JP5199177B2 - Plant virus inoculation method - Google Patents
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- JP5199177B2 JP5199177B2 JP2009114776A JP2009114776A JP5199177B2 JP 5199177 B2 JP5199177 B2 JP 5199177B2 JP 2009114776 A JP2009114776 A JP 2009114776A JP 2009114776 A JP2009114776 A JP 2009114776A JP 5199177 B2 JP5199177 B2 JP 5199177B2
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Description
本発明は、植物の苗に植物ウイルスを接種する方法に関するものである。 The present invention relates to a method for inoculating plant seedlings with a plant virus.
本発明者らは、これまでウイルスによる農作物や花卉への病害を防除するため、これら植物ウイルスの弱毒ウイルスや当該弱毒ウイルスを用いたウイルスによる病害を防除する方法を開発し開示した。 The present inventors have developed and disclosed a method for controlling diseases caused by attenuated viruses of these plant viruses and viruses using the attenuated viruses in order to control diseases caused by viruses on crops and flowers.
また、本発明者らは、弱毒ウイルスを農作物や花卉に、高い感染率で簡便に接種する方法として、これら植物体の表面に植物ウイルスと研磨剤を介在させて、ローラーを圧接回転させ、当該植物体を被傷すると共に植物ウイルスの接種を行う植物ウイルスの接種方法（特許文献１参照）や、セルトレイを用いて苗生産を行うトレイ育苗法において、植物体表面にブラシを当接して、当該植物体表面を被傷させる共に植物ウイルスの接種を行う、リンドウ等のロゼッタ状態で生育し、比較的外力等に対して丈夫な苗に適した、植物ウイルスの接種方法（特許文献２参照）を開発し開示した。 In addition, as a method of simply inoculating the attenuated virus to crops and flowers with a high infection rate, the present inventors interposed a plant virus and an abrasive on the surface of these plants, and rotated the rollers by pressure. In a plant virus inoculation method (see Patent Document 1) for injuring a plant body and inoculating a plant virus, or in a tray raising seedling method for producing seedlings using a cell tray, the surface of the plant body is brought into contact with the brush, A plant virus inoculation method (see Patent Document 2) suitable for seedlings that grow on a rosetta state such as gentian and injure the plant surface and inoculate with a plant virus and are relatively strong against external forces. Developed and disclosed.
また、一方、セル成型育苗トレイにて育苗された植物苗の葉の表皮に、弱毒ウイルスを含有する処理剤を噴霧する際に、当該植物苗の葉の下方位置において該植物苗を支持する支持手段を備えることを特徴とする植物苗の処理装置が開示されている（特許文献３参照）。 On the other hand, when spraying the treatment agent containing the attenuated virus onto the skin of the leaves of the plant seedlings grown in the cell-shaped seedling raising tray, support for supporting the plant seedlings in the lower position of the leaves of the plant seedlings An apparatus for treating plant seedlings characterized by comprising means is disclosed (see Patent Document 3).
また、植物ウイルスを分散した植物ウイルス液を植物体に接触させて行う植物ウイルスの接種方法において、接木を行う際に、穂木の胚軸の切断面又は台木の茎の切断面に植物ウイルスを接触させてから、これら切断面同士を密着させることを特徴とする植物ウイルスの接種方法、又は挿木を行う際に、挿し穂の胚軸の切断面に植物ウイルスを接触させてから、苗床又は培地に差し込むことを特徴とする植物ウイルスの接種方法が開示されている（特許文献４参照）。 Further, in the plant virus inoculation method in which a plant virus solution in which a plant virus is dispersed is brought into contact with the plant body, the plant virus is applied to the cut surface of the hypocotyl or the stem of the rootstock when grafting. The plant virus inoculation method characterized by bringing the cut surfaces into close contact with each other, or when cutting, when the plant virus is brought into contact with the cut surface of the hypocotyl of the cutting ear, A plant virus inoculation method characterized by being inserted into a medium is disclosed (see Patent Document 4).
