分子画分と１２，０００以上の高分子画分を取得し、これらのB.cinerea分生胞子に対する抗菌活性を調査した。
【０１３７】
図７（ｃ）には高分子画分における結果を、図８（ｃ）には低分子画分における結果を示す。また、低分子画分および高分子画分をそれぞれ７０℃、３０分間加熱処理を行ったときの抗菌活性についても検討した。このうち高分子画分の結果を図９（ｃ）に示す。
【０１３８】
〔比較例２〕
揮発成分未処理のシロイヌナズナの葉租汁液を透析し、分子量１２，０００以下の低分子画分と１２，０００以上の高分子画分を取得し、これらのB.cinerea分生胞子に対する抗菌活性を調査した。
【０１３９】
図７（ｄ）には高分子画分における結果を、図８（ｄ）には高分子画分における結果を示す。また、低分子画分および高分子画分をそれぞれ７０℃、３０分間加熱処理を行ったときの抗菌活性についても検討した。このうち高分子画分の結果を図９（ｄ）に示す。
【０１４０】
図７、８は、実施例１３、１４、１５、および比較例２で得た高分子画分（非加熱）および低分子画分（非加熱）をB.cinerea分生胞子懸濁液に添加し、２４時間後のB.cinerea分生胞子の様子を示している。実施例１３、１４、１５では、高分子画分及び低分子画分ともに、比較例２に比して明らかに菌糸生育が抑制されていることが分かった。特に、実施例１３（(E)-2-hexenal）における低分子画分では、分生胞子の発芽率が比較例２の３分の１という劇的な抑制効果が示された。
【０１４１】
また図９では、各実施例における高分子画分の抗菌活性は、７０℃、３０分の加熱処理で失活することがわかった。一方低分子画分は、各実施例における低分子画分の抗菌活性は、加熱処理によって失活はしなかった（図示せず）。よって一つの揮発成分を曝露で、複数の抗菌性物質が誘導されているということがわかった。
【０１４２】
実施例１０から１５および比較例１，２の結果をまとめると、各実施例のシロイヌナズナでは比較例に比して、明らかにB.cinereaの病徴進展が抑制された。また、このとき顕微鏡観察の結果、曝露後の葉では、B.cinerea菌糸の感染が抑制されていた。さらに、曝露後の葉租汁液高分子画分と低分子画分では抗菌活性の増強が認められ、複数の抗菌性物質の誘導が示唆された。
【０１４３】
よって、植物に短鎖アルデヒドやイソプレノイドを曝露することによって、複数の生体防御関連遺伝子の誘導、およびこれらにコードされた抗菌性物質が誘導発現されることが確認された。この事実は、本実施例の結果から、短鎖アルデヒドやイソプレノイドが植物用抵抗性誘導剤として作用するということを示すものである。そして、この結果、当該植物の抗菌性が向上し、病害を有効に予防することが可能となる。
【０１４４】
【発明の効果】
本発明にかかる植物用抗菌性物質誘導剤は、植物由来情報伝達物質と同一物質を含んでなるものであるため、植物に曝露されると、ＪＡシグナル伝達経路等を経てその情報伝達物質が伝達され、植物体内で複数の生体防御関連遺伝子を誘導する。その結果、植物体内で抗菌性物質が発現し、当該抗菌性物質の作用によって、植物体の抗菌性が向上し、種々の病害等を予防することができる。
【０１４５】
つまり、本発明の植物用抵抗性誘導剤によれば、植物が本来有する生体防御機構を利用して、植物自身の害虫（細菌、ウイルスを含む）などに対する抵抗性を向上させることができる。それゆえ、農薬などの使用による病害虫の駆除とは異なり、人体や環境に安全な方法で植物の病害や食害などを予防することができる。
【０１４６】
さらに、本発明の植物用抵抗性誘導剤は、揮発性を有する情報伝達物質が風媒性シグナル（air-borne signal)として機能することによって植物の生体防御機構に作用するものであるため、未解明の部分の多い上記生体防御機構のメカニズムの解明にも利用することができる。
【０１４７】
また、本発明の抗菌性物質誘導方法および植物の病害および／または食害の予防方法によれば、これまで種々の病害によって被害を受けていた植物、特に野菜等の食用作物を栽培する際に、農薬等の使用量を低下することができ、人体や環境等に対して安全かつ有効に植物の病害等を予防することができるという効果を奏する。
【図面の簡単な説明】
【図１】 (E)-2-hexenal、(Z)-3-hexenalおよびallo-ocimeneを、それぞれシロイナズナに曝露させて、４時間後、１２時間後、２４時間後のＣＯＭＴ遺伝子、ＣＨＳ遺伝子、ＤＧＫ１遺伝子、ＬＯＸ２遺伝子の発現量をActinの転写量との相対値で示した結果を示すグラフである。なお、本図は、実施例１，２，３の結果を示すものである。
【図２】 (E)-2-hexenal、(Z)-3-hexenalおよびallo-ocimeneを、それぞれシロイナズナに曝露させて、４時間後、１２時間後、２４時間後のＰＲ２遺伝子、ＰＤＦ１．２遺伝子、ＶＳＰ２遺伝子、ＧＳＴ１遺伝子の発現量をActinの転写量との相対値で示した結果を示すグラフである。なお、本図は、実施例１，２，３の結果を示すものである。
【図３】 (E)-2-hexenal、(Z)-3-hexenalおよびallo-ocimeneを、それぞれシロイナズナ（（ａ）および（ｄ））、ＪＡ非感受性シロイナズナ変異体（ｊａｒ１変異体）（（ｂ）および（ｅ））、またはエチレン非感受性白イナズナ変異体（ｅｔｒ１）（（ｃ）および（ｆ））に曝露させて、４時間後、１２時間後、２４時間後のＣＨＳ遺伝子およびＧＳＴ１遺伝子の発現量をActinの転写量との相対値で示した結果である。なお、本図は、実施例４〜９の結果を示すものである。
【図４】実施例１０〜１２、および比較例１において、各揮発性成分を曝露した２４時間後のシロイヌナズナに、２．５％（ｗ／ｖ）グルコースを含んだ１×１０⁵個／ｍｌB.cinereaの分生胞子懸濁液を接種して二日後のシロイヌナズナの状態を示す模式図である。なお、（ａ）は(E)-2-hexenalを曝露したもの、（ｂ）は(Z)-3-hexenalを曝露したもの、（ｃ）はallo-ocimeneを曝露したもの、（ｄ）はコントロールをそれぞれ示している。
【図５】実施例１０〜１２、および比較例１において、各揮発性成分を曝露した２４時間後のシロイヌナズナに、２．５％（ｗ/ｖ）グルコースを含んだ１×１０⁵個／ｍｌB.cinereaの分生胞子懸濁液を接種して二日後の病徴の程度の割合示す円グラフである。なお、（ａ）は(E)-2-hexenalを曝露したもの、（ｂ）は(Z)-3-hexenalを曝露したもの、（ｃ）はallo-ocimeneを曝露したもの、（ｄ）はコントロールをそれぞれ示している。
【図６】実施例１０〜１２、および比較例１において、各揮発性成分を曝露した２４時間後のシロイヌナズナに、２．５％（ｗ／ｖ）グルコースを含んだ１×１０⁵個／ｍｌB.cinereaの分生胞子懸濁液を接種して二日後の葉の様子を示模式図である。なお、（ａ）は(E)-2-hexenalを曝露したもの、（ｂ）は(Z)-3-hexenalを曝露したもの、（ｃ）はallo-ocimeneを曝露したもの、（ｄ）はコントロールをそれぞれ示している。
【図７】実施例１３〜１５、および比較例２において、各揮発性成分を曝露した２４時間後のシロイヌナズナ葉租汁液の高分子画分の、B.cinerea分生胞子に対する抗菌活性を調査した結果を示す模式図である。なお、（ａ）は(E)-2-hexenalを曝露したもの、（ｂ）は(Z)-3-hexenalを曝露したもの、（ｃ）はallo-ocimeneを曝露したもの、（ｄ）はコントロールをそれぞれ示している。
【図８】実施例１３〜１５、および比較例２において、各揮発性成分を曝露した２４時間後のシロイヌナズナ葉租汁液の低分子画分の、B.cinerea分生胞子に対する抗菌活性を調査した結果を示す模式図である。なお、（ａ）は(E)-2-hexenalを曝露したもの、（ｂ）は(Z)-3-hexenalを曝露したもの、（ｃ）はallo-ocimeneを曝露したもの、（ｄ）はコントロールをそれぞれ示している。
【図９】実施例１３〜１５、および比較例２において、各揮発性成分を曝露した２４時間後のシロイヌナズナ葉租汁液の高分子画分を、さらに７０℃、３０分間加熱処理を行ったときのB.cinerea分生胞子に対する抗菌活性を調査した結果を示す模式図である。なお、（ａ）は(E)-2-hexenalを曝露したもの、（ｂ）は(Z)-3-hexenalを曝露したもの、（ｃ）はallo-ocimeneを曝露したもの、（ｄ）はコントロールをそれぞれ示している。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a resistance inducer that suppresses the propagation of bacteria and the like that cause various diseases in plants, and suppresses food damage such as pests, and a resistance inducer using the resistance inducer. The present invention relates to an induction method and a method for preventing disease and food damage of plants using them.
