JP2011155903A - Attenuated strain of melon yellow spot virus and method for producing the same, and method for preventing yellow spot disease in cucurbitaceous plant by inoculating the same - Google Patents
Attenuated strain of melon yellow spot virus and method for producing the same, and method for preventing yellow spot disease in cucurbitaceous plant by inoculating the same Download PDFInfo
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Abstract
Description
本発明は、メロン黄化えそウイルス弱毒株およびその製造方法、ならびに該弱毒株の接種によるウリ科植物における黄化えそ病の防除方法に関する。 The present invention relates to a melon yellow wilt virus attenuated strain, a method for producing the same, and a method for controlling yellow rot in cucurbitaceae plants by inoculation with the attenuated strain.
キュウリやメロンなどのウリ科植物の栽培において、メロン黄化えそウイルス（Melon yellow spot virus；以下、ＭＹＳＶという場合がある）の感染によって引き起こされる黄化えそ病の発生は、作物の収量や品質の低下を招き、生産者に深刻な打撃を与えている。我が国では、現在までに関東以西の各地で発生が確認されている。高知県では、キュウリ露地栽培の９０％以上、施設栽培の約８０％の圃場で黄化えそ病が発生しており、その被害が大きくなっている。 In the cultivation of Cucurbitaceae plants such as cucumbers and melons, the occurrence of yellow rot caused by infection with Melon yellow spot virus (hereinafter sometimes referred to as MYSV) is related to crop yield and The quality is degraded and the producers are severely hit. In Japan, outbreaks have been confirmed in various places west of Kanto. In Kochi Prefecture, yellow wilt has occurred in over 90% of cucumber field cultivation and about 80% of institutional cultivation, and the damage is increasing.
黄化えそ病の発生を防ぐためには、ＭＹＳＶの媒介虫であるミナミキイロアザミウマの防除が一般的に行われている。殺虫剤の散布や防虫ネットなどによる侵入防止対策が行われているものの、ミナミキイロアザミウマは殺虫剤に対する感受性の低下が著しく、殺虫剤を散布しても十分な防除効果が得られない場合が多い。また、ミナミキイロアザミウマは微小な昆虫であるので、防虫ネットを利用して侵入防止対策を施したとしても、多くの場合、防虫ネットの隙間などから侵入してＭＹＳＶを媒介する。 In order to prevent the occurrence of yellow spot rot, the control of Southern Thrips thrips that is a vector of MYSV is generally performed. Although invasion prevention measures such as spraying of insecticides and insect nets are being carried out, southern thrips have a marked decrease in sensitivity to insecticides, and even when sprayed with insecticides, sufficient control effects are often not obtained. . In addition, since Southern Thrips thrips are minute insects, even if an anti-intrusion measure is taken using an insect repellent net, in many cases, it enters through the gap of the insect repellent net and mediates MYSV.
本発明者らは、上記事情に鑑みて、ウリ科植物に予防的に接種することによって黄化えそ病に対する抵抗性を付与し、黄化えそ病の発生を抑制するＭＹＳＶ弱毒株を開発するべく、鋭意研究を行ってきた。ＭＹＳＶ強毒株を感染させたタバコ属植物を高温処理し、接種葉からＭＹＳＶを回収、選抜したところ、強毒株に対して干渉効果を有するＭＹＳＶ弱毒株を取得し、本発明を完成させた。 In view of the above circumstances, the present inventors have developed a MYSV attenuated strain that imparts resistance to yellow wilt disease by prophylactic inoculation of cucurbitaceae plants and suppresses yellow wilt disease occurrence. In order to do so, I have been conducting intensive research. Tobacco plants infected with MYSV highly toxic strains were treated at high temperature, and MYSV was collected and selected from the inoculated leaves. As a result, MYSV attenuated strains having an interference effect on toxic strains were obtained, and the present invention was completed. .
すなわち、本発明は、下記に関するものである：
［１］以下の工程を含む、メロン黄化えそウイルス（ＭＹＳＶ）弱毒株の製造方法：
（ａ）ＭＹＳＶを自然界での宿主植物以外の植物の葉に接種する工程、
（ｂ）ＭＹＳＶを接種した植物を高温処理する工程、および
（ｃ）接種葉からＭＹＳＶ弱毒株を選抜、回収する工程；
［２］自然界での宿主植物以外の植物がニコチアナ グルチノサである、［１］記載の製造方法；
［３］［１］または［２］記載の製造方法によって得ることができるＭＹＳＶ弱毒株；
［４］配列番号１〜３で示すアミノ酸配列をそれぞれコードするＲＮＡまたはその相補鎖を有する、［３］記載のＭＹＳＶ弱毒株；
［５］配列番号１〜３で示すアミノ酸配列をそれぞれコードするＲＮＡまたはその相補鎖の１つまたは複数が、
（ａ）配列番号１〜３で示すアミノ酸配列において、１または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列をコードするＲＮＡまたはその相補鎖；
（ｂ）配列番号１〜３で示すアミノ酸配列または（ａ）のアミノ酸配列をコードするＲＮＡまたはその相補鎖と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし得るＤＮＡと相補的な塩基配列を有するＲＮＡまたはその相補鎖；および
（ｃ）配列番号１〜３で示すアミノ酸配列と８０％以上の同一性を有するアミノ酸配列をコードするＲＮＡまたはその相補鎖
からなる群より選択されるＲＮＡまたはその相補鎖である、［４］記載のＭＹＳＶ弱毒株；
［６］ＲｄＲｐのＮ末端から１８９番目のアミノ酸がイソロイシン、２５５番目のアミノ酸がアラニン、１６４０番目のアミノ酸がグリシン、１６９２番目のアミノ酸がバリン、２０１５番目のアミノ酸がプロリンであり、ＧＮ／ＧＣタンパク質のＮ末端から８６番目のアミノ酸がグルタミン酸、１６３番目のアミノ酸がプロリン、３３６番目のアミノ酸がバリン、３７１番目のアミノ酸がフェニルアラニン、６２１番目のアミノ酸がリシン、９３５番目のアミノ酸がロイシンであり、ＮＳｓタンパク質のＮ末端から１５番目のアミノ酸がフェニルアラニンである、［５］記載のＭＹＳＶ弱毒株；
［７］配列番号６〜８で示す塩基配列を含むＲＮＡまたはその相補鎖を有する、［３］記載のＭＹＳＶ弱毒株；
［８］配列番号６〜８で示す塩基配列を含むＲＮＡまたはその相補鎖の１つまたは複数が、
（ａ）配列番号６〜８で示す塩基配列において、１または数個の塩基が欠失、置換または付加された塩基配列を含むＲＮＡまたはその相補鎖；
（ｂ）配列番号６〜８で示す塩基配列または（ａ）の塩基配列を含むＲＮＡまたはその相補鎖と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし得るＤＮＡと相補的な塩基配列を有するＲＮＡまたはその相補鎖；および
（ｃ）配列番号６〜８で示す塩基配列と６０％以上の同一性を有する塩基配列を含むＲＮＡまたはその相補鎖
からなる群より選択されるＲＮＡまたはその相補鎖である、［７］記載のＭＹＳＶ弱毒株；
［９］前記ＲＮＡまたはその相補鎖の塩基配列が、ＲｄＲｐのＮ末端から１８９番目のアミノ酸がイソロイシン、２５５番目のアミノ酸がアラニン、１６４０番目のアミノ酸がグリシン、１６９２番目のアミノ酸がバリン、２０１５番目のアミノ酸がプロリンであり、ＧＮ／ＧＣタンパク質のＮ末端から８６番目のアミノ酸がグルタミン酸、１６３番目のアミノ酸がプロリン、３３６番目のアミノ酸がバリン、３７１番目のアミノ酸がフェニルアラニン、６２１番目のアミノ酸がリシン、９３５番目のアミノ酸がロイシンであり、ＮＳｓタンパク質のＮ末端から１５番目のアミノ酸がフェニルアラニンとなるような塩基配列である、［８］記載のＭＹＳＶ弱毒株；
［１０］ＳＡ０８−８である、［３］記載のＭＹＳＶ弱毒株；
［１１］［３］〜［１０］のいずれかに記載のＭＹＳＶ弱毒株をウリ科植物に接種することを特徴とする、ウリ科植物における黄化えそ病の防除方法；
［１２］ウリ科植物がキュウリ、メロン、スイカ、シロウリ、トウガン、ユウガオまたはニガウリから選択される、［１１］記載の防除方法；
［１３］［３］〜［１０］のいずれかに記載のＭＹＳＶ弱毒株を接種することによって得ることができる、黄化えそ病抵抗性のウリ科植物；ならびに
［１４］キュウリ、メロン、スイカ、シロウリ、トウガン、ユウガオまたはニガウリから選択される、［１３］記載のウリ科植物。
That is, the present invention relates to the following:
[1] A method for producing a melon yellow spot virus (MYSV) attenuated strain comprising the following steps:
(A) inoculating MYSV into leaves of plants other than host plants in nature;
(B) a step of high-temperature treatment of the plant inoculated with MYSV, and (c) a step of selecting and recovering a MYSV attenuated strain from the inoculated leaves;
[2] The production method according to [1], wherein the plant other than the host plant in nature is Nicotiana glutinosa;
[3] MYSV attenuated strain obtainable by the production method according to [1] or [2];
[4] The MYSV attenuated strain according to [3], which has an RNA encoding the amino acid sequence represented by SEQ ID NOs: 1 to 3 or a complementary strand thereof;
[5] One or more of RNAs encoding the amino acid sequences represented by SEQ ID NOs: 1 to 3 or their complementary strands,