弱毒ウイルス接種苗を大量生産する際に、本発明者らが開発した特許文献１の植物ウイルスの接種方法によって、セルトレイ上の苗に弱毒ウイルスと研磨剤を介在させてローラーを圧接回転し、これら苗を被傷すると共に弱毒ウイルスを接種すると、接種した苗の茎が折れ、さらに茎や葉の損傷も甚だしく、弱毒ウイルス接種苗生産の歩留まりを下げてしまうという問題があった。 When mass-producing seedlings inoculated with attenuated virus, the method of inoculating plant viruses of Patent Document 1 developed by the present inventors, the seedlings on the cell tray were pressed against the seedlings on the seedlings on the cell tray, and the roller was pressed and rotated. When the seedlings were injured and the attenuated virus was inoculated, the stems of the inoculated seedlings were broken, and the stems and leaves were severely damaged, resulting in a decrease in yield of attenuated virus inoculated seedling production.
そこで本発明者らは、上記課題を解決するため、弱毒ウイルスを接種する際に苗を圧接回転するローラー部分について、種々検討を重ねた結果、このローラー表面が柔らかい性状であると、接種によって苗の茎が折れることが激減し、その葉茎の損傷も大きく緩和されることを見出して本発明を完成した。 Therefore, in order to solve the above problems, the present inventors have conducted various studies on the roller portion that press-rotates the seedling when inoculating the attenuated virus. As a result, when the roller surface is soft, The present invention was completed by finding that the stalks of stalks were greatly reduced and the damage to the leaves and stems was greatly reduced.
即ち本発明は、植物体の表面に植物ウイルスと研磨材とを介在させて、柔らかい表面のローラーを圧接回転し、当該植物体を被傷すると共に当該植物ウイルスを接種することを特徴とする植物ウイルスの接種方法である。 That is, the present invention is a plant characterized in that a plant virus and an abrasive are interposed on the surface of a plant body, a roller having a soft surface is pressed and rotated, the plant body is damaged, and the plant virus is inoculated. Virus inoculation method.
本発明によれば、茎が折れたり葉茎が損傷することの少ない弱毒ウイルス接種苗を生産でき、そして、このような茎の折れや葉茎の損傷等の接種苗へのダメージが少ないため、当該接種苗の生育速度が速くなり、所定期間内での弱毒ウイルスの感染率も高い。したがって、簡便な方法で高品質の弱毒ウイルス接種苗を、短期間に高歩留まりで大量生産することができる。 According to the present invention, it is possible to produce attenuated virus-inoculated seedlings with less stem breakage or leaf stem damage, and less damage to the inoculated seedlings such as stem breakage and leaf stem damage, The growth rate of the inoculated seedling is increased, and the infection rate of the attenuated virus within a predetermined period is high. Therefore, high-quality attenuated virus-inoculated seedlings can be mass-produced with a high yield in a short time by a simple method.
本発明の植物ウイルスの接種法は、植物体に植物ウイルスを接種する方法であり、植物苗の表面に植物ウイルスと研磨材とを介在させて、表面の柔らかいローラーを圧接回転し、当該植物体を被傷すると共に植物ウイルスを接種する方法である。 The plant virus inoculation method of the present invention is a method of inoculating a plant body with a plant virus, wherein the plant body and a polishing material are interposed on the surface of a plant seedling, and a soft roller on the surface is pressed and rotated. And inoculating a plant virus.
本発明において、植物体上に弱毒ウイルスと研磨材を介在させる方法は、如何なる方法でもよく、例えば、植物ウイルス液と研磨材の混合液をスプレーで植物体に噴霧したり、植物ウイルス液を植物体にスプレーした後に研磨材を振り掛けたり、研磨材を植物体に振り掛けた後に植物ウイルス液をスプレーしたり、又は接着剤等で研磨材を表面に固定したローラーを、植物ウイルス液を植物体にスプレーする前、若しくはスプレーした後に用いること等が挙げられる。 In the present invention, any method may be used to intervene the attenuated virus and the abrasive on the plant body. For example, a mixed solution of the plant virus liquid and the abrasive is sprayed on the plant body, or the plant virus liquid is applied to the plant. Sprinkle the abrasive after spraying on the body, spray the plant virus liquid after sprinkling the abrasive on the plant body, or use a roller with the abrasive fixed on the surface with an adhesive etc. It may be used before spraying or after spraying.