[0002]
[Prior art]
More than 80% of plant infections are caused by filamentous fungi (fungi, fungi), and the rest are caused by bacteria, viruses, viroids, phytoplasma, rickettsia-like microorganisms, nematodes, protozoa and the like. Taking fungal diseases as an example, there are more than tens of thousands of filamentous fungi on the earth, most of which are saprophytes, of which about 8,000 are plant pathogens. For example, in the case of rice, there are about 50 types of pathogenic bacteria that can harm rice to varying degrees, and among them, less than 10 types cause serious damage to rice. Thus, plants are inherently resistant to the majority of pathogens and are damaged by a handful of pathogenic species. Plants do not have biological defense mechanisms by immune systems such as antibody reactions and phagocytosis found in vertebrates, but they protect themselves against attack from pathogens by a different mechanism. As one of them, inducible resistance expression, so-called plant immune system is known (see Non-Patent Document 1).
[0003]
In this system, when some tissue of a plant is infected by pathogenic bacteria, some signal substance is released from the infected site, it reaches the site not yet infected through the plant body, Therefore, the expression of genes involved in resistance expression is induced. Substances involved in this resistance include PR proteins (Pathogenesis-related proteins) having antibacterial activity themselves, glucanases and chitinases that dissolve cell walls, and phytoalexins that are toxic to pathogenic bacteria.
[0004]
Until now, it has been considered that the above-described induction of resistance occurs only within the same plant individual. However, it has been discovered that plant-derived volatile components such as methyl jasmonate, short chain aldehydes and isoprenoids have a function as an air-borne signal that induces plant resistance, It has been known that a physically distant homologous or heterogeneous plant recognizes it to activate a biological defense mechanism (see, for example, Non-Patent Documents 2, 3, and 4). For example, there is a report that the induction efficiency of natural enemies of tomato pests is increased by administering volatile methyl jasmonate to tomato (see Non-Patent Document 2).
[0005]
Non-Patent Document 4 includes C ₆ It has been reported that aldehyde induces some genes (hereinafter referred to as biological defense-related genes) involved in plant biological defense. However, this non-patent document 4 includes C ₆ The causal relationship between aldehydes and antibacterial properties has not been investigated.
[0006]
In addition, the present inventors ₆ It has been reported that exposure of aldehydes and isoprenoids to plants induces biological defense-related genes and clearly enhances antibacterial activity against pathogenic bacteria (Non-patent Document 5).
[0007]
[Non-Patent Document 1]
"Plant resistance from the molecular level" Cell engineering separate volume, Plant cell engineering series 8, pp. 131-140, Shujunsha, published October 1, 1997
[0008]
[Non-Patent Document 2]
Jennifer S. Thaler, NATURE (1999) Vol.399, pp. 686-688
[0009]
[Non-Patent Document 3]
Gregg A. Howe, PANAS (October 23, 2001) vol.98 no.22,
12317-12319 pages
[0010]
[Non-Patent Document 4]
Nicholas J. Bate et al., The Plant Journal (1998) 16 (5),
561-569
[0011]
[Non-Patent Document 5]
Proceedings of the 2003 Annual Meeting of the Japanese Society for Plant Pathology, 172
[0012]
[Problems to be solved by the invention]
By the way, food crops, fruits and vegetables, horticultural crops including flowers and trees, and other useful trees, craft crops, feed crops, etc. are greatly damaged by pests, herbivores, fungal diseases, natural disasters, etc. May receive. In particular, damage caused by pests and fungi is a serious problem, and in order to mitigate it, agricultural chemical spraying is routinely performed. Certainly, the effects of pest control and disease prevention with agricultural chemicals are enormous.
[0013]
However, on the other hand, problems such as human damage caused by eating vegetables and the like attached with agricultural chemicals, environmental pollution such as soil pollution and water pollution have been pointed out. In addition, organic farming methods are carried out without using these pesticides, but this is disadvantageous in terms of yield and cost.
[0014]
The above-described expression of induced resistance is a biological defense mechanism inherent in plants. Therefore, if this mechanism can be applied to pest control and disease prevention, the amount of agricultural chemicals used can be reduced. In addition, because volatile components such as methyl jasmonate, short-chain aldehydes, and isoprenoids are used to induce the biological defense mechanism of plants, there is also an advantage of low persistence compared to conventional agricultural chemicals. Have. However, conventionally, such a biological defense mechanism has not yet been put to practical use for pest control and disease prevention.
[0015]
The present invention has been made in view of the above problems, and an object thereof is to prevent plant disease and pest damage in a safe manner against the human body, the environment, and the like, by using the above-described biological defense mechanism of the plant. is there. That is, the present invention provides a method for preventing plant disease and / or food damage, a plant resistance inducer used in the method, and a resistance induction method.
[0016]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have exposed a volatile component such as a short-chain aldehyde or isoprenoid that is expressed when a plant is infected with a pest or a disease caused by a pest, It was confirmed that the expression of genes related to biodefense was induced and that there were differences in the genes induced by the types of volatile components to be exposed. Moreover, when the antibacterial activity against the pathogenic bacteria of the plants to which these volatile components were actually exposed was examined, it was confirmed that the antibacterial properties were certainly improved as compared with the plants that did not act on the volatile components. Furthermore, it was suggested that the induced expression of these genes involves the jasmonate signaling pathway (hereinafter referred to as the JA signaling pathway). The present invention has been completed based on these findings.
[0017]
That is, the present invention relates to a plant resistance-inducing agent characterized in that it contains a plant-derived information transmitting substance that is synthesized when a plant receives an external stimulus and is responsible for transmitting information related to the external stimulus.
[0018]
Here, the “plant-derived information transmission substance” means a substance synthesized by a plant body under the influence of the external environment. The plant-derived information transmission substance functions to induce a gene involved in the biological defense of the plant in the plant body by being exposed to the plant. The plant-derived information transmitting substance contained in the plant resistance inducer of the present invention is not limited to those synthesized by the plant body, but is artificially synthesized having the same structure as the plant-derived information transmitting substance. It may be a material. Therefore, the “plant-derived information transmitting substance” referred to in the present invention may be a “natural” compound produced by a plant, or a compound artificially synthesized by a known method.