(A) RNA encoding the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NOs: 1 to 3 or a complementary strand thereof;
(B) Complementary to DNA that can hybridize under stringent conditions with DNA having the amino acid sequence shown in SEQ ID NOs: 1 to 3 or RNA having the nucleotide sequence complementary to RNA encoding the amino acid sequence of (a) or its complementary strand (C) selected from the group consisting of RNA encoding an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NOs: 1 to 3 or a complementary strand thereof. The MYSV attenuated strain according to [4], which is RNA or a complementary strand thereof;
[6] RdRp N-terminal 189 amino acids are isoleucine, 255th amino acid alanine, 1640 amino acid is glycine, 1692 th amino acids valine, a 2015 amino acid is proline, G N / G C The 86th amino acid from the N-terminal of the protein is glutamic acid, the 163rd amino acid is proline, the 336th amino acid is valine, the 371st amino acid is phenylalanine, the 621st amino acid is lysine, the 935th amino acid is leucine, NSs MYSV attenuated strain of [5], wherein the 15th amino acid from the N-terminal of the protein is phenylalanine;
[7] The MYSV attenuated strain according to [3], which has an RNA comprising the nucleotide sequence represented by SEQ ID NOs: 6 to 8 or a complementary strand thereof;
[8] One or more of RNAs containing the base sequences represented by SEQ ID NOs: 6 to 8 or complementary strands thereof are
(A) RNA or a complementary strand thereof comprising a base sequence in which one or several bases have been deleted, substituted or added in the base sequences shown in SEQ ID NOs: 6 to 8;
(B) Complementary to DNA that can hybridize under stringent conditions with DNA having the base sequence shown in SEQ ID NOs: 6 to 8 or RNA having the base sequence of (a) or a complementary base sequence thereof. (C) an RNA comprising a base sequence having 60% or more identity with the base sequence shown in SEQ ID NOs: 6 to 8, or an RNA selected from the group consisting of the complementary strand; Or the MYSV attenuated strain of [7], which is a complementary strand thereof;
[9] The base sequence of the RNA or its complementary strand is such that the 189th amino acid from the N-terminus of RdRp is isoleucine, the 255th amino acid is alanine, the 1640th amino acid is glycine, the 1692nd amino acid is valine, the 2015th amino acid amino acid is proline, G N / G C 86 amino acids from the N-terminus of the protein glutamate, 163 amino acid proline, 336 th amino acid valine, 371 th amino acid phenylalanine, 621 amino acid lysine MYSV attenuated strain according to [8], wherein the 935th amino acid is leucine and the 15th amino acid from the N-terminal of the NSs protein is phenylalanine;
[10] The MYSV attenuated strain of [3], which is SA08-8;
[11] A method for controlling yellow spot rot in cucurbitaceae plants, which comprises inoculating the cucurbitaceae plants with the MYSV attenuated strain according to any one of [3] to [10];
[12] The pest control method according to [11], wherein the cucurbitaceae plant is selected from cucumber, melon, watermelon, shirori, togan, yugao or bitter gourd;
[13] Yellow wilt resistant Cucurbitaceae plant obtained by inoculating the MYSV attenuated strain according to any one of [3] to [10]; and [14] Cucumber, melon, watermelon The cucurbitaceae plant according to [13], which is selected from Shirouri, Togan, Yugao or Nigari.
本発明により、ＭＹＳＶ強毒株に対して干渉効果を有するＭＹＳＶ弱毒株を提供することができる。また、本発明のＭＹＳＶ弱毒株をウリ科植物に接種することによって、ウリ科植物に黄化えそ病に対する抵抗性を付与することができ、媒介虫の防除といった間接的な手段ではない、直接的な手段による黄化えそ病の防除が可能となる。 According to the present invention, a MYSV attenuated strain having an interference effect with respect to a MYSV highly toxic strain can be provided. In addition, by inoculating a cucurbitaceae plant with the MYSV attenuated strain of the present invention, the cucurbitaceae plant can be imparted with resistance to yellow rot, and it is not an indirect means such as control of a vector worm. It is possible to control yellow wilt disease by a conventional means.
本発明は、１つの態様において、メロン黄化えそウイルス（ＭＹＳＶ）弱毒株の製造方法に関するものである。本発明のＭＹＳＶ弱毒株の製造方法は、
（ａ）ＭＹＳＶを自然界での宿主植物以外の植物の葉に接種する工程、
（ｂ）ＭＹＳＶを接種した植物を高温処理する工程、および
（ｃ）接種葉からＭＹＳＶ弱毒株を選抜、回収する工程
を含んでもよい。本発明において、ＭＹＳＶは、ブニヤウイルス科に属するトスポウイルス（tospovirus）属に分類されるものであって、ミナミキイロアザミウマなどのアザミウマ類によって媒介されるものをいう。本明細書において、宿主植物に感染することによってＭＹＳＶによる病害、例えば、子葉にてえそ斑点、本葉にて葉脈透過、葉脈えそ、モザイク、果実にてモザイク、奇形などの病徴を生じる黄化えそ病などを引き起こすものをＭＹＳＶ強毒株といい、宿主植物に感染しても病害を引き起こさないか、または病害が抑制されているものをＭＹＳＶ弱毒株という。また、本発明により製造されたＭＹＳＶ弱毒株は、ＭＹＳＶ強毒株に対して干渉効果を有する。本発明によれば、宿主植物に感染することによって黄化えそ病などの病害を引き起こし得るＭＹＳＶ強毒株などのＭＹＳＶからＭＹＳＶ弱毒株を製造することができる。
In one aspect, the present invention relates to a method for producing a melon yellow spot virus (MYSV) attenuated strain. The method for producing the MYSV attenuated strain of the present invention comprises:
(A) inoculating MYSV into leaves of plants other than host plants in nature;
(B) A step of high-temperature treatment of the plant inoculated with MYSV, and (c) a step of selecting and collecting a MYSV attenuated strain from the inoculated leaf. In the present invention, MYSV is classified into the genus tospovirus belonging to the Bunyaviridae family and is mediated by thrips such as the southern thrips. In this specification, infection with a host plant causes diseases caused by MYSV, such as necrotic spots in cotyledons, vein penetration in true leaves, leaf veins, mosaic, fruit, mosaic, malformation, etc. Those that cause yellow wilt or the like are called MYSV highly virulent strains, and those that do not cause disease even when infected with a host plant or whose diseases are suppressed are called MYSV attenuated strains. Moreover, the MYSV attenuated strain produced according to the present invention has an interference effect on the MYSV highly virulent strain. According to the present invention, a MYSV attenuated strain can be produced from MYSV such as a MYSV highly toxic strain that can cause diseases such as yellow wilt by infecting a host plant.
