Evolutionary Characterization of Two Lily Isolates of Cucumber mosaic virus Isolated in Japan and Korea

We analyzed the evolutionary histories of two lily strains of Cucumber mosaic virus (CMV) isolated in Japan and Korea (HL- and Ly2-CMVs). They share common biological characteristics in that their host ranges are very restricted perhaps from a unique adaptation to lily plants. Although HL and Ly2 were isolated independently from different lily species in separate countries, their RNA3 sequences had a very high sequence similarity (97%). The evolutionary relationships between the two isolates were characterized by comparing their phylogenetic trees for the 3a and CP genes. The two lily CMVs always formed a distinct cluster within subgroup IB in 3a, but within IA in CP. Together, the phylogenetic tree topology and the sequence identity between the two lily CMVs suggest that they evolved from a common progenitor. Received 5 November 2001/ Accepted in revised form 11 January 2002     

烟草品种对烟草花叶病毒和黄瓜花叶病毒的抗性鉴定

 2010-2011年采用大田人工接种鉴定的方法,对生产中大面积推广使用的24份烟草品种进行了烟草花叶病毒（TMV）和黄瓜花叶病毒（CMV）的抗性鉴定。结果表明,供试品种对TMV和CMV的抗病性存在较大差异,对TMV表现抗病的有Coker86、吉烟5号、Coker176、CV87、辽烟8号、CV91、中烟90等7份材料;表现中抗的有秦烟98、双抗70、C151等3份材料;表现中感的有秦烟96、G80、金星6007、龙江981、K326、秦烟201、NC89等7份材料;表现感病的有G28、云烟97、净叶黄、红花大金元、RG11、云烟85、云烟87等7份材料。对CMV表现抗病的有Coker86、龙江981、C151、秦烟201、云烟87等5份材料;表现中抗的材料是金星6007;表现中感的有CV91、RG11、Coker176、中烟90、K326、红花大金元、净叶黄、G80、G28等9份材料;表现感病的有秦烟98、云烟85、秦烟96、NC89、双抗70、云烟97、CV87、辽烟8号、吉烟5号等9份材料。其中兼抗TMV和CMV两种病毒病的材料有2份,分别是Coker86和C151。同时研究还发现,抗病性不同的烟草品种在受到病毒危害以后,对烟叶的产量和品质的影响也不同。明确了中国24个烟草品种的抗病性水平,为抗病品种的利用与品种合理布局提供科学依据,同时为烟草抗病毒病育种的亲本选择提供抗源信息。     

侵染紫松果菊的黄瓜花叶病毒分离物鉴定

从表现黄花叶症状的紫松果菊病株上获得分离物2-1-2,电镜下可见直径约30 nm的球状病毒粒体,其与黄瓜花叶病毒抗体呈强的阳性反应,ds-RNA的谱带类型与本实验室保存的标准黄瓜花叶病毒株系相同。通过生物学、病毒粒体观察、血清学以及病毒核酸双链试验结果,确定该病毒分离物为黄瓜花叶病毒。     

烟草品种对烟草花叶病毒病和黄瓜花叶病毒病的抗性鉴定

由烟草普通花叶病毒(TMV)和烟草黄瓜花叶病毒(CMV)引致的烟草病毒病是世界烟草主产区普遍发生且危害严重的侵染性病害,每年给烟叶生产造成了重大的经济损失。本文采用温室苗期接种鉴定的方法,对16份烟草种质进行了TMV和CMV的抗病性鉴定。结果表明:不同的烟草品种对TMV和CMV的抗病性存在较大差异。在供试种质中,对TMV表现免疫的有‘牛耳烟’、‘8301’、‘台烟7号’、‘三生-NN’共4份材料;表现抗病的有‘吉烟5号’、‘双抗70’、‘大护脖香’、‘秦烟95’共4份材料;表现中抗的有‘铁把子’、‘中烟15’、‘秦烟98’、‘中烟98’共4份材料;表现中感的有‘NC89’、‘翠碧1号’、‘云烟97’共3份材料;表现感病的只有‘秦烟97’。对CMV表现中抗的材料有1份,是‘铁把子’;表现中感的有‘秦烟95’、‘三生-NN’、‘8301’、‘牛耳烟’、‘翠碧1号’共5份材料;表现感病的有‘秦烟98’、‘云烟97’、‘中烟98’、‘NC89’、‘大护脖香’、‘双抗70’、‘秦烟97’、‘中烟15’、‘台烟7号’、‘吉烟5号’共10份材料。研究发现,‘铁把子’是兼抗这两种病毒病的材料。本研究明确了我国16个烟草品种资源的抗病性水平,为抗耐病品种的利用与品种合理布局提供科学依据,同时为烟草抗病毒病育种的亲本选择提供抗源信息。     