[0019]
Therefore, according to the plant resistance inducer of the present invention, it is possible to improve the resistance of the plant itself against pests (including bacteria and viruses) by utilizing the biological defense mechanism inherent in the plant. Therefore, unlike the control of pests by using pesticides and the like, it is possible to prevent plant diseases and food damages in a safe manner for the human body and the environment. The resistance to plant pests and the like will be described in detail in the embodiments.
[0020]
As described above, the “plant resistance-inducing agent” of the present invention improves resistance to pests by inducing resistance to pests and diseases caused by exposure to plants by exposure to plants. Means a drug.
[0021]
In the plant resistance inducer, the plant-derived information transmitting substance is preferably a volatile substance.
[0022]
If the plant-derived information transfer substance is a volatile substance, when the plant resistance inducer is exposed to the plant, it can be sprayed evenly over a wide range of plants, effectively preventing plant diseases and food damage. Can be used. Furthermore, since the surplus plant resistance inducer after acting on plants evaporates in the air, there is no accumulation in soil, rivers, etc., and the risk to the environment can be reduced.
[0023]
In addition, in the above-described plant resistance inducer, the plant-derived information transmission substance is a short-chain aldehyde and / or isoprenoid and / or a plant hormone because of its high ability to induce plant resistance. Is preferred.
[0024]
Furthermore, in the above-mentioned plant resistance inducer, the short-chain aldehyde is C ₆ More preferably, it is an aldehyde. In addition, the above C ₆ Even more preferably, the aldehyde is (E) -2-hexenal and / or (Z) -3-hexenal.
[0025]
In the plant resistance inducer, the plant-derived information transmitting substance is preferably (Z) -3-hexen-1-ol because of its high ability to induce plant resistance. .
[0026]
Moreover, when the plant-derived information transmission substance is an isoprenoid, the isoprenoid is more preferably allo-ocimene. Furthermore, when the plant-derived information transmission substance is a plant hormone, the plant hormone may be jasmonic acid (hereinafter referred to as JA).
[0027]
In addition, the plant resistance inducer is preferably such that the plant resistance inducer induces an antibacterial substance. According to this, it is possible to suppress the growth of bacteria or the like that cause disease in plants. Furthermore, the above-described plant resistance inducer is preferably used as an antibacterial agent against bacteria of the genus Botrytis, in order to induce an antibacterial substance against bacteria of the genus Botrytis.
[0028]
Moreover, since the said plant-resistance induction agent improves resistance more effectively in a Brassicaceae plant, and shows an antibacterial action, it is preferable to be used with respect to a Brassicaceae plant.
[0029]
The plant resistance inducing method according to the present invention is characterized by exposing any of the above plant resistance inducing agents to a plant.
[0030]
That is, in the plant resistance-inducing method according to the present invention, a plant defense-related gene is induced by exposing the plant resistance-inducing agent to a plant, and further, an antibacterial substance is induced by the gene induction. Is expressed.
[0031]
According to said resistance induction method, the resistance with respect to a disease and a food damage can be improved in a plant by the above effects, and the damage can be prevented.
[0032]
Furthermore, the present invention also provides a method for preventing plant diseases and / or food damage, characterized by using the above-described resistance induction method. The above-described method for preventing plant diseases and / or food damage can be suitably used for the prevention of diseases caused by infection with bacteria of the genus Botrytis.
[0033]
DETAILED DESCRIPTION OF THE INVENTION
More specifically, the present invention is as follows. Needless to say, the present invention is not limited to this description.
[0034]
(I) Resistance inducer for plants
The plant resistance inducer of the present invention is characterized in that it contains the same substance as the plant-derived information transmitting substance. Before describing the plant resistance inducer of the present invention, first, “plant resistance” will be described.
[0035]
“Plant resistance” means a defense mechanism against plant pathogens or pests. The resistance can be roughly classified into static (passive) resistance and dynamic (active) resistance. Static resistance means resistance due to the potential physical and chemical properties that the plant has before being infected with pathogenic bacteria. Examples of static resistance include hardness and thickness of plant cell walls, accumulation of polyphenol substances, and the like. On the other hand, dynamic resistance means a series of defense reactions in which plant cells / tissues are actively activated against attacks by external enemies.
[0036]
Classifying the dynamic resistance of higher plants based on temporal and spatial differences
(1) Generation of active oxygen and initiation of hypersensitive reaction
(2) Phenylalanine ammonia lyase (PAL), phenylpropanoid synthase such as caffeic acid dehydrogenase, or induction of genes involved in the synthesis of isoflavonoid / phytoalexin such as chalcone acid synthase (CHS) and gene products Accumulation
(3) Synthesis of kinases, glucanases, and other PR (pathogenesis-related) proteins that not only break down the cell walls of invading bacteria but also liberate elicitor molecules from the invading bacteria or autologous cell walls.
It is classified as follows.
[0037]
The plant resistance-inducing agent of the present invention refers to a drug that works to induce the resistance of the plant as described above in the body of the plant and improve the tolerance of the plant against pests, pathogens and the like.
[0038]
Next, the plant-derived information transmitting substance contained in the plant resistance inducer will be described in more detail.
[0039]
For example, when a stress is applied to a certain part of a plant body, the plant has a mechanism that transmits this information to the whole plant body and activates a self-protection mechanism against the stress systemically, that is, a systemic acquired resistance. On the other hand, when a plant is infested by an insect or infected with a disease, the information is transmitted to the same or another plant that grows next to it, so as to induce insect resistance and reduce damage. There is a mechanism. These mechanisms are called plant biological defense mechanisms.
[0040]
In the biological defense mechanism, a plurality of substances are involved and the information is transmitted in multiple stages. In this way, a substance that is synthesized when an external stimulus is received in a plant, and a substance that is responsible for information transmission regarding the external stimulus is called a plant-derived information transfer substance.
[0041]
Examples of such plant-derived information transmission substances include aldehydes, isoprenoids, plant hormones and the like.
[0042]
The aldehyde here is not particularly limited, but is preferably a short chain aldehyde because of its high ability to induce plant resistance, for example, C ₉ Aldehydes (E) -2-nonenal (see formula (1)), (Z) -3-nonenal (see formula (2)), (E, Z) -2,6-nonadienal (see formula (3)) ), (Z, Z) -3,6-nonadienal (see formula (4)), (E) -3-nonenal (see formula (5)), and the like. Furthermore C ₆ It is preferably an aldehyde, for example, (E) -2-hexenal (see formula (6)), (Z) -3-hexenal (see formula (7)), (E) -3-hexenal (formula (8) And (Z) -3-hexen-1-ol (see formula (9)).
[0043]
[Chemical 1]

[0044]
[Chemical formula 2]

[0045]
[Chemical 3]

[0046]
[Formula 4]

[0047]
[Chemical formula 5]

[0048]
[Chemical 6]

[0049]
[Chemical 7]

[0050]
[Chemical 8]

[0051]
[Chemical 9]

[0052]
As shown in Examples 1 to 3 to be described later, several biological defense-related genes were induced by exposing (E) -2-hexenal and (Z) -3-hexenal, respectively, to Arabidopsis thaliana. Therefore, it can be said that these substances functioned as information transmission substances and induced ecological defense system genes in plants. It was also found that the type of gene to be induced and the amount of induction differ depending on the type of plant-derived information transmitter to be acted on. Furthermore, when the signal transduction pathway was examined as shown in Examples 4 to 9, it was found that some genes were induced via the JA signal transduction pathway.
[0053]
The isoprenoid referred to here is a natural organic compound having isoprene as a structural unit, also called terpene or terpenoid. Examples of the isoprenoid include steroids, carotenoids, dolichol, and natural rubber. The substances classified as isoprenoids include various compounds such as allo-ocimene (see formula (10)), β-ocimene (see formula (11)), limonene, myrcene, and pinene. Allo-ocimene and β-ocimene are preferred because of their high ability to induce plant resistance.
