First Report of Cucumber mosaic virus infecting Metaplexis japonica (Thunb.) Makino in China
First Report of Cucumber mosaic virus infecting Metaplexis japonica (Thunb.) Makino in China
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我国首次报道黄瓜花叶病毒感染Metaplexis japonica (Thunb.) Makino
DOI：
10.1094/pdis-08-17-1237-pdn
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发表时间：
2018
期刊：
影响因子：
4.5
通讯作者：
Quan
中科院分区：
文献类型：
--
作者：
Lei Yang;Xin Sun;Quan
日本山萝木通属夹竹桃科（Apocynaceae）植物，是我国著名的传统中草药，在我国分布广泛。2016年9月，对M.在江苏省镇江市南瓜田附近生长的一株粳稻植株表现出病毒感染特征的深浅绿色花叶和变形症状。有症状的M.通过将500 μl提取缓冲液（0.01 M磷酸盐缓冲盐水[pH 7.4]，1% wt/vol Na 2SO 3和0.05% vol/vol Tween-20）加入到50 mg在液氮中研磨的叶组织中制备日本叶。在6叶期本氏烟草植物的完全展开的幼叶上用汁液进行机械接种，导致接种后5天（dpi）的叶变形和30 dpi的花叶和植物矮化症状。模拟接种N.在5或30 dpi时，本塞姆氏植物没有表现出任何症状。使用先前描述的方法（Morris等，1979）从症状叶片中提取的双链（ds）RNA进行6%聚丙烯酰胺凝胶电泳。经银染后，观察到大小约为4.0、3.5、2.7和1.7 kb的四种dsRNA。这些结果表明，M.具有花叶和变形症状的粳稻植株最可能被RNA病毒感染。为了更好地了解该病毒的基因组信息，从M.日本植物用作序列非依赖性扩增（SIA）的模板（Agindotan等人，2010; Bohlander等人，1992）。将聚合酶链反应（PCR）产物连接到pUCm-T Vector（Sangon Biotech，Shanghai）中并测序（GenScript，Nanjing，China）。BLAST分析表明，630-nt序列与黄瓜花叶病毒（CMV）中国分离物（KC 218822）的核苷酸同源性最高（98%），1，151-nt序列与黄瓜花叶病毒（CMV）IB亚组中国分离物（FJ 268745）的核苷酸同源性最高（96%），表明CMV是致病因子。该分离物暂命名为CMV-Met。使用根据获得的外壳蛋白（CP）核苷酸序列设计的CMV特异性引物（正向，5′-TAACTTTAGAGTCCTGTCGC-3′;反向，5′-ATGAAGTACTAGCTCATCCG-3′）进行逆转录（RT）PCR检测。从有症状的N.本塞姆氏烟草植株中扩增出了特异性条带，但无症状烟草植株中没有扩增出。本塞姆氏植物或阴性对照。随后，通过对使用简并CMV引物和从SIA获得的序列设计的CMV-Met特异性引物获得的RT-PCR产物进行测序来确定CMV-Met（GenBank KY 794710）的RNA 3的完整序列。基于CMV-Met和其他CMV毒株的CP氨基酸序列和RNA 3 5′-非翻译区，使用MEGA 5.1软件进行系统发育分析，结果表明CMV-Met聚为IB亚组（Roossinck et al. 1999）。这一发现有助于制定控制策略，在农业地区种植M。日本。据我们所知，这是第一次报告的感染M。日本的CMV。
Metaplexis japonica (Thunb.) Makino, a species of family Apocynaceae, is well known as a traditional herbal medicine and has a wide distribution in China. In September 2016, leaves of M. japonica plants growing near a pumpkin field in Zhenjiang, Jiangsu province, exhibited light and dark green mosaic and deformation symptoms characteristic of virus infection. Crude sap of symptomatic M. japonica leaves was prepared by adding 500 μl of extraction buffer (0.01 M phosphate buffered saline [pH 7.4], 1% wt/vol Na 2 SO 3, and 0.05% vol/vol Tween-20) to 50 mg of leaf tissue ground in liquid nitrogen. Mechanical inoculation with the sap was performed on the young, fully expanded leaves of the 6-leaf stage Nicotiana benthamiana plants, resulting in leaf distortion at 5 days postinoculation (dpi) and mosaic and plant stunting symptoms at 30 dpi. Mock-inoculated N. benthamiana plants did not exhibit any symptoms at 5 or 30 dpi. Double-stranded (ds) RNAs extracted from the symptomatic leaves using a previously described method (Morris et al. 1979) were subjected to 6% polyacrylamide gel electrophoresis. After silver staining, four dsRNA approximately 4.0, 3.5, 2.7, and 1.7 kb in size were observed. These results suggested that the M. japonica plants with mosaic and deformation symptoms were most likely infected by an RNA virus. To better understand the genomic information of this virus, dsRNA extracted from the symptomatic leaves of M. japonica plants served as a template for sequence-independent amplification (SIA)(Agindotan et al. 2010; Bohlander et al. 1992). Polymerase chain reaction (PCR) products were ligated into pUCm-T Vector (Sangon Biotech, Shanghai) and sequenced (GenScript, Nanjing, China). BLAST analysis showed that a 630-nt sequence shared the highest (98%) nucleotide identity with a Cucumber mosaic virus (CMV) isolate from China (KC218822), and a 1,151-nt sequence shared the highest (96%) nucleotide identity with a CMV subgroup IB isolate from China (FJ268745), suggesting CMV was the causal agent. This isolate was tentatively named CMV-Met. Reverse transcription (RT) PCR detection was performed using the CMV-specific primer (forward, 5′-TAACTTTAGAGTCCTGTCGC-3′; and reverse, 5′-ATGAAGTACTAGCTCATCCG-3′) designed using the obtained coat protein (CP) nucleotide sequence. The expected RT-PCR product was amplified from symptomatic N. benthamiana plants inoculated with sap but was not amplified from the asymptomatic N. benthamiana plants or negative controls. Subsequently, the complete sequence of RNA3 of CMV-Met (GenBank KY794710) was determined by sequencing RT-PCR products obtained using degenerate CMV primers and CMV-Met-specific primers designed from sequences obtained by SIA. Phylogenetic analysis based on the CP amino acid sequence and the RNA3 5′-nontranslated region of CMV-Met and other CMV strains using MEGA 5.1 software indicated that CMV-Met was clustered in subgroup IB (Roossinck et al. 1999). This finding is helpful in developing strategies for controlling disease caused by CMV in agricultural areas growing M. japonica. To our knowledge, this is the first report of infection in M. japonica by CMV.