また、上記植物ウイルス液は、植物ウイルスの感染葉の搾汁液を滅菌水若しくは中性付近の０．１Ｍリン酸緩衝液等で、当該感染葉の重量の５〜５０倍希釈して調製するか、又は通常の方法によって得られた純化ウイルス液を滅菌水若しくは適当な緩衝液で希釈して調製する。当該植物ウイルス液の濃度は、当該植物ウイルスによる病徴等の接種植物への作用が発現するものであるならば、如何なる濃度でもよいが、経済上の理由から、より低い濃度のものが好ましい。例えば、弱毒ウイルスの接種における接種液のウイルス濃度は２５〜５００μｇ／ｍｌである。 Also, is the plant virus solution prepared by diluting the squeezed solution of plant virus infected leaves with sterilized water or near neutral 0.1 M phosphate buffer, etc. 5 to 50 times the weight of the infected leaves? Alternatively, a purified virus solution obtained by a usual method is prepared by diluting with sterilized water or an appropriate buffer solution. The concentration of the plant virus solution may be any concentration as long as the effect on the inoculated plant such as disease symptoms by the plant virus is expressed, but a lower concentration is preferable for economic reasons. For example, the virus concentration of the inoculum in inoculation with attenuated virus is 25 to 500 μg / ml.
また、使用する研磨材は、植物を被傷して、その傷口からウイルスを侵入させて、当該ウイルスを感染するために用い、例えば、カーボランダム、セライト、ベントナイト、石英砂、海砂、セラミックパウダー、金剛砂及びガラス粉末等が挙げられ、電動スプレーを使用して弱毒ウイルス接種液を噴霧する場合は、そのノズルが詰まらない粒度５〜３５μのものを１〜３重量％添加する。 In addition, the abrasive used is used to infect a plant by injuring the virus through the wound and infecting the virus. For example, carborundum, celite, bentonite, quartz sand, sea sand, ceramic powder In the case of spraying the attenuated virus inoculum using an electric spray, 1 to 3% by weight of a particle having a particle size of 5 to 35 μm that does not clog the nozzle is added.
また、植物ウイルスを接種する植物の種類には、なんら制限はなく、如何なる種類の植物に接種することができるが、好ましくは育苗過程における植物に適し、さらに好ましくはトマトやピーマン、パプリカ等の傷付き易く折れ易い、外力等に非常に弱い苗に最適である。 In addition, there is no limitation on the type of plant inoculated with the plant virus, and any type of plant can be inoculated. However, it is preferably suitable for plants in the seedling raising process, more preferably wounds such as tomatoes, peppers and paprika. It is most suitable for seedlings that are easy to attach and break, and are very sensitive to external forces.
また、接種する植物ウイルスの種類にも、なんら制限はなく、如何なる種類の植物ウイルスを植物に接種することが可能であり、例えば、ウイルスによる植物の病徴や植物の耐病性等の試験研究にウイルスを植物に接種したり、また、ウイルス耐病性を有する植物苗の生産するため、作物及び花卉等に病徴をもたらす植物ウイルスの弱毒ウイルスを、野菜苗又は花卉苗等に接種したりすることができる。 In addition, there are no restrictions on the type of plant virus to be inoculated, and any type of plant virus can be inoculated into a plant. For example, for plant symptom by virus or disease resistance of plants. Inoculating a plant with an attenuated virus of a plant virus that causes symptoms of crops and flower buds in order to inoculate a plant with a virus or to produce a plant seedling with virus disease resistance Can do.
また、植物ウイルスを接種する接種器は、表面の柔らかいローラーを有するものであり、例えば、通常の表面の硬いローラー（図１）の表面に、エアーキャップ（登録商標）若しくはプチプチ（登録商標）又は飲料用紙パック、クラフト紙若しくはスポンジ等をシュレットしたものを袋詰めしたもの等の梱包用緩衝材やスポンジシート又は発泡ポリエチレンシート等のクッション性のあるものを貼り付けたローラーや、ポリエステル（繊維）、アクリル（繊維）若しくは発泡ウレタン等の柔らかい素材からなるローラーを有するものを使用することができる。図２にローラー表面にエアーキャップ（登録商標）を貼り付けたものを示す。 Moreover, the inoculator which inoculates a plant virus has a soft roller of a surface, for example, an air cap (registered trademark) or bubble wrap (registered trademark) or the surface of a normal hard roller (FIG. 1) or Rollers with cushioning materials such as beverage paper packs, kraft paper or sponge shredded bags, cushioning materials such as sponge sheets or foamed polyethylene sheets, polyester (fiber), A roller having a roller made of a soft material such as acrylic (fiber) or urethane foam can be used. FIG. 2 shows a roller surface with an air cap (registered trademark) attached thereto.