[0054]
[Chemical Formula 10]

[0055]
Embedded image

[0056]
In Examples 1 to 3 to be described later, several types of biological defense-related genes were induced by exposing allo-ocimene to Shiroina. Therefore, it can be said that allo-ocimene functions as a signal transmitter and induces an ecological defense gene in the plant. Further, as described above, as shown in Examples 4 to 9, when signal transduction pathways were examined, it was found that some genes were induced via the JA signal transduction pathway.
[0057]
The plant hormone is an organic compound synthesized in a plant body under the influence of an external environment and controlling growth and various physiological actions in a small amount. Usually, it acts at a site away from the production site, but it does not have a specific target organ or exhibit a specific physiological function, and it changes quantitatively and qualitatively according to the growth of the plant and reacts differently. As shown, it is an extremely important substance in considering the morphogenesis of plants. Examples of plant hormones include JA, salicylic acid, abscisic acid (ABA), ethylene, auxin, gibberenin, cytokinin and the like. Moreover, ester compounds, such as the methyl ester of the said plant hormone, may be sufficient. For example, jasmonic acid ester, salicylic acid ester and the like.
[0058]
Moreover, as a plant-derived information transmission substance other than the above, a proteolytic enzyme group such as MAP kinase, catalase, polyphenol oxidase, prosystemamine, lipoxygenase or a signal for inducing systemic resistance of tomato induced by injury. Examples thereof include peptides such as cystemin known as a substance.
[0059]
As shown in the examples described later, since the types of biological defense-related genes induced by the plant-derived information transmission substance to be acted are different, the above-described plant-derived information transmission substances are appropriately combined to form a plant resistance-inducing substance. This is preferable in that resistance to a wide range of diseases and food damages is improved.
[0060]
Therefore, the plant resistance inducer of the present invention may contain a plurality of the above-mentioned plant-derived information transmission substances. In addition to the plant-derived information transmission substance, other components may be included. As said other component, a pH buffer solution, a stabilizer, etc. can be mentioned, for example. Moreover, in order to assist disease prevention and food damage prevention, you may add agrochemicals etc. suitably.
[0061]
(II) Method for obtaining plant-derived information transmission substance / Method for preparing plant resistance inducer As a method for obtaining the plant-derived information transmission substance, a method of purifying from a plant according to a conventionally known method can be used. . Furthermore, the information transmission substance contained in the plant resistance inducer is not limited to those collected from plants, and may be the same substance as the plant-derived information transmission substance. Therefore, in order to acquire the information transmission substance, it may be collected from microorganisms using a conventionally known method, or a substance having the same composition and structure as a plant-derived information transmission substance may be obtained according to a conventionally known synthesis method. You may use the method of synthesize | combining artificially.
[0062]
For example, when (E) -2-hexenal, (Z) -3-hexenal, allo-ocimene is included in a plant resistance inducer as a plant-derived signal transmitter, these substances are isolated from plants. However, what is already on the market can be purchased and used. Further, (Z) -3-hexenal and the like that are not commercially available may be obtained by an oxidation reaction as shown in Examples described later, or may be obtained by other known methods (for example, see Non-Patent Document 4). .
[0063]
Next, a preparation method (manufacturing method) of a plant resistance inducer containing the above-mentioned plant-derived information transmitting substance or the same substance as the plant-derived information transmitting substance will be described.
[0064]
The plant resistance inducer may be prepared by diluting these plant-derived information transmitting substances to an appropriate concentration. With respect to the medium to be diluted, there are no particular limitations on the aqueous system and organic system, but in the case of an organic system, a substance that does not adversely affect the growth of the plant body is desirable. Specific examples of the medium include methanol and ethanol. It is preferable to use a highly volatile solvent such as methanol or ethanol because there is no accumulation in soil after using the plant resistance inducer.
[0065]
Moreover, it is the density | concentration of the plant-derived information transmission substance contained in the said plant resistance inducer, For example, when exposing to a plant by evaporating 10 microliters of plant resistance inducers in a 1L container, It is preferable that the plant-derived information transmission substance is contained in the resistance inducer at a concentration of 0.01 M or more and 1 M or less, and more preferably a concentration of 0.1 M. In other words, it is preferable to treat the plant under conditions such that the plant-derived signal transmitting substance is present at a concentration of 0.1 μmol or more and 10 μmol or less with respect to a gas volume of 1 L, so that it exists at a concentration of 1 μmol. More preferably, the conditions are satisfied. If a plant-derived information transmission substance is contained within such a concentration range, resistance can be reliably induced when exposed to plants under the above conditions.
[0066]
However, when the plant-derived information transmitting substance is (E) -2-hexenal, the concentration is preferably not more than 1M in the plant resistance inducer. In other words, it is preferable to treat the plant under a concentration condition where (E) -2-hexenal does not exceed 10 μmol per 1 L of gas volume. When 10 μl of a plant resistance inducer containing (E) -2-hexenal at a concentration higher than this concentration is used and exposed to plants in a 1 L container, the expression of biological defense-related genes is significantly observed, but leaf necrosis is observed. Occurs, and part of the leaves may die, which is not preferable.
[0067]
These substances may be used alone, but are preferable because resistance can be induced more widely by combining them appropriately as described above.
[0068]
For example, if (E) -2-hexenal and allo-ocimene are included as plant-derived information transmitters, the expression of a variety of biological defense-related genes can be induced compared to the case where each is used alone. it can. The same applies when (E) -2-hexenal and (Z) -3-hexenal are combined, (Z) -3-hexenal and allo-ocimene are combined, or (E) -2 This is also possible when -hexenal, (Z) -3-hexenal, and allo-ocimene are combined.
[0069]
Note that these plant-derived information transmission substances, if the above-mentioned plant-derived information transmission substance is a volatile substance, the excess plant resistance inducer after acting on the plant evaporates in the air, so that・ There is little risk to the environment, such as no accumulation in rivers, etc., which is more preferable.
[0070]
(III) Effects of the present invention
Then, the effect | action of the resistance inducer for plants of this invention is demonstrated.
[0071]
The plant resistance-inducing agent can induce plant resistance by being exposed to the plant. That is, a plant exposed to the plant resistance inducer generates a substance in its body to acquire resistance to pathogenic bacteria and pests.
[0072]
Examples of substances generated when a plant acquires resistance to pathogenic bacteria include, for example, phytoalexin synthesizing enzymes, proteins involved in cell wall reinforcement, PR proteins, transcriptional control proteins, polygalacturonase inhibitory proteins (PGIP) ), S-adenosylmethionine synthase (SAMS), ACC synthase and the like.
[0073]
Further, examples of the phytoalexin synthesizing enzyme include phenylalanine ammonia lyase (PAL), 4 coumaryl CoA ligase (4CL), chalcone synthase (CHS), 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). ) Etc. Examples of proteins involved in cell wall strengthening include hydroxyproline rich glycoprotein (HRGP), glycine rich protein (GRP), peroxidase (POX) and the like.
[0074]
In addition, caffeic acid-O-methyltransferase (COMT), which is a key enzyme in the phenylpropanoid synthesis pathway, low temperature stress-inducible diacylglycerol kinase (DGK1), JA and C, which were examined in Examples 1 to 9 described later. ₆ -Lipoxygenase (LOX2), β-1,3-glucanase (PR2), which are key enzymes of the oxylipin pathway in which aldehyde is synthesized, antibacterial protein encoded by PDF1.2 gene, and nutrition encoded by VSP2 gene gene There are storage proteins, glutathione-S-transferase (GST1) and the like.
[0075]
When these substances are generated in the body of the plant, the plant can acquire resistance to pests, prevent infection with pathogenic bacteria, and prevent feeding damage caused by pests.