侵染观赏南瓜的黄瓜绿斑驳花叶病毒的初步鉴定

从广西某农业展示中心观赏南瓜上分离到一种直杆状病毒,长约300nm,该病毒分离物P-3(NN)在测试的葫芦科作物上表现为系统花叶、褪绿斑及明脉症状,在曼陀罗上为局部褪绿斑,在测试的其它茄科作物及豆科和藜科作物上无任何症状反应.其致死温度为95～100℃.经DAC-ELISA测定,P-3(NN)与黄瓜绿斑驳花叶病毒有密切的血清学关系.根据以上结果,初步鉴定P-3(NN)为黄瓜绿斑驳花叶病毒(CGMMV)的一个分离物.     

利用黄瓜花叶病毒M和Fny株系 的假重组体分析致病关键因子

 用RT-PCR扩增黄瓜花叶病毒M株系（CMV-M）全长基因组cDNA,成功构建CMV-M RNA2和RNA3侵染性克隆后,与CMV-Fny基因组RNA交换得到3个假重组型病毒 （F1M2F3、F1F2M3、F1M2M3）。用F1M2F3、F1F2M3、F1M2M3分别侵染白肋烟,产生坏死环斑、轻微绿斑驳、明脉、黄白化和叶尖线性化等症状。根据假重组型病毒和野生型病毒的表观症状,分析引起各种症状的关键因子,初步判定：CP基因是诱导花叶症状的关键因子,CMV-Fny RNA2是诱导叶尖线性化的关键因子,CMV-M RNA2是诱导叶尖坏死斑关键因子。实时荧光定量PCR结果显示：野生株CMV-M、CMV-Fny和假重组体F1M2F3、F1F2M3、F1M2M3侵染烟草后引起的症状差异与病毒基因组RNA累积没有直接关系。     

黄瓜绿斑驳花叶病毒单克隆抗体的研制

将纯化的黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus, CGMMV)制剂免疫BALB/c小鼠,最后一次免疫后第3天取其脾细胞与SP2/0细胞融合,经采用选择性培养基、有限稀释法克隆和间接ELISA方法进行筛选,成功获得了3株分泌CGMMV特异性单克隆抗体的杂交瘤细胞株并分别命名为3C9,3F7,2G10。用ELISA方法对所获得的3个杂交瘤细胞株进行亚型鉴定均为IgG2a,kappa链。 间接ELISA效价测定结果分别为3C9：1.024×107,3F7：2.56×106,2G10：1.28×106。此3株杂交瘤细胞所分泌的单克隆抗体均能与本研究室保存的其他3种不同的CGMMV分离物发生特异性反应,而不与其他3种同属成员病毒 烟草花叶病毒(Tobacco mosaic virus, TMV)、辣椒轻斑驳病毒(Pepper mild mottle virus, PMMoV)、齿瓣兰环斑病毒(Odontoglossum ringspot virus,ORSV) 发生反应。     

Effect of Figaren, an Antiviral Protein from Cucumis figarei, on Cucumber mosaic virus Infection