そして、当該植物ウイルス接種器は、回転軸方向を長くしたローラー（図３）の表面にエアーキャップ（登録商標）を貼り付けたもの（図４）によって、セルトレイに育苗した大量の苗に植物ウイルスを短時間で接種することができ、弱毒ウイルス接種苗を大量生産する際には、植物苗を育苗した複数枚のセルトレイを密着させて矩形に並べ、当該矩形の短辺に等しい長さのローラーを作成して、各セルトレイ上の大量の苗に弱毒ウイルスを大量接種することができる（図５）。 And the said plant virus inoculator is a plant virus to a large amount of seedlings grown on the cell tray by attaching an air cap (registered trademark) to the surface of a roller (FIG. 3) having a longer rotation axis direction (FIG. 3). When mass-producing attenuated virus-inoculated seedlings, a plurality of cell trays with plant seedlings are brought into close contact with each other and arranged in a rectangle, and a roller having a length equal to the short side of the rectangle Can be used to inoculate a large number of seedlings on each cell tray with a large amount of attenuated virus (FIG. 5).
（弱毒ウイルス接種器の作成）
片手で支持しながら転がして用いるローラーを（図１）を利用して、当該ローラー部分を４つ連結した、両手で支持しながら用いる従来法による弱毒ウイルス接種器を作成した（図３）。そして、当該接種器のローラー表面に気泡入り合成樹脂製包装用緩衝材のエアーキャップ（登録商標、宇部フィルム（株）製）を貼付して、本発明で用いるウイルス接種器を作成した（図４）。
(Making an attenuated virus inoculator)
Using a roller that is rolled while being supported with one hand (FIG. 1), an attenuated virus inoculator according to a conventional method that was used while being supported with both hands was created (FIG. 3). Then, an air cap (registered trademark, manufactured by Ube Film Co., Ltd.), a cushioning material made of a synthetic resin containing bubbles, was attached to the roller surface of the inoculator to create a virus inoculator used in the present invention (FIG. 4). ).
（トマト苗の育苗）
１２８穴セルトレイ（トレイサイズ：５９０mm×３００mm、セルの配列：８×１６、セルサイズ：３０mm角×高さ４０mm）３枚にトマト用培養土（日本デルモンテ（株）製）を充填して、トマト種子（品種：サマーキッス、日本デルモンテ（株）製）を１セルに１粒ずつ播種し、慣行の方法で育苗を行った。
(Tomato seedling raising)
Three 128-well cell trays (tray size: 590 mm x 300 mm, cell arrangement: 8 x 16, cell size: 30 mm square x height 40 mm) are filled with tomato culture soil (manufactured by Del Monte Japan) and tomato Seeds (variety: Summer Kiss, manufactured by Nippon Del Monte Co., Ltd.) were sown one cell at a time, and seedlings were raised by a conventional method.