[0076]
(IV) Use of the present invention
The plant resistance inducing method according to the present invention is achieved by exposing the above-described plant resistance inducing agent to a target plant.
[0077]
The target plant is not particularly limited, and examples thereof include edible crops, horticultural crops including fruit and vegetables, flowers / trees and other effective trees, craft crops, and fertilizer crops. As for the specific crops included in the above-mentioned food crops, horticultural crops, craft crops, and fertilizer crops, for example, the table of contents of the Agricultural University Dictionary 4th edition (Yokendo, issued April 30, 1997) It is also described in detail on pages 8-16.
[0078]
Moreover, in the Example mentioned later, resistance improved effectively in Arabidopsis thaliana which is a plant of the Brassicaceae family. Therefore, the plant resistance-inducing agent of the present invention is preferably used for cruciferous plants, and accordingly, the method for inducing resistance to cruciferous plants according to the present invention also induces resistance to cruciferous plants. It is preferable to be used for. Specific examples of cruciferous plants include Arabidopsis, Brassica, cabbage, turnip, Chinese cabbage, Japanese radish, horseradish, mustard, and tuna.
[0079]
The exposure method of the plant resistance inducer according to the present invention is not particularly limited. For example, the plant resistance inducer is filled in a spray bottle, a spray container or the like, and directly sprayed onto the target plant. Etc. In addition, when the plant-derived information transfer substance is a volatile substance, an absorbent cotton / sponge, etc., infiltrated with the plant resistance inducer is placed in the vicinity of the target plant and exposed to the plant by evaporating from it. It is preferable to employ the method of causing Alternatively, a method may be employed in which the plant resistance inducer and the gelling agent are mixed and gelled, and the gel is placed in the vicinity of the target plant and evaporated from them to be exposed to the plant. . In the case where the target plant is cultivated in a closed system (semi-closed system) such as a vinyl house or a greenhouse, it is preferable to employ a method of exposing the plant resistance inducer from air conditioning.
[0080]
In the examples described later, the cotton-resistant cotton impregnated with the plant resistance-inducing agent was used to expose the silkworms in a sealed container. However, induction of a plurality of biological defense-related genes has been confirmed, and the present invention is applied. It was found that the method of inducing plant resistance is effective. In addition, as shown in Examples 10 to 12, the antibacterial activity of Botrytis cinerea (hereinafter referred to as B. cinerea), which is a causative bacterium of gray mold disease of plants exposed to the plant resistance inducer, was examined. Antibacterial activity was improved, and the expression of antibacterial substances was confirmed. From this result, the plant resistance inducer of the present invention is preferably used as an antibacterial agent against bacteria of the genus Botrytis. Moreover, it is preferable that the method for inducing plant resistance of the present invention is used for preventing infection by bacteria of the genus Botrytis.
[0081]
In addition, as shown in Examples 13 to 15, in the examination of the antibacterial properties of the fraction obtained by molecular weight fractionation of leaf juice, the presence of antibacterial substances with clearly different properties was confirmed, and the expression of a plurality of antibacterial substances was confirmed. It was done.
[0082]
As described above, according to the plant resistance inducing method of the present invention, an antibacterial substance can be generated by the action of the plant itself by exposing the plant resistance inducing agent to the plant, You can acquire resistance to pathogenic bacteria. Therefore, for example, if the plant resistance inducing method of the present invention is used for edible crops, it is considered that the spraying amount of pesticides for preventing diseases can be reduced, which is safe for the human body. Can be harvested. It also has the potential to realize agriculture with less adverse effects on the environment.
[0083]
The plant disease and food damage prevention method of the present invention uses a plant resistance inducer containing the same substance as the above-mentioned plant-derived information transmission substance, and induces plant resistance, thereby causing pathogens and pests from It is to prevent damage.
[0084]
Diseases to which the present invention can be applied are not particularly limited. For example, diseases caused by bacteria of the genus Botrytis including B. cinerea, Fusarium including Fusarium oxysporum (Fusarium ) Diseases caused by fungi of the genus, powdery mildew, downy mildew, rust, smut, black peach, leaf blast, rice blast, rice sesame leaf blight, black spot, wheat spot, etc. .
[0085]
By using the above-described plant resistance inducing method, as described in Examples 1 to 15 described later, by using the plant resistance inducing agent according to the present invention and the plant resistance inducing method, biological defense-related Not only gene expression but also induced expression of antibacterial substances has been confirmed in the plant body, which can effectively prevent plant diseases and the like.
[0086]
In particular, in the examples described later, the antibacterial action against B. cinerea, a bacterium of the genus Botrytis, has been confirmed by using (E) -2-hexenal as a plant resistance inducer. Therefore, it is preferable that the method for preventing disease and / or food damage of the plant of the present invention is used for prevention of disease caused by infection with bacteria of the genus Botrytis.
[0087]
【Example】
EXAMPLES Hereinafter, although an Example and a comparative example demonstrate this invention further in detail, this invention is not limited to this. Before describing each example, the test materials and experimental methods used in each example will be described below.
[0088]
[Pretreatment of test plants]
The surface of Arabidopsis thaliana seeds was sterilized, and allowed to stand in 0.1% agar at 4 ° C. for 48 hours, and then spread on MS plates containing 2% sucrose. The plants were grown under conditions of a light period of 23 ° C. for 16 hours and a dark period of 21 ° C. for 8 hours. It was grown under these conditions for 21 days before pretreatment. For pretreatment, the petri dish lid was removed, transferred to a 1 L sealed jar, and incubated for 24 hours before the introduction of the test volatile components.
[0089]
[Exposure conditions for volatile components]
(E) -2-hexenal (manufactured by Wako Pure Chemical Industries, Ltd.), (Z) -3-hexenal, and allo-ocimene (manufactured by Tokyo Kasei Kogyo Co., Ltd.) are used as plant resistance inducers. Got ready.
[0090]
Since (Z) -3-hexenal is not commercially available, (Z) -3-hexen-1-ol was produced by oxidation by desmartin oxidation and used in the test.
[0091]
(E) -2-hexenal, (Z) -3-hexenal, and allo-ocimene obtained by the above method were each diluted with methanol so as to be 0.1 M, and 10 μl of a solution cooled to 4 ° C. was added to the plant. After placing on a sterile swab, the container was sealed. As a control, the same treatment was performed only with methanol not containing various drugs.
[0092]
[Obtain plants, chemicals, and DNA probes to be used for testing]
Shiroizuna JA-insensitive mutants (see Staswick PE, Su W, Howell SH., Proc Natl Acad Sci USA (1992.8.1) Vol.89 no.15, pages 6837-6340) and Shiroizuna ethylene-insensitive mutants (Chang C, Kwok SF, Bleecker AB, Meyerowitz EM., Science (1993.10.22) Vol.262 no.5133, pp. 539-544) were obtained from Ohio State University The Arabidopsis Biological Resource Center (ABRC).
[0093]
NAD was purchased from Boehringer Mannheim. The other chemicals used were those with a purity of 95% or more, and they were purchased from Sigma Aldrich.
[0094]
DGK1 is a gene for diacylglycerol kinase.
[0095]
LOX2 and VSP2 are genes encoding a lipoxygenase gene and a vegetative strange protein, respectively.
[0096]
CHS is a potash clone synthase gene.
[0097]
GST1 is a glutathione-S-transferase gene.
[0098]
COMT is a caffeic acid-O-methyltransferase gene.
[0099]
PDF1.2 is a gene encoding an antibacterial protein.
[0100]
PR2 is a β-1,3-glucanase gene.
[0101]
Information on the above genes was obtained by searching from the GenBank database of the National Center for Biological Information. In addition, these genes were obtained by PCR by designing primers uniquely based on the published gene sequence information.