本発明において、ＭＹＳＶ弱毒株を製造するためにＭＹＳＶを接種する植物は、ＭＹＳＶの自然界での宿主植物以外の植物であり、その種類は特に限定されるものではない。例示すれば、キュウリ、メロン、スイカ、シロウリ、ニガウリなどのウリ科植物以外の植物が挙げられ、例えば、ニコチアナ グルチノサ（Nicotiana glutinosa）などのタバコ属植物であってもよい。また、ＭＹＳＶの接種は、自然界での宿主植物以外の植物のいずれの部位に対して行ってもよく、例えば、葉に対して接種してもよい。また、上記植物へのＭＹＳＶの接種は、当該分野で通常用いられる手段を用いて接種してもよく、例えば、カーボンランダム法などを用いて汁液接種してもよい。 In the present invention, a plant inoculated with MYSV to produce a MYSV attenuated strain is a plant other than the host plant in the natural world of MYSV, and the type thereof is not particularly limited. For example, plants other than the Cucurbitaceae plants such as cucumber, melon, watermelon, white wax, and bitter gourd are mentioned, and for example, tobacco plants such as Nicotiana glutinosa may be used. Moreover, inoculation of MYSV may be performed on any part of plants other than host plants in nature, for example, on leaves. Moreover, inoculation of the said plant to MYSV may be inoculated using the means normally used in the said field | area, for example, you may inoculate juice using the carbon random method etc.
ＭＹＳＶを接種した上記植物は、次いで、高温処理に供される。高温処理は、当該分野において病原性ウイルスから当該ウイルスの弱毒株を作出する場合に通常使用され得る条件下で行ってもよく、例えば、３０〜３８℃で５〜２０日間、好ましくは３２〜３６℃で１０〜１８日間、より好ましくは３４℃で１４日間栽培してもよい。 The plant inoculated with MYSV is then subjected to high temperature treatment. The high-temperature treatment may be performed under conditions that can be normally used when an attenuated strain of the virus is produced from a pathogenic virus in the art, for example, at 30 to 38 ° C. for 5 to 20 days, preferably 32-36. You may grow at 10 degreeC for 10 to 18 days, More preferably at 34 degreeC for 14 days.
通常、タバコモザイクウイルス（ＴＭＶ）をニコチアナ グルチノサに接種すると、植物の抵抗性反応によって感染は接種部分のみに留まり、局部病斑を形成する。しかし、ＴＭＶを接種したニコチアナ グルチノサに高温処理を行うと抵抗性反応が発揮されず、全身に感染が広がることが知られている（Oguni et al. 1983. Induction of attenuated strain of tobacco mosaic virus by serial passage through Nicotiana glutinosa plants alternately infected with the virus systemically at 30-38C and locally at 25C）。しかしながら、ＭＹＳＶをニコチアナ グルチノサの葉に接種し、高温処理に供したところ、ＭＹＳＶの感染は全身に広がらず、接種葉に局部病斑を形成するのみであることを本発明者らは確認した。本発明において、高温処理に供した上記植物のいずれの部位からＭＹＳＶ弱毒株を選抜、回収してもよいが、好ましくは、この接種葉からＭＹＳＶ弱毒株を選抜、回収する。ＭＹＳＶ弱毒株の選抜、回収は、当該分野にて通常用いられる方法に従ってもよく、例えば、ニコチアナ グルチノサの接種葉から回収したウイルスを、局部病斑を形成することが知られているセンニチコウに接種することによって、ウイルス株ごとに単離してもよい。さらに、センニチコウの局部病斑部分からウイルスを回収し、キュウリなどの宿主植物に接種し、ＭＹＳＶの感染が確認された宿主植物について、黄化えそ病の病徴の有無またはその程度を肉眼で確認し、ＭＹＳＶ弱毒株の選抜を行ってもよい。ＭＹＳＶ感染の有無は、当該分野において通常用いられる手段に従って確認すればよく、例えば、ＭＹＳＶに対する血清を用いたＥＬＩＳＡなどによって確認してもよい。また、ＭＹＳＶ弱毒株の選抜・回収する工程は、１回行うだけでもよいが、ＭＹＳＶによる病害を全く示さないか、または病害がより抑制された所望のＭＹＳＶ株が得られるまで適宜繰り返してもよい。 Normally, when Nicotiana glutinosa is inoculated with tobacco mosaic virus (TMV), the infection remains only in the inoculated part due to the resistance reaction of the plant, forming a local lesion. However, it is known that the high temperature treatment of TMV-inoculated Nicotiana glutinosa does not exert a resistance reaction and spreads the infection throughout the body (Oguni et al. 1983. Induction of attenuated strain of tobacco mosaic virus by serial passage through Nicotiana glutinosa plants alternately infected with the virus systemically at 30-38C and locally at 25C). However, when the MYSV was inoculated into Nicotiana glutinosa leaves and subjected to high-temperature treatment, the present inventors confirmed that the infection of MYSV did not spread throughout the body, but only formed local lesions on the inoculated leaves. In the present invention, the MYSV attenuated strain may be selected and recovered from any part of the plant subjected to the high temperature treatment, but preferably, the MYSV attenuated strain is selected and recovered from this inoculated leaf. The selection and recovery of MYSV attenuated strains may be in accordance with methods commonly used in the art. For example, a virus recovered from the inoculated leaves of Nicotiana glutinosa is inoculated into Sennicitikous known to form local lesions. Depending on the situation, each virus strain may be isolated. In addition, the virus was collected from the local lesions of P. nigrass and inoculated into a host plant such as cucumber. The MYSV attenuated strain may be selected after confirmation. The presence or absence of MYSV infection may be confirmed according to means commonly used in the art, and may be confirmed by, for example, ELISA using serum against MYSV. Further, the process of selecting and recovering the MYSV attenuated strain may be performed only once, but may be repeated as appropriate until a desired MYSV strain that exhibits no disease due to MYSV or has a further suppressed disease is obtained. .
また、本発明は、別の態様において、本発明の製造方法によって得ることができるＭＹＳＶ弱毒株に関する。本発明のＭＹＳＶ弱毒株は、宿主植物に感染してもＭＹＳＶによる病害、例えば、子葉でのえそ斑点、本葉での葉脈透過、葉脈えそ、モザイク、もしくは果実でのモザイク、奇形などの黄化えそ病に特有の病徴を引き起こさないか、または病害が抑制されているので、本発明のＭＹＳＶ弱毒株をウリ科植物などの植物に接種した場合、植物の収量および品質に重大な影響を及ぼさない。一方で、本発明のＭＹＳＶ弱毒株はＭＹＳＶ強毒株に対して干渉効果を有するので、本発明のＭＹＳＶ弱毒株は、接種した植物にＭＹＳＶによる病害に対する抵抗性を付与することができる。 Moreover, this invention relates to the MYSV attenuated strain which can be obtained with the manufacturing method of this invention in another aspect. The MYSV attenuated strain of the present invention is a disease caused by MYSV even if it infects a host plant, such as variegation spots in cotyledons, leaf vein penetration in true leaves, leaf veins, mosaics, or mosaics in fruits, malformations, etc. Since the symptom peculiar to yellow wilt is not caused or the disease is suppressed, when the MYSV attenuated strain of the present invention is inoculated into a plant such as a cucurbitaceae plant, it is important for the yield and quality of the plant. Has no effect. On the other hand, since the MYSV attenuated strain of the present invention has an interference effect on the MYSV highly virulent strain, the MYSV attenuated strain of the present invention can impart resistance to diseases caused by MYSV to the inoculated plant.