（弱毒ウイルスの接種）
キュウリモザイクウイルスの弱毒ウイルスＮＤＭ−３（特許文献５参照）の純化液を５０ｍＭリン酸緩衝液（ｐＨ７．０）に１３５μｇ／ｍｌの濃度で希釈して、これに食紅を０．２％となるように、また、セライトを２％となるように添加して均一に混合し、弱毒ウイルス接種液を作成した。そして、育苗したトマト苗のセルトレイ３枚を密着して並べ、簡易ハンドスプレーでセルトレイ上のトマト苗に当該弱毒ウイルス接種液を、食紅による着色度合いを確認ながら少量均一に噴霧した。次いでウイルス接種器（図４）をトマト苗に押し付けるようにセルトレイの縦方向に転がして（図５）、トマト苗表面を被傷した後、さらに７日間育苗した。
(Inoculation of attenuated virus)
A purified solution of attenuated cucumber mosaic virus NDM-3 (see Patent Document 5) is diluted to a concentration of 135 μg / ml in 50 mM phosphate buffer (pH 7.0), resulting in 0.2% redness. In addition, Celite was added to 2% and mixed uniformly to prepare an attenuated virus inoculum. Then, three cell trays of the tomato seedlings that were raised were closely arranged and sprayed on the tomato seedlings on the cell tray with a simple hand spray, and the small amount of the attenuated virus inoculum was sprayed uniformly while confirming the degree of coloring by the food color. Next, the virus inoculator (FIG. 4) was rolled in the vertical direction of the cell tray so as to press against the tomato seedling (FIG. 5), and the surface of the tomato seedling was damaged, and then the seedling was further grown for 7 days.
（従来法による弱毒ウイルスの接種）
実施例と同様にセルトレイ３枚にトマト苗を育苗して、上記弱毒ウイルス接種液を均一に噴霧し、従来法による弱毒ウイルス接種器（図３）をトマト苗に押し付けるように転がしてトマト苗表面を被傷した後、さらに７日間育苗した。
(Inoculation of attenuated virus by conventional method)
Tomato seedlings are grown on three cell trays in the same manner as in the Examples, and the attenuated virus inoculum is sprayed uniformly, and the attenuated virus inoculator (FIG. 3) according to the conventional method is rolled to press against the tomato seedlings. After being injured, seedlings were further raised for 7 days.
（接種方法によるトマト苗の品質）
実施例と比較例とで弱毒ウイルスを接種したトマト苗の品質について調査した。調査項目としては、それぞれの接種法による苗への弱毒ウイルスの感染率をＥＬＩＳＡ法で調べた結果を表１に示し、茎が途中で折れた苗を茎折れ率として表２に示した。また、それぞれの接種法によるトマト苗が受けた損傷の程度を、その葉が無傷の苗（図６）、弱い損傷を受けた苗（図７）及び強い損傷を受けた苗（図８）に分類して表３に示した。
(Quality of tomato seedling by inoculation method)
The quality of tomato seedlings inoculated with the attenuated virus was examined in Examples and Comparative Examples. As the survey items, the results of examining the infection rate of the attenuated virus to the seedlings by each inoculation method by the ELISA method are shown in Table 1, and the seedlings whose stems broke in the middle are shown in Table 2 as the stem breaking rate. In addition, the degree of damage to the tomato seedlings by each inoculation method was determined for the seedlings whose leaves were intact (FIG. 6), weakly damaged seedlings (FIG. 7) and strong damaged seedlings (FIG. 8). The results are shown in Table 3.
表1の結果より、本発明の弱毒ウイルスの接種方法で接種したトマト苗群は、従来法で接種したものに比べ、弱毒ウイルスの感染率が非常に高く、また、表２の結果より、本発明によるトマト苗群は、接種による茎の折れが少ないものであった。 From the results in Table 1, the tomato seedling group inoculated by the method of inoculating the attenuated virus of the present invention has a much higher infection rate of the attenuated virus than that inoculated by the conventional method. The tomato seedling group according to the invention had few stem breakage due to inoculation.
また、表３の結果より、本発明によって接種したトマト苗群は、従来法で接種したものに比べ、無傷のトマト苗が大変多く、そして、強い損傷を受けた苗が大変少ないものであった。 Moreover, from the results in Table 3, the tomato seedling group inoculated according to the present invention had much more intact tomato seedlings and very few seedlings that were severely damaged than those inoculated by the conventional method. .
さらに本発明によって接種したトマト苗群は、従来法で接種したものに比べ、胚軸が太く子葉がしっかりした充実したものであり、生育も１日ほど早かった（図９）。 Furthermore, the tomato seedling group inoculated according to the present invention had a rich hypocotyl, a solid cotyledon, and a fast growth of about 1 day, compared to those inoculated by the conventional method (FIG. 9).
１ ウイルス接種器
２ 植物苗
３ セルトレイ
1 Virus inoculator 2 Plant seedling 3 Cell tray
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