[0102]
[Semi-quantitative RT-PCR]
The quantification principle of gene expression is based on the fluorescence of cyber green in the reaction solution. As the PCR reaction proceeds, the fluorescence of cyber green increases according to the amount of transcript, and Gene Amp measures the luminance. That's it.
[0103]
Specifically, RNA was extracted from leaves treated with a plant resistance inducer using TRIzol (Invitrogen). Next, this RNA was converted into cDNA using THERMOSCRIPT RT-PCR kit (Invitrogen). This cDNA was amplified using Cyber Green PCR Core Reagent Kit (Applied Biosystem). At this time, PCR and PCR product quantification were simultaneously performed using Gene Amp 5700 (Applied Biosystem). The primers were designed using Primer Express (Applied Biosystem). Other detailed RT-PCR conditions were tested according to the attached manual.
[0104]
[Example 1]
(E) -2-hexenal was exposed to S. thaliana under the above-mentioned conditions, and 8 types of biological defense-related genes (COMT gene, CHS gene, DGK1 gene, LOX2 gene 4 hours later, 12 hours later, 24 hours later) , PR2 gene, PDF1.2 gene, VSP2 gene, GST1 gene) were evaluated as relative values to the amount of Actin transcription. The results are shown in FIGS. In addition, each graph of FIGS. 1 and 2 shows various volatile components ((E) -2-hexenal, (Z) -3-hexenal, allo-ocimene) for the relative values of the expression levels of the above eight types of biological defense-related genes. It shows the change over time after exposure. The expression level of the gene when (E) -2-hexenal of Example 1 is exposed is indicated by a solid line in each graph.
[0105]
[Example 2]
Eight types of biological defense-related genes (COMT gene, CHS gene, DGK1 gene, LOX2 gene) after 4 hours, 12 hours, and 24 hours after exposure to (Z) -3-hexenal under the above-mentioned conditions. , PR2 gene, PDF1.2 gene, VSP2 gene, GST1 gene) were evaluated as relative values to the amount of Actin transcription. The results are shown in FIGS. In addition, the expression level of the gene when (Z) -3-hexenal of Example 2 is exposed is indicated by a broken line in each graph shown in FIGS.
[0106]
Example 3
Allo-ocimene was exposed to S. thaliana under the above-mentioned conditions, and 8 biological defense-related genes (COMT gene, CHS gene, DGK1 gene, LOX2 gene, PR2 gene, 4 hours later, 12 hours later, 24 hours later) The expression levels of PDF1.2 gene, VSP2 gene, and GST1 gene were evaluated relative to the amount of Actin transcription. The results are shown in FIGS. In addition, the expression level of the gene at the time of exposing allo-ocimene of this Example 3 is shown with the dashed-dotted line in each graph shown to FIG.
[0107]
The results of Examples 1 to 3 will be described below.
[0108]
In FIG. 1, the key enzyme of the phenylpropanoid synthesis pathway, kafine acid-O-methyltransferase (COMT), chalcone synthase (CHS) that synthesizes flavonoids from its derivatives, low-temperature stress-inducible diacylglycerol kinase (DGK1) ), Induction of lipoxygenase (LOX2), which is a key enzyme of the oxypine pathway in which JA and C6-aldehyde are synthesized. As a result, (E) -2-hexenal and allo-ocimene significantly induced the expression of each gene encoding the protein. On the other hand, (Z) -3-hexenal hardly induced the expression of CHS gene and DGK1 gene.
[0109]
In FIG. 2, expression levels of β-1,3-glucanase (PR2), PDF1.2 gene encoding antibacterial protein, VSP2 gene encoding nutrient storage protein, and glutathione-S-transferase (GST1) gene were observed. All of these genes are known to induce gene expression by infection with pathogenic bacteria. However, in this case, expression induction of PR2 gene and VSP2 gene was not observed. On the other hand, the PDF1.2 gene and the GST1 gene were significantly induced in any of the examples.
[0110]
Example 4
(E) -2-hexenal was exposed to a JA-insensitive syleuthna mutant (jar1 mutant) under the above-described conditions, and 8 types of biological defense-related genes (4 hours, 12 hours, 24 hours later) The expression level of COMT gene, CHS gene, DGK1 gene, LOX2 gene, PR2 gene, PDF1.2 gene, VSP2 gene, GST1 gene) was evaluated as a relative value to the transcription amount of Actin. The results for the CHS gene and GST1 gene are shown in FIGS. 3 (b) and 3 (e). The expression level of the gene when (E) -2-hexenal of Example 4 is exposed is indicated by a solid line in each graph shown in FIGS. 3 (b) and 3 (e).
[0111]
Example 5
(E) -2-hexenal was exposed to an ethylene-insensitive syleudon mutant (etr1 mutant) under the above-mentioned conditions, and 8 types of biological defense-related genes (4 hours, 12 hours, 24 hours later) ( The expression level of COMT gene, CHS gene, DGK1 gene, LOX2 gene, PR2 gene, PDF1.2 gene, VSP2 gene, GST1 gene) was evaluated as a relative value to the transcription amount of Actin. The results for the CHS gene and GST1 gene are shown in FIGS. 3 (c) and 3 (f). The expression level of the gene when (E) -2-hexenal of Example 5 is exposed is indicated by a solid line in each graph shown in FIGS. 3 (c) and 3 (f).
[0112]
Example 6
(Z) -3-hexenal was exposed to the jar1 mutant under the above-mentioned conditions, and 8 types of biological defense-related genes (COMT gene, CHS gene, DGK1 gene, The expression level of LOX2 gene, PR2 gene, PDF1.2 gene, VSP2 gene, GST1 gene) was evaluated as a relative value to the amount of Actin transcription. The results for the CHS gene and GST1 gene are shown in FIGS. 3 (b) and 3 (e). The expression level of the gene when (Z) -3-hexenal of Example 6 is exposed is indicated by a broken line in each graph shown in FIGS. 3 (b) and 3 (e).
[0113]
Example 7
(Z) -3-hexenal was exposed to the etr1 mutant under the above-mentioned conditions, and 8 types of biological defense-related genes (COMT gene, CHS gene, DGK1 gene, The expression level of LOX2 gene, PR2 gene, PDF1.2 gene, VSP2 gene, GST1 gene) was evaluated as a relative value to the amount of Actin transcription. The results for the CHS gene and GST1 gene are shown in FIGS. 3 (c) and 3 (f). The expression level of the gene when exposed to (Z) -3-hexenal of Example 7 is indicated by a broken line in each graph shown in FIGS. 3 (c) and 3 (f).
[0114]
Example 8
Allo-ocimene was exposed to the jar1 mutant under the above-mentioned conditions, and 8 biological defense-related genes (COMT gene, CHS gene, DGK1 gene, LOX2 gene, PR2 gene, 4 hours, 12 hours, and 24 hours later) Gene, PDF1.2 gene, VSP2 gene, GST1 gene) expression level was evaluated relative to the amount of Actin transcription. The results for the CHS gene and GST1 gene are shown in FIGS. 3 (b) and 3 (e). The expression level of the gene when the allo-ocimene of Example 8 is exposed is indicated by a one-dot chain line in each graph shown in FIGS. 3 (b) and 3 (e).
[0115]
Example 9
Allo-ocimene was exposed to the etr1 mutant under the above-mentioned conditions, and 8 biological defense-related genes (COMT gene, CHS gene, DGK1 gene, LOX2 gene, PR2 gene, 4 hours, 12 hours, and 24 hours later) Gene, PDF1.2 gene, VSP2 gene, GST1 gene) expression level was evaluated relative to the amount of Actin transcription. The results for the CHS gene and GST1 gene are shown in FIGS. 3 (c) and 3 (f). The expression level of the gene when allo-ocimene of Example 9 is exposed is indicated by a one-dot chain line in each graph shown in FIGS. 3 (c) and 3 (f).