また、本発明のＭＹＳＶ弱毒株は、配列番号１〜３で示すアミノ酸配列をそれぞれコードするＲＮＡまたはその相補鎖を有するものでもよい。あるいは、本発明のＭＹＳＶ弱毒株は、配列番号１〜３で示すアミノ酸配列をそれぞれコードするＲＮＡまたはその相補鎖の１つまたは複数が、（ａ）配列番号１〜３で示すアミノ酸配列において、１または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列をコードするＲＮＡまたはその相補鎖、（ｂ）配列番号１〜３で示すアミノ酸配列または（ａ）のアミノ酸配列をコードするＲＮＡまたはその相補鎖と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし得るＤＮＡと相補的な塩基配列を有するＲＮＡまたはその相補鎖、および（ｃ）配列番号１〜３で示すアミノ酸配列と８０％以上の同一性を有するアミノ酸配列をコードするＲＮＡまたはその相補鎖からなる群より選択されるＲＮＡまたはその相補鎖である、上記ＭＹＳＶ弱毒株でもよい。
詳細には、本発明のＭＹＳＶ弱毒株は、配列番号１〜３で示すアミノ酸配列をそれぞれコードするＲＮＡまたはその相補鎖を有するものであるか、あるいは、配列番号１〜３で示すアミノ酸配列をそれぞれコードするＲＮＡまたはその相補鎖の１つまたは複数（例えば、２もしくは３）が、（ａ）配列番号１〜３で示すアミノ酸配列において、１または数個（例えば、２、３、４、５、６、７、８もしくは９個）のアミノ酸が欠失、置換または付加されたアミノ酸配列をコードするＲＮＡまたはその相補鎖；（ｂ）配列番号１〜３で示すアミノ酸配列または（ａ）のアミノ酸配列をコードするＲＮＡまたはその相補鎖と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし得るＤＮＡと相補的な塩基配列を有するＲＮＡまたはその相補鎖；および、（ｃ）配列番号１〜３で示すアミノ酸配列と８０％以上（好ましくは、９０％、９３％、９５％、９６％、９７％、９８％、９９％または９９．５％）の同一性を有するアミノ酸配列をコードするＲＮＡまたはその相補鎖からなる群より選択されるＲＮＡまたはその相補鎖である、上記ＭＹＳＶ弱毒株でもよい。なお、上記ＭＹＳＶ弱毒株において、ＲＮＡ依存性ＲＮＡポリメラーゼ（ＲｄＲｐ）のＮ末端から１８９番目のアミノ酸はイソロイシン、２５５番目のアミノ酸はアラニン、１６４０番目のアミノ酸はグリシン、１６９２番目のアミノ酸はバリン、２０１５番目のアミノ酸はプロリンであり、ＧＮ／ＧＣタンパク質のＮ末端から８６番目のアミノ酸はグルタミン酸、１６３番目のアミノ酸はプロリン、３３６番目のアミノ酸はバリン、３７１番目のアミノ酸はフェニルアラニン、６２１番目のアミノ酸はリシン、９３５番目のアミノ酸はロイシンであり、ＮＳｓタンパク質のＮ末端から１５番目のアミノ酸はフェニルアラニンであってもよい。
Moreover, the MYSV attenuated strain of the present invention may have an RNA encoding the amino acid sequence shown in SEQ ID NOs: 1 to 3 or a complementary chain thereof. Alternatively, in the MYSV attenuated strain of the present invention, one or a plurality of RNAs encoding the amino acid sequences represented by SEQ ID NOs: 1 to 3 or their complementary strands are (a) Or an RNA encoding an amino acid sequence in which several amino acids are deleted, substituted or added, or a complementary strand thereof, (b) an amino acid sequence represented by SEQ ID NOs: 1 to 3 or an RNA encoding an amino acid sequence of (a) RNA having a base sequence complementary to DNA capable of hybridizing under stringent conditions with a DNA having a base sequence complementary to the complementary strand, or a complementary strand thereof, and (c) the amino acid sequence represented by SEQ ID NOs: 1 to 3 Or an RNA selected from the group consisting of an RNA encoding an amino acid sequence having at least 80% identity or its complementary strand, or A complementary strand, or above MYSV attenuated strain.
Specifically, the MYSV attenuated strain of the present invention has an RNA encoding the amino acid sequence represented by SEQ ID NO: 1 to 3 or a complementary strand thereof, or the amino acid sequence represented by SEQ ID NO: 1 to 3, respectively. One or more (eg, 2 or 3) of the encoding RNA or its complementary strand is (a) one or several (eg, 2, 3, 4, 5, An RNA encoding an amino acid sequence in which 6, 7, 8 or 9 amino acids have been deleted, substituted or added, or a complementary chain thereof; (b) the amino acid sequence shown in SEQ ID NOs: 1 to 3 or the amino acid sequence of (a) Having a base sequence complementary to DNA that can hybridize under stringent conditions to DNA having a base sequence complementary to RNA encoding DNA or its complementary strand NA or its complementary strand; and (c) 80% or more (preferably 90%, 93%, 95%, 96%, 97%, 98%, 99% or 99) with the amino acid sequence shown in SEQ ID NOs: 1 to 3 The MYSV attenuated strain may be an RNA selected from the group consisting of an RNA encoding an amino acid sequence having 5% identity or a complementary strand thereof, or a complementary strand thereof. In the MYSV attenuated strain, the 189th amino acid from the N-terminus of RNA-dependent RNA polymerase (RdRp) is isoleucine, the 255th amino acid is alanine, the 1640th amino acid is glycine, the 1692nd amino acid is valine, and the 2015th position. amino acid is proline, 86 th amino acid from the N-terminus of G N / G C protein glutamic acid, 163 amino acid proline, 336 th amino acids valine, 371 th amino acid phenylalanine, 621 th amino acids Lysine, the 935th amino acid may be leucine, and the 15th amino acid from the N-terminal of the NSs protein may be phenylalanine.