[0116]
The results of Examples 4 to 9 will be described below with reference to FIG. In addition, in FIG. 3 (a) and (d), the result about the CHS gene and GST1 gene in Examples 1-3 is shown as a comparison.
[0117]
As shown in FIG. 3 (b), in the jar1 mutant, induction of the CHS gene by (E) -2-hexenal was suppressed. Although not shown in the figure, the DGK1 gene was another gene that showed the same behavior. Moreover, as shown in FIG.3 (e), as for GST1 gene, the gene expression level by all the volatile components fell in jar1 mutant. Although not shown in the figure, LOX2 gene, COMT gene and PDF1.2 gene expression induction was not suppressed in the jar1 mutant. On the other hand, as shown in FIGS. 3 (c) and (f), in any of the genes, the induction of the gene was not suppressed in the etr1 mutant.
[0118]
Summarizing the results of Examples 1 to 9 so far, expression of various biological defense-related genes is induced by (E) -2-hexenal, (Z) -3-hexenal, and allo-ocimene. confirmed. It was also found that the expression level of the same gene may vary depending on the type of volatile component. Furthermore, the analysis using the jar1 mutant and the etr1 mutant suggested that the JA signaling pathway is involved in the induction of several gene expressions by each volatile component.
[0119]
Example 10
Arabidopsis thaliana 24 hours after exposure to (E) -2-hexenal contained 1 x 10 containing 2.5% (w / v) glucose ^Five After inoculation with a conidial spore suspension of 1 cell / ml B. cinerea, the symptom was observed 2 days later. The result is shown in FIG.
[0120]
In addition, the degree of the symptom was determined by the method of Berrocal-Lobo et al. (Berrocal-Lobo, M., Molina, A., Solano, R., Plant J. (2002) Vol.29 no.1, pp.23-32). FIG. 5 (a) shows a graph. Furthermore, the state of the leaves at this time is shown in FIG.
[0121]
Example 11
1 x 10 containing 2.5% (w / v) glucose in Arabidopsis thaliana 24 hours after exposure to (Z) -3-hexenal ^Five After inoculation with a conidial spore suspension of 1 cell / ml B. cinerea, the symptom was observed 2 days later. The result is shown in FIG.
[0122]
FIG. 5 (b) shows a graph of the degree of the disease symptoms by the method of Berrocal-Lobo et al. Furthermore, the state of the leaves at this time is shown in FIG.
[0123]
Example 12
Arabidopsis thaliana 24 hours after exposure to allo-ocimene, 1 × 10 5 containing 2.5% (w / v) glucose ^Five After inoculation with a conidial spore suspension of 1 cell / ml B. cinerea, the symptom was observed 2 days later. The result is shown in FIG.
[0124]
In addition, FIG. 5 (c) shows a graph of the degree of the disease symptoms by the method of Berrocal-Lobo et al. Furthermore, the state of the leaves at this time is shown in FIG.
[0125]
[Comparative Example 1]
1 x 10 containing 2.5% (w / v) glucose in Arabidopsis treated with methanol alone ^Five After inoculation with a conidial spore suspension of 1 cell / ml B. cinerea, the symptom was observed 2 days later. The result is shown in FIG.
[0126]
FIG. 5D shows a graph of the degree of the disease symptoms. Furthermore, the state of the leaves at this time is shown in FIG.
[0127]
As shown in FIG. 4, it was confirmed that the necrotic lesions were clearly less in Examples 10, 11 and 12 than in Comparative Example 1. In particular, in Example 10 ((E) -2-hexenal), a high symptom suppression effect was observed.
[0128]
Moreover, in FIG. 5, the ratio of the disease symptom in 120 strain | stump | stock in Examples 10-12 and the comparative example 1 is shown with the pie chart. In each pie chart, the white area indicates no diseased strain, the shaded area indicates 1 to 4 leaves with mild necrotic lesions, and the shaded area indicates 5 or more. Severe symptomatic strains with necrotic spots on the leaves, black areas indicate the percentage of completely dead strains.
[0129]
As a result, in Examples 10, 11, and 12, it was found that the ratio of the strain that withered was about 3 times higher than that of Comparative Example 1. In particular, the case of Example 10 ((E) -2-hexenal) shown in FIG. 5A was a result that the ratio of severe disease symptoms was smaller than that of the other Examples.
[0130]
Moreover, as shown in FIG. 6, in Comparative Example 1, the hyphae grew thickly on the leaves of B. cinerea, and vigorous growth of the invading hyphae was recognized. On the other hand, in Examples 10, 11, and 12, hyphal growth and hyphal invasion were suppressed.
[0131]
From the above results, by exposing (E) -2-hexenal, (Z) -3-hexenal, and allo-ocimene in Examples to Syleuzens, some antibacterial substances are produced, and mycelial growth is suppressed. I found out.
[0132]
Example 13
(E) -2-hexenal was exposed to dialyzed Arabidopsis leaf juice 24 hours later to obtain a low molecular fraction having a molecular weight of 12,000 or less and a high molecular fraction having a molecular weight of 12,000 or more. The antibacterial activity against B. cinerea conidia was investigated.
[0133]
FIG. 7A shows the result in the high molecular fraction, and FIG. 8A shows the result in the low molecular fraction. In addition, the antibacterial activity when the low molecular fraction and the high molecular fraction were each subjected to heat treatment at 70 ° C. for 30 minutes was also examined. Among these, the result of the polymer fraction is shown in FIG.
[0134]
Example 14
(Z) -3-hexenal was exposed to dialyzed Arabidopsis thaliana leaf juice 24 hours later to obtain a low molecular fraction having a molecular weight of 12,000 or less and a high molecular fraction having a molecular weight of 12,000 or more. The antibacterial activity against B. cinerea conidia was investigated.
[0135]
FIG. 7B shows the result in the high molecular fraction, and FIG. 8B shows the result in the low molecular fraction. In addition, the antibacterial activity when the low molecular fraction and the high molecular fraction were each subjected to heat treatment at 70 ° C. for 30 minutes was also examined. Among these, the result of the polymer fraction is shown in FIG.
[0136]
Example 15
Dialyzing Arabidopsis thaliana leaves 24 hours after exposure to allo-ocimene, low molecular fractions with a molecular weight of 12,000 or less and high molecular fractions with a molecular weight of 12,000 or more are obtained. The antibacterial activity against live spores was investigated.
[0137]
FIG. 7C shows the result in the high molecular fraction, and FIG. 8C shows the result in the low molecular fraction. In addition, the antibacterial activity when the low molecular fraction and the high molecular fraction were each subjected to heat treatment at 70 ° C. for 30 minutes was also examined. Among these, the result of the polymer fraction is shown in FIG.
[0138]
[Comparative Example 2]
Dialyze the untreated arabic leaves of Arabidopsis to obtain a low molecular weight fraction with a molecular weight of 12,000 or less and a high molecular fraction with a molecular weight of 12,000 or more, and have antibacterial activity against these B.cinerea conidia. investigated.
[0139]
FIG. 7 (d) shows the result in the polymer fraction, and FIG. 8 (d) shows the result in the polymer fraction. In addition, the antibacterial activity when the low molecular fraction and the high molecular fraction were each subjected to heat treatment at 70 ° C. for 30 minutes was also examined. Among these, the result of the polymer fraction is shown in FIG.
[0140]
7 and 8 show that the high molecular fraction (non-heated) and the low molecular fraction (non-heated) obtained in Examples 13, 14, 15 and Comparative Example 2 were added to the B. cinerea conidia spore suspension. And the state of B.cinerea conidia after 24 hours is shown. In Examples 13, 14, and 15, it was found that hyphal growth was clearly suppressed as compared with Comparative Example 2 in both the high molecular fraction and the low molecular fraction. In particular, the low molecular fraction in Example 13 ((E) -2-hexenal) showed a dramatic inhibitory effect that the germination rate of conidia was one-third that of Comparative Example 2.
[0141]
Moreover, in FIG. 9, it turned out that the antimicrobial activity of the polymer fraction in each Example is deactivated by the heat processing for 30 minutes at 70 degreeC. On the other hand, the antimolecular activity of the low molecular fraction in each Example was not inactivated by the heat treatment (not shown). Therefore, it was found that multiple antibacterial substances were induced by exposure to one volatile component.
[0142]
When the results of Examples 10 to 15 and Comparative Examples 1 and 2 are summarized, the development of disease symptoms of B. cinerea was clearly suppressed in the Arabidopsis thaliana of each Example as compared with the Comparative Example. At the same time, as a result of microscopic observation, infection with B. cinerea mycelia was suppressed in the leaves after exposure. Furthermore, enhanced antibacterial activity was observed in the high molecular fraction and low molecular fraction of leaf juice after exposure, suggesting the induction of multiple antibacterial substances.
[0143]
Therefore, it was confirmed that by exposing short-chain aldehydes and isoprenoids to plants, induction of a plurality of biological defense-related genes and antibacterial substances encoded by them were induced. This fact shows that the short-chain aldehydes and isoprenoids act as plant resistance inducers from the results of this Example. As a result, the antibacterial properties of the plant are improved and the disease can be effectively prevented.
[0144]
【The invention's effect】
Since the antibacterial substance inducer for plants according to the present invention comprises the same substance as a plant-derived information transmission substance, the information transmission substance is transmitted via the JA signal transmission pathway when exposed to a plant. And induces multiple biological defense-related genes in plants. As a result, an antibacterial substance is expressed in the plant body, and the antibacterial property of the plant body is improved by the action of the antibacterial substance, and various diseases can be prevented.
[0145]
That is, according to the plant resistance inducer of the present invention, it is possible to improve the resistance of a plant itself to pests (including bacteria and viruses) by utilizing a biological defense mechanism inherent in the plant. Therefore, unlike the control of pests by using pesticides and the like, it is possible to prevent plant diseases and food damages in a safe manner for the human body and the environment.
[0146]
Further, the plant resistance inducer of the present invention acts on a plant's biological defense mechanism by functioning as an air-borne signal by a volatile information-transmitting substance. It can also be used to elucidate the mechanism of the above-mentioned biological defense mechanism that has many elucidated parts.
[0147]
In addition, according to the antibacterial substance induction method and the plant disease and / or food damage prevention method of the present invention, when cultivating edible crops such as vegetables, particularly vegetables, which have been damaged by various diseases so far, It is possible to reduce the amount of agricultural chemicals used and to prevent plant diseases and the like safely and effectively against the human body and the environment.
[Brief description of the drawings]
[FIG. 1] (E) -2-hexenal, (Z) -3-hexenal, and allo-ocimene were each exposed to tyrosine, and the COMT gene, CHS gene after 4 hours, 12 hours, and 24 hours, It is a graph which shows the result which showed the expression level of the DGK1 gene and the LOX2 gene by the relative value with the transcription amount of Actin. In addition, this figure shows the result of Example 1,2,3.
[Fig. 2] (E) -2-hexenal, (Z) -3-hexenal and allo-ocimene were respectively exposed to syleudon, and PR4 gene, PDF1.2 after 4 hours, 12 hours and 24 hours. It is a graph which shows the result which showed the expression level of the gene, VSP2 gene, and GST1 gene by the relative value with the transcription amount of Actin. In addition, this figure shows the result of Example 1,2,3.
FIG. 3 shows that (E) -2-hexenal, (Z) -3-hexenal, and allo-ocimene were respectively converted to syleudon ((a) and (d)), JA-insensitive syleuthna mutant (jar1 mutant) (( b) and (e)), or CHS gene and GST1 gene after 4 hours, 12 hours and 24 hours after exposure to ethylene-insensitive white locust mutants (etr1) ((c) and (f)) It is the result which showed the expression level of as a relative value with the transcription | transfer amount of Actin. In addition, this figure shows the result of Examples 4-9.
FIG. 4 shows that Arabidopsis thaliana 24 hours after exposure to each volatile component in Examples 10 to 12 and Comparative Example 1 contains 1 × 10 2 containing 2.5% (w / v) glucose. ^Five It is a schematic diagram which shows the state of Arabidopsis thaliana two days after inoculating a conidial spore suspension of pcs / ml B. cinerea. (A) is exposed to (E) -2-hexenal, (b) is exposed to (Z) -3-hexenal, (c) is exposed to allo-ocimene, (d) is Each control is shown.
FIG. 5 shows that Arabidopsis thaliana 24 hours after exposure to each volatile component in Examples 10 to 12 and Comparative Example 1 contained 1 × 10 5 containing 2.5% (w / v) glucose. ^Five 2 is a pie chart showing the proportion of disease symptoms two days after inoculation with a conidial spore suspension of individuals / ml B. cinerea. (A) is exposed to (E) -2-hexenal, (b) is exposed to (Z) -3-hexenal, (c) is exposed to allo-ocimene, (d) is Each control is shown.
FIG. 6 shows that Arabidopsis thaliana 24 hours after exposure to each volatile component in Examples 10 to 12 and Comparative Example 1 contained 1 × 10 5 containing 2.5% (w / v) glucose. ^Five It is a schematic diagram showing the state of leaves two days after inoculation with a conidial spore suspension of pcs / ml B. cinerea. (A) is exposed to (E) -2-hexenal, (b) is exposed to (Z) -3-hexenal, (c) is exposed to allo-ocimene, (d) is Each control is shown.
FIG. 7: In Examples 13 to 15 and Comparative Example 2, antibacterial activity against B. cinerea conidia spores of a polymer fraction of Arabidopsis thaliana juice 24 hours after exposure to each volatile component was investigated. It is a schematic diagram which shows a result. (A) is exposed to (E) -2-hexenal, (b) is exposed to (Z) -3-hexenal, (c) is exposed to allo-ocimene, (d) is Each control is shown.
FIG. 8: In Examples 13 to 15 and Comparative Example 2, antibacterial activity against B. cinerea conidia spores of a low molecular fraction of Arabidopsis leaf juice 24 hours after exposure to each volatile component was investigated. It is a schematic diagram which shows a result. (A) is exposed to (E) -2-hexenal, (b) is exposed to (Z) -3-hexenal, (c) is exposed to allo-ocimene, (d) is Each control is shown.
FIG. 9 shows a case where a polymer fraction of Arabidopsis thaliana juice 24 hours after exposure to each volatile component in Examples 13 to 15 and Comparative Example 2 was further heat-treated at 70 ° C. for 30 minutes. It is a schematic diagram which shows the result of having investigated the antibacterial activity with respect to B.cinerea conidia. (A) is exposed to (E) -2-hexenal, (b) is exposed to (Z) -3-hexenal, (c) is exposed to allo-ocimene, (d) is Each control is shown.

Claims (7)

A plant resistance inducer comprising allo-ocimene . Plant resistance inducing agent according to claim 1, characterized in that induces anti-fungal agent. The plant resistance inducer according to claim 2 , which is used as an antibacterial agent against a bacterium of the genus Botrytis. The plant resistance inducer according to any one of claims 1 to 3 , which is used for cruciferous plants. A plant resistance inducing agent according to any one of claims 1 to 4 , wherein the plant resistance inducing agent is exposed to a plant. A method for preventing plant disease and / or food damage, comprising using the method for inducing resistance according to claim 5 . Prevention of disease damage of plants according to claim 6, characterized in that to prevent diseases caused by infection with fungi Botrytis spp.

Images