また、本発明のＭＹＳＶ弱毒株は、配列番号６〜８で示す塩基配列含むＲＮＡまたはその相補鎖を有するものであってもよい。あるいは、本発明のＭＹＳＶ弱毒株は、配列番号６〜８で示す塩基配列を含むＲＮＡまたはその相補鎖の１つまたは複数が、（ａ）配列番号６〜８で示す塩基配列において、１または数個の塩基が欠失、置換または付加された塩基配列を含むＲＮＡまたはその相補鎖、（ｂ）配列番号６〜８で示す塩基配列または（ａ）の塩基配列を含むＲＮＡまたはその相補鎖と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし得るＤＮＡと相補的な塩基配列を有するＲＮＡまたはその相補鎖、および（ｃ）配列番号６〜８で示す塩基配列と６０％以上の同一性を有する塩基配列を含むＲＮＡまたはその相補鎖からなる群より選択されるＲＮＡまたはその相補鎖である、上記ＭＹＳＶ弱毒株でもよい。
詳細には、本発明のＭＹＳＶ弱毒株は、配列番号６〜８で示す塩基配列含むＲＮＡまたはその相補鎖を有するものであるか、あるいは、配列番号６〜８で示す塩基配列を含むＲＮＡまたはその相補鎖の１つまたは複数（例えば、２もしくは３）が、（ａ）配列番号６〜８で示す塩基配列において、１または数個（例えば、６、９、１２、１５、１８、２１、２４もしくは２７個）の塩基が欠失、置換または付加された塩基配列を含むＲＮＡまたはその相補鎖、（ｂ）配列番号６〜８で示す塩基配列または（ａ）の塩基配列を含むＲＮＡまたはその相補鎖と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし得るＤＮＡと相補的な塩基配列を有するＲＮＡまたはその相補鎖、および（ｃ）配列番号６〜８で示す塩基配列と６０％以上（好ましくは、７０％、８０％、９０％、９３％、９５％、９６％、９７％、９８％、９９％または９９．５％）の同一性を有する塩基配列を含むＲＮＡまたはその相補鎖からなる群より選択されるＲＮＡまたはその相補鎖である、上記ＭＹＳＶ弱毒株でもよい。なお、上記ＭＹＳＶ弱毒株において、前記ＲＮＡまたはその相補鎖の塩基配列は、ＲｄＲｐのＮ末端から１８９番目のアミノ酸がイソロイシン、２５５番目のアミノ酸がアラニン、１６４０番目のアミノ酸がグリシン、１６９２番目のアミノ酸がバリン、２０１５番目のアミノ酸がプロリンであり、ＧＮ／ＧＣタンパク質のＮ末端から８６番目のアミノ酸がグルタミン酸、１６３番目のアミノ酸がプロリン、３３６番目のアミノ酸がバリン、３７１番目のアミノ酸がフェニルアラニン、６２１番目のアミノ酸がリシン、９３５番目のアミノ酸がロイシンであり、ＮＳｓタンパク質のＮ末端から１５番目のアミノ酸がフェニルアラニンとなるような塩基配列であってもよい。
Moreover, the MYSV attenuated strain of the present invention may have an RNA containing the nucleotide sequence represented by SEQ ID NOs: 6 to 8 or a complementary strand thereof. Alternatively, in the MYSV attenuated strain of the present invention, one or more of RNAs containing the nucleotide sequences represented by SEQ ID NOs: 6 to 8 or complementary strands thereof are (a) one or more of the nucleotide sequences represented by SEQ ID NOs: 6 to Complementary RNA or its complementary strand containing a base sequence in which one base has been deleted, substituted or added, (b) complementary to RNA shown in SEQ ID NO: 6-8 or RNA containing (a) the base sequence or its complementary strand RNA having a complementary base sequence to DNA capable of hybridizing under stringent conditions with a DNA having a basic base sequence or a complementary strand thereof, and (c) 60% or more of the base sequence shown in SEQ ID NOs: 6-8 The MYSV attenuated strain may be an RNA selected from the group consisting of an RNA comprising a nucleotide sequence having the same identity or a complementary strand thereof, or an complementary strand thereof.
Specifically, the MYSV attenuated strain of the present invention has an RNA comprising the nucleotide sequence represented by SEQ ID NO: 6-8 or a complementary strand thereof, or an RNA comprising the nucleotide sequence represented by SEQ ID NO: 6-8 or One or a plurality of complementary strands (for example, 2 or 3) are (a) one or several (for example, 6, 9, 12, 15, 18, 21, 24) in the base sequences represented by SEQ ID NOs: 6 to 8. Or an RNA comprising a nucleotide sequence in which 27 bases have been deleted, substituted or added, or a complementary strand thereof, (b) a nucleotide sequence represented by SEQ ID NOs: 6 to 8, or an RNA comprising a nucleotide sequence of (a) or a complement thereof RNA having a base sequence complementary to DNA that can hybridize under stringent conditions with DNA having a base sequence complementary to the strand, or a complementary strand thereof, and (c) a salt represented by SEQ ID NOs: 6 to 8 A nucleotide sequence having 60% or more (preferably 70%, 80%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 99.5%) identity with the sequence The MYSV attenuated strain may be an RNA selected from the group consisting of RNA or a complementary strand thereof or a complementary strand thereof. In the MYSV attenuated strain, the base sequence of the RNA or its complementary strand is that the 189th amino acid from the N-terminus of RdRp is isoleucine, the 255th amino acid is alanine, the 1640th amino acid is glycine, and the 1692th amino acid is valine, a 2015 amino acid is proline, G N / G C 86 amino acids from the N-terminus of the protein glutamate, 163 amino acid proline, 336 th amino acid valine, 371 th amino acid phenylalanine, 621 The nucleotide sequence may be such that the first amino acid is lysine, the 935th amino acid is leucine, and the 15th amino acid from the N-terminal of the NSs protein is phenylalanine.
上記ストリンジェントな条件とは、ハイブリダイゼーションおよび／またはハイブリダイゼーション後の洗浄に用いる溶液の組成、温度などによって決定され得る条件であって、当業者によって適宜設定され得る。例えば、J. Sambrook et al.,“Molecular Cloning: A Laboratory Manual, Second Edition”, 1989, Cold Spring Harbor Laboratory Pressに記載されるような条件が挙げられる。例えば、６×ＳＳＣ、５×デンハルト溶液（Denhardt's solution）、０．５％ ＳＤＳ、１００μｇ／ｍｌ変性サケ精子ＤＮＡを含む溶液中、５０℃、より好ましくは６８℃にてプローブとハイブリダイズさせた後で、２×ＳＳＣ、０．１％ ＳＤＳの溶液中で室温にて洗浄してもよいが、ストリンジェンシーを増大させるために、好ましくは０．１×ＳＳＣ、０．５％ＳＤＳの溶液中で６８℃にて洗浄してもよい。あるいは、ハイブリダイズさせた後で、６５℃で、２×ＳＳＰＥ（Frederick M. Ausubel et al., “Current Protocols in Molecular Biology”, 1987, John Wiley & Sons, Hoboken NJ.に記載）および０．１％ ＳＤＳを含む溶液中で１５分を２回、次いで０．５×ＳＳＰＥおよび０．１％ ＳＤＳを含む溶液中でさらに１５分を２回、さらに０．１×ＳＳＰＥおよび０．１％ ＳＤＳを含む溶液中で１５分を２回洗浄してもよく、あるいは、６５℃で、２×ＳＳＰＥ、０．１％ ＳＤＳおよびホルムアミド（５〜５０％）を含む溶液中で１５分を２回、０．５×ＳＳＰＥ、０．１％ ＳＤＳおよびホルムアミド（５〜５０％）を含む溶液中でさらに１５分を２回、次いで０．１×ＳＳＰＥ、０．１％ ＳＤＳおよびホルムアミド（５〜５０％）を含む溶液中で１５分を２回洗浄を行ってもよい。 The stringent conditions are conditions that can be determined by the composition, temperature, and the like of the solution used for hybridization and / or washing after hybridization, and can be appropriately set by those skilled in the art. For example, conditions as described in J. Sambrook et al., “Molecular Cloning: A Laboratory Manual, Second Edition”, 1989, Cold Spring Harbor Laboratory Press. For example, after hybridization with a probe at 50 ° C., more preferably 68 ° C. in a solution containing 6 × SSC, 5 × Denhardt's solution, 0.5% SDS, 100 μg / ml denatured salmon sperm DNA. May be washed at room temperature in a solution of 2 × SSC, 0.1% SDS, but preferably in a solution of 0.1 × SSC, 0.5% SDS to increase stringency. You may wash | clean at 68 degreeC. Alternatively, after hybridization, at 65 ° C., 2 × SSPE (described in Frederick M. Ausubel et al., “Current Protocols in Molecular Biology”, 1987, John Wiley & Sons, Hoboken NJ.) And 0.1 Twice for 15 minutes in a solution containing% SDS, then twice for another 15 minutes in a solution containing 0.5 × SSPE and 0.1% SDS, and further 0.1 × SSPE and 0.1% SDS. You may wash 15 minutes twice in the solution containing, or twice at 15 minutes in a solution containing 2 × SSPE, 0.1% SDS and formamide (5-50%) at 65 ° C. .5 × SSPE, 0.1% SDS and formamide (5-50%) twice for another 15 minutes, then 0.1 × SSPE, 0.1% SDS and formamide (5-50%) Wash twice for 15 minutes in a solution containing It may be.
上記アミノ酸配列および塩基酸配列の相同性は、例えば、ＦＡＳＴＡ、ＢＬＡＳＴ、ＤＮＡＳＩＳ（日立ソフトウェアエンジニアリング（株）製）、ＧＥＮＥＴＹＸ（（株）ジェネティクス製）を用いて測定することができる。 The homology between the amino acid sequence and the base acid sequence can be measured using, for example, FASTA, BLAST, DNASIS (manufactured by Hitachi Software Engineering Co., Ltd.), GENETYX (manufactured by Genetics Co., Ltd.).
また、本発明のＭＹＳＶ弱毒株は、本明細書においてＳＡ０８−８と命名するウイルス株であってもよい。なお、このＳＡ０８−８は、特許法施行規則第２７条の３の規定に従い、本出願人が分譲可能であることを保証する。 Further, the MYSV attenuated strain of the present invention may be a virus strain named SA08-8 in the present specification. This SA08-8 guarantees that the applicant can be sold in accordance with Article 27-3 of the Patent Law Enforcement Regulations.
また、本発明は、さらなる態様において、本発明のＭＹＳＶ弱毒株をウリ科植物に接種することを特徴とする、ウリ科植物における黄化えそ病の防除方法に関する。上述のように、本発明のＭＹＳＶ弱毒株は、接種によって、接種した植物に黄化えそ病などのＭＹＳＶによる病害に対する抵抗性を付与することができる。そのため、本発明のＭＹＳＶ弱毒株を予防的に接種することによって、黄化えそ病などのＭＹＳＶによる病害を予防または軽減することができる。ＭＹＳＶ弱毒株を接種する植物としては、キュウリ、メロン、スイカ、シロウリ、トウガン、ユウガオ、ニガウリなどのウリ科植物が挙げられる。 Moreover, this invention relates to the control method of the yellow spot disease in a cucurbitaceae plant characterized by inoculating a cucurbitaceae plant with the MYSV attenuated strain of this invention in the further aspect. As described above, the MYSV attenuated strain of the present invention can confer resistance to diseases caused by MYSV, such as yellow wilt, to the inoculated plant by inoculation. Therefore, disease caused by MYSV such as yellow wilt can be prevented or reduced by prophylactic inoculation with the MYSV attenuated strain of the present invention. Examples of plants inoculated with the MYSV attenuated strain include Cucurbitaceae plants such as cucumber, melon, watermelon, shirori, Togan, Yugao and Nigauri.
さらに、本発明は、もう１つの態様において、本発明のＭＹＳＶ弱毒株を接種することによって得ることができる、黄化えそ病抵抗性のウリ科植物に関する。上述のように、本発明のＭＹＳＶ弱毒株はＭＹＳＶ強毒株に対して干渉効果を有するので、ＭＹＳＶを接種することによって、接種した植物に黄化えそ病などのＭＹＳＶによる病害に対する抵抗性を付与することができる。本発明によってＭＹＳＶによる病害に対する抵抗性を付与し得る植物としては、キュウリ、メロン、スイカ、シロウリ、トウガン、ユウガオ、ニガウリなどのウリ科植物が挙げられる。 Furthermore, the present invention, in another aspect, relates to a yellow wilt resistant Cucurbitaceae plant obtainable by inoculating the MYSV attenuated strain of the present invention. As described above, since the MYSV attenuated strain of the present invention has an interference effect on the MYSV highly virulent strain, inoculating the MYSV makes the inoculated plant resistant to diseases caused by MYSV such as yellow wilt. Can be granted. Examples of plants that can impart resistance to diseases caused by MYSV according to the present invention include cucumbers such as cucumbers, melons, watermelons, shirouri, togan, yugao, and bitter gourd.
以下に実施例を示して本発明をさらに具体的かつ詳細に説明するが、実施例はあくまで例示説明であって、本発明を限定するものではない。 EXAMPLES The present invention will be described more specifically and in detail below with reference to examples. However, the examples are merely illustrative and do not limit the present invention.
ＭＹＳＶ弱毒株の作出
植物ウイルスから当該ウイルスの弱毒株を作出する方法として、低温処理、紫外線照射または局部病斑形成植物への連続接種などの方法が当該分野において知られている。ＭＹＳＶ弱毒株を作成するにあたり、これらの方法を試みた。しかしながら、病原性の低い弱毒株を選抜することはできなかった。植物ウイルス弱毒株の作出方法として、高温処理を用いる方法も知られている。しかし、通常行われている全身感染寄主植物（ＭＹＳＶの場合、キュウリが該当する）を利用した高温処理では、病原性の低いＭＹＳＶ弱毒株を選抜することはできなかった。また、タバコモザイクウイルス弱毒株の作出方法として知られている局部感染寄主植物（ニコチアナ グルチノサ（Nicotiana glutinosa））に接種して高温処理をし、その上位葉に移行したウイルスから選抜する方法は、ＭＹＳＶが上位葉へ移行しないので適用不可能であった。
Production of MYSV Attenuated Strain As a method for producing an attenuated strain of the virus from a plant virus, methods such as low-temperature treatment, ultraviolet irradiation, or continuous inoculation of local lesion-forming plants are known in the art. These methods were attempted in creating the MYSV attenuated strain. However, it was not possible to select attenuated strains with low pathogenicity. As a method for producing a plant virus attenuated strain, a method using high-temperature treatment is also known. However, the MYSV-attenuated strain with low pathogenicity could not be selected by high-temperature treatment using a systemically infected host plant (in the case of MYSV, which corresponds to cucumber). In addition, a method for inoculating a locally infected host plant (Nicotiana glutinosa), which is known as a method for producing an attenuated strain of tobacco mosaic virus, and subjecting it to high-temperature treatment, and selecting from a virus that has migrated to its upper leaves is MYSV. Was not applicable because it did not move to the upper leaves.
そこで、キュウリに黄化えそ症状を発現するＭＹＳＶ強毒株（Ｃ９５Ｓ）をタバコ属植物の一種であるニコチアナ グルチノサにカーボンランダム法によって汁液接種し、３４℃で１４日間高温処理した。高温処理した後で、接種葉を磨砕することによって、接種葉からウイルスを回収した。回収したウイルスを含む汁液を、センニチコウに汁液接種した。接種葉に形成された局部病斑から単離したウイルスをキュウリに接種して、病原性の低いＭＹＳＶ弱毒株の選抜を繰り返した。センニチコウを用いたウイルス単離を４回、キュウリによる選抜を１３回行った。子葉でのえそ斑点、および本葉での葉脈透過、葉脈えそ、モザイクなどの黄化えそ病に特有の病徴を呈さないキュウリについて、ＭＹＳＶ感染の有無を、ＥＬＩＳＡ法によって確認したところ、キュウリに対して病原性を示さないＭＹＳＶ弱毒株（ＳＡ０８−８）を選抜した。なお、上記ウイルス株は、特許法施行規則第２７条の３の規定に従い、本出願人が分譲可能であることを保証する。 Therefore, MYSV highly toxic strain (C95S) that develops yellowing symptom in cucumber was inoculated by sap into Nicotiana glutinosa, a kind of tobacco genus plant, by carbon random method and treated at 34 ° C. for 14 days at high temperature. After high temperature treatment, the virus was recovered from the inoculated leaves by grinding the inoculated leaves. The juice containing the collected virus was inoculated into the sap. Virus isolated from local lesions formed on the inoculated leaves was inoculated into cucumbers, and selection of MYSV attenuated strains with low pathogenicity was repeated. Virus isolation using Sensenchikou was performed 4 times and cucumber selection 13 times. The presence or absence of MYSV infection of cucumbers that do not exhibit symptom peculiar to yellow spot disease, such as variegation spots in cotyledons and leaf vein penetration, leaf veins, and mosaics in true leaves, was confirmed by ELISA. A MYSV attenuated strain (SA08-8) that does not show pathogenicity against cucumber was selected. The virus strains are guaranteed to be sold by the applicant in accordance with Article 27-3 of the Patent Law Enforcement Regulations.
ＭＹＳＶ弱毒株の接種による黄化えそ病抵抗性の付与
１次接種としてＭＹＳＶ弱毒株（ＳＡ０８−８）をキュウリ（品種：ＺＱ−７）の子葉に汁液接種し、その２２日後（本葉３〜４枚展開期）にＭＹＳＶ弱毒株の感染を確認した。次いで、２次接種として強毒株のＭＹＳＶ（Ｃ０５Ｔ）を最頂葉に接種した。比較のために、１次接種のみで２次接種を行わなかったものと、１次接種せずに２次接種のみ行ったものも調製した。
その結果を、表１に示す。１次接種としてＭＹＳＶ弱毒株を接種したものでは、２次接種の有無に関わらず、一部の株で軽微なモザイク症状が認められたにとどまったのに対し、１次接種せずに２次接種のみ行ったものでは、ほとんどの株に強い黄化えそ症状が認められた。
Giving yellow spot wilt resistance by inoculation with MYSV attenuated strain As a primary inoculation, MYSV attenuated strain (SA08-8) was inoculated into cucumber (variety: ZQ-7) cotyledons and 22 days later (main leaf 3) The infection of the MYSV attenuated strain was confirmed in the ~ 4 sheet development stage). Next, as a secondary inoculation, the highly virulent strain MYSV (C05T) was inoculated on the topmost leaf. For comparison, a primary inoculation alone and no secondary inoculation were prepared, and a primary inoculation without secondary inoculation was also prepared.
The results are shown in Table 1. In the case of inoculating MYSV attenuated strain as the primary inoculation, minor mosaic symptoms were observed in some strains regardless of the presence or absence of the secondary inoculation, whereas the secondary inoculation without the primary inoculation In the strains that received only inoculation, strong yellowing symptoms were observed in most strains.
ＭＹＳＶ弱毒株のゲノムＲＮＡ配列決定
Takeuchi et al. (2009) First report of Melon yellow spot virus infecting balsam pear ( Momordica charantia L.) in Japan，Journal of General Plant Pathology Volume 75, 154-156に記載された方法に準じてＭＹＳＶ弱毒株（ＳＡ０８−８）のゲノムＲＮＡ配列を決定し、アミノ酸配列を推定した。親ウイルス株Ｃ９５Ｓおよび強毒野生株Ｃ０５Ｔと比較した結果、ＲＮＡ依存ＲＮＡ複製酵素（ＲｄＲｐ）で５か所、ＧＮ／ＧＣタンパク質で６か所およびＮＳｓタンパク質で１か所、ＳＡ０８−８に特徴的なアミノ酸が確認された。
Genomic RNA sequencing of MYSV attenuated strains
According to the method described in Takeuchi et al. (2009) First report of Melon yellow spot virus infecting balsam pear (Momordica charantia L.) in Japan, Journal of General Plant Pathology Volume 75, 154-156 (SA08) The genomic RNA sequence of -8) was determined, and the amino acid sequence was deduced. Results compared to the parental virus strain C95S and virulent wild strain C05T, 5 places with RNA-dependent RNA replication enzyme (RdRp), 1 place at six and NSs protein G N / G C protein, the SA08-8 Characteristic amino acids were identified.
現地実証試験における干渉効果の確認（ハウスでの防除試験）
弱毒ウイルス（ＳＡ０８−８）を子葉に汁液接種したキュウリ（品種：ＺＱ−７）の苗と、無接種のキュウリの苗とをビニルハウスで栽培し、強毒ウイルスに対する干渉効果を調査した。ハウスには伝染源として強毒系統のＭＹＳＶ（Ｔ１−１３）を接種したキュウリ苗を同時に定植した。葉の症状について０〜４の５段階（０：無病徴、１：生育に影響を及ぼさない程度のモザイク・退緑、２：えそ症状、３：葉全面のえそ、４：枯死）で調査し、発病の推移をみた。その結果を、表３に示す。無接種キュウリでは９月上旬より病徴が認められ始め、生育に悪影響を及ぼすと考えられる指数２以上の症状が徐々に増加したが、弱毒ウイルス接種キュウリでは、初期から軽度の症状が見られたものの、栽培終了時まで指数２以上の症状はほとんど認められなかった。なお、強毒ウイルスを接種したキュウリでは、栽培初期から強いウイルス症状が認められた。このことから、弱毒ウイルスを接種することで強毒ウイルスに対する干渉効果が発揮され、黄化えそ病の予防が可能であることが明らかとなった。
Confirmation of interference effect in field verification test (control test in house)
Cucumber seedlings (variety: ZQ-7) inoculated into the cotyledons of the attenuated virus (SA08-8) and non-inoculated cucumber seedlings were cultivated in a vinyl house, and the interference effect on the virulent virus was investigated. In the house, cucumber seedlings inoculated with MYSV (T1-13), a highly toxic strain, were simultaneously planted as an infectious source. About 0 to 4 leaf symptoms (0: no symptom, 1: mosaic or discoloration to the extent that it does not affect growth, 2: symptom symptoms, 3: full-leaf burrow, 4: dead) We investigated and looked at the development of disease. The results are shown in Table 3. In non-inoculated cucumbers, symptoms began to be observed from early September, and symptoms of index 2 or higher, which are thought to have an adverse effect on growth, gradually increased, but attenuated virus-inoculated cucumbers had mild symptoms from the beginning. However, almost no symptoms with index 2 or higher were observed until the end of cultivation. In addition, in the cucumber inoculated with the virulent virus, strong viral symptoms were observed from the beginning of cultivation. From this, it was clarified that inoculation with the attenuated virus exerted an interference effect against the virulent virus and can prevent yellow rot.
現地実証試験における干渉効果の確認（強毒発生圃場での暴露試験）
弱毒ウイルス（ＳＡ０８−８）を子葉に汁液接種したキュウリ（品種：ＺＱ−７）の苗と、無接種のキュウリの苗とを、強毒系統のＭＹＳＶが発生している圃場に持ち込み、７日後に回収して施設内で栽培し、回収２０日後の葉の症状を０〜４の５段階（０：無病徴、１：生育に影響を及ぼさない程度のモザイク・退緑、２：えそ症状、３：葉全面のえそ、４：枯死）で調査した。その結果を、表４に示す。無接種のキュウリでは全株で程度３の激しい症状が発現したが、弱毒ウイルス接種株では生育に問題ないと考えられる程度１の症状が認められたのみであった。このことから、弱毒ウイルスを接種することで強毒ウイルスに対する干渉効果が発揮され、強毒ウイルスによる症状の発現を抑えることが可能であることが明らかとなった。
Confirmation of interference effect in field verification test (exposure test in highly toxic field)
Bring cucumber (variety: ZQ-7) seedlings inoculated with the attenuated virus (SA08-8) into the cotyledons and uninoculated cucumber seedlings to the field where MYSV of the virulent strain is generated, 7 days It was later collected and cultivated in the facility, and after 20 days of collection, the leaf symptom was classified into 5 stages from 0 to 4 (0: no symptom, 1: mosaic that does not affect growth, 2: green symptom) (3: The entire bottom of the leaf, 4: Dead). The results are shown in Table 4. In the uninoculated cucumber, about 3 severe symptoms were observed in all the strains, but in the attenuated virus-inoculated strain, only about 1 symptoms that were considered to have no problem with growth were observed. From this, it was clarified that inoculation with the attenuated virus exerts an interference effect on the highly toxic virus and can suppress the expression of symptoms due to the highly toxic virus.
本発明により、ＭＹＳＶ強毒株に対して干渉効果を有するＭＹＳＶ弱毒株を提供することができる。また、本発明のＭＹＳＶ弱毒株をウリ科植物に接種することによって、ウリ科植物に黄化えそ病に対する抵抗性を付与することができ、媒介虫の防除といった間接的な手段ではない、直接的な手段による黄化えそ病の防除が可能となる。 According to the present invention, a MYSV attenuated strain having an interference effect with respect to a MYSV highly toxic strain can be provided. In addition, by inoculating a cucurbitaceae plant with the MYSV attenuated strain of the present invention, the cucurbitaceae plant can be imparted with resistance to yellow rot, and it is not an indirect means such as control of a vector worm. It is possible to control yellow wilt disease by a conventional means.
SEQ ID NO: 1: RNA dependent RNA polymerase amino acid sequence of Melon yellow spot virus
SEQ ID NO: 2: GN/GC protein amino acid sequence of Melon yellow spot virus
SEQ ID NO: 3: NSs amino acid sequence of Melon yellow spot virus
SEQ ID NO: 4: NSm amino acid sequence of Melon yellow spot virus
SEQ ID NO: 5: Nucleocapsid amino acid sequence of Melon yellow spot virus
SEQ ID NO: 6: RNA dependent RNA polymerase gene-encoding sequence of Melon yellow spot virus
SEQ ID NO: 7: GN/GC protein gene-encoding sequence of Melon yellow spot virus
SEQ ID NO: 8: NSs gene-encoding sequence of Melon yellow spot virus
SEQ ID NO: 9: NSm gene-encoding sequence of Melon yellow spot virus
SEQ ID NO: 10: Nucleocapsid gene-encoding sequence of Melon yellow spot virus
SEQ ID NO: 1: RNA dependent RNA polymerase amino acid sequence of Melon yellow spot virus
SEQ ID NO: 2: G N / G C protein amino acid sequence of Melon yellow spot virus
SEQ ID NO: 3: NSs amino acid sequence of Melon yellow spot virus
SEQ ID NO: 4: NSm amino acid sequence of Melon yellow spot virus
SEQ ID NO: 5: Nucleocapsid amino acid sequence of Melon yellow spot virus
SEQ ID NO: 6: RNA dependent RNA polymerase gene-encoding sequence of Melon yellow spot virus
SEQ ID NO: 7: G N / G C protein gene-encoding sequence of Melon yellow spot virus
SEQ ID NO: 8: NSs gene-encoding sequence of Melon yellow spot virus
SEQ ID NO: 9: NSm gene-encoding sequence of Melon yellow spot virus
SEQ ID NO: 10: Nucleocapsid gene-encoding sequence of Melon yellow spot virus
Claims (14)
（ａ）ＭＹＳＶを自然界での宿主植物以外の植物の葉に接種する工程、
（ｂ）ＭＹＳＶを接種した植物を高温処理する工程、および
（ｃ）接種葉からＭＹＳＶ弱毒株を選抜、回収する工程。 A method for producing a melon yellow spot virus (MYSV) attenuated strain comprising the following steps:
(A) inoculating MYSV into leaves of plants other than host plants in nature;
(B) a step of high-temperature treatment of the plant inoculated with MYSV, and (c) a step of selecting and recovering a MYSV attenuated strain from the inoculated leaf.
（ａ）配列番号１〜３で示すアミノ酸配列において、１または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列をコードするＲＮＡまたはその相補鎖；
（ｂ）配列番号１〜３で示すアミノ酸配列または（ａ）のアミノ酸配列をコードするＲＮＡまたはその相補鎖と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし得るＤＮＡと相補的な塩基配列を有するＲＮＡまたはその相補鎖；および
（ｃ）配列番号１〜３で示すアミノ酸配列と８０％以上の同一性を有するアミノ酸配列をコードするＲＮＡまたはその相補鎖
からなる群より選択されるＲＮＡまたはその相補鎖である、請求項４記載のＭＹＳＶ弱毒株。 One or more of the RNAs encoding the amino acid sequences shown in SEQ ID NOs: 1 to 3 or their complementary strands,
(A) RNA encoding the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NOs: 1 to 3 or a complementary strand thereof;
(B) Complementary to DNA that can hybridize under stringent conditions with DNA having the amino acid sequence shown in SEQ ID NOs: 1 to 3 or RNA having the nucleotide sequence complementary to RNA encoding the amino acid sequence of (a) or its complementary strand (C) selected from the group consisting of RNA encoding an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NOs: 1 to 3 or a complementary strand thereof. The MYSV attenuated strain according to claim 4, which is RNA or a complementary strand thereof.
（ａ）配列番号６〜８で示す塩基配列において、１または数個の塩基が欠失、置換または付加された塩基配列を含むＲＮＡまたはその相補鎖；
（ｂ）配列番号６〜８で示す塩基配列または（ａ）の塩基配列を含むＲＮＡまたはその相補鎖と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし得るＤＮＡと相補的な塩基配列を有するＲＮＡまたはその相補鎖；および
（ｃ）配列番号６〜８で示す塩基配列と６０％以上の同一性を有する塩基配列を含むＲＮＡまたはその相補鎖
からなる群より選択されるＲＮＡまたはその相補鎖である、請求項７記載のＭＹＳＶ弱毒株。 One or more of RNA or its complementary strand comprising the nucleotide sequence shown in SEQ ID NOs: 6 to 8,
(A) RNA or a complementary strand thereof comprising a base sequence in which one or several bases have been deleted, substituted or added in the base sequences shown in SEQ ID NOs: 6 to 8;
(B) Complementary to DNA that can hybridize under stringent conditions with DNA having the base sequence shown in SEQ ID NOs: 6 to 8 or RNA having the base sequence of (a) or a complementary base sequence thereof. (C) an RNA comprising a base sequence having 60% or more identity with the base sequence shown in SEQ ID NOs: 6 to 8, or an RNA selected from the group consisting of the complementary strand; Alternatively, the MYSV attenuated strain according to claim 7, which is a complementary strand thereof.
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| CN104805219A (en) * | 2015-04-28 | 2015-07-29 | 上海市农业科学院 | Specific RT-LAMP primer groups for detecting melon yellow spot virus as well as RT-LAMP detection kit and RT-LAMP detection method of specific RT-LAMP primer group |
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| CN104247623A (en) * | 2014-09-02 | 2014-12-31 | 谢振西 | Soilless cultivation method for increasing bitter gourd yield |
| CN104805219A (en) * | 2015-04-28 | 2015-07-29 | 上海市农业科学院 | Specific RT-LAMP primer groups for detecting melon yellow spot virus as well as RT-LAMP detection kit and RT-LAMP detection method of specific RT-LAMP primer group |
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