Local lesion formation on cowpea leaves was more than 50% inhibited by treatment with a 23 kDa RNase-like glycoprotein from Cucumis figarei, figaren, from 24 hr before to 1 hr after inoculation with Cucumber mosaic virus (CMV). CMV accumulation detected by ELISA in tobacco leaves treated with figaren 6 or 0 hr before inoculation with CMV was suppressed. When upper leaves of tobacco plants were treated with figaren and inoculated 10 min later with CMV, mosaic symptoms were delayed for 5–7 days on most of the tobacco plants, and some plants remained asymptomatic. From fluorescence in situ hybridization, infection sites were present in figaren-treated cowpea or melon leaves after inoculation with CMV, though the sites were reduced in number and size compared with those in water-treated control leaves. The amount of CMV RNAs and CMV antigen in melon protoplasts inoculated with CMV and subsequently incubated with figaren similarly increased with time as did that in the control. ELISA and local lesion assays indicated that CMV infection on the upper surfaces of the leaves of tobacco, melon, cowpea and C. amaranticolor whose lower surfaces had been treated with figaren 5–10 min before CMV inoculation was almost completely inhibited. Figaren did not inhibit CMV infection on the opposite untreated leaf halves of melon, cowpea and C. amaranticolor, whereas it almost completely inhibited CMV infection on the untreated halves of leaves of tobacco. CMV infection was not inhibited in the untreated upper or lower leaves of the four plants. These data suggest that figaren does not completely prevent CMV invasion but does inhibit the initial infection processes. It may also induce localized acquired resistance in host plants. Received 10 October 2000/ Accepted in revised form 6 February 2001     

黄瓜绿斑驳花叶病毒病防治研究进展

黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)主要危害葫芦科作物,已被世界上许多国家和地区列为检疫性有害生物。CGMMV目前在我国23个省、市、区已有报道发生和危害,严重影响葫芦科作物的生产;近年来该病害在国内外呈现迅猛扩展的趋势并对生产造成危害。本文综述了防治该病害的种子处理、化学及生物防治、嫁接以及转基因等分子生物学方法;分析了CGMMV与寄主黄瓜互作研究的最新进展,对小分子RNA参与调控寄主对CGMMV病毒的防控策略提出了展望,并概述了下一代测序技术、基因编辑技术在植物新病毒的检测、鉴定以及培育抗病新品种等方面的应用。     

葫芦种子传黄瓜绿斑驳花叶病毒的检测*

从感染黄瓜绿斑驳花叶病毒（Cucumber green mottle mosaic virus, CGMMV）的葫芦植株上收取种子,通过苗期症状观察法、双抗体夹心酶联免疫吸附法（DAS-ELISA）、免疫捕获反转录PCR（IC-RT-PCR）法测定葫芦种子的带毒情况,并用生物学接种方法测定葫芦种子携带病毒的侵染活性。苗期症状观察法结果表明,199株幼苗有2株表现花叶斑驳症状,种子传毒率为1.01%;而利用DAS-ELISA和IC-RT-PCR法随机检测30粒葫芦病株种子,CGMMV检出率为100%。种子各部位携带的CGMMV接种葫芦表现典型的花叶斑驳症状,表明葫芦种子携带的CGMMV具有侵染活性。DAS-ELISA检测葫芦种子CGMMV的灵敏度为1/5120种子研磨液。     

白术矮化病毒病病原的分子鉴定和部分序列分析

 本试验在生物学接种的基础上,利用非序列依赖性PCR扩增(sequence-independent amplification,SIA)对白术矮化病毒病病原进行了分子鉴定,序列测定及分析。结果发现,具有矮化症状的白术为黄瓜花叶病毒(Cucumber mosaic virus,CMV)所侵染。为明确CMV白术分离物(CMV-Am)的分类地位,本研究进一步克隆了CMV-Am的外壳蛋白基因(CP)和移动蛋白基因(MP)全序列。序列比对分析表明,CMV-Am与CMV亚组ⅠB中株系XJ2序列同源性最高,核苷酸、氨基酸序列同源性分别为98.9%、99.1%。氨基酸序列同源性聚类分析表明,CMV-Am与中国大多数CMV分离物一样,属于CMV亚组ⅠB。     

卫星RNA SatC382对辅助病毒的影响

 通过体外转录将黄瓜花叶病毒(CMV)卫星RNASatC382与不带卫星的黄瓜花叶病毒CNa株系进行假重组,获取带卫星的CMV重组株(CNa-SatC382)。经dsRNA提取及RT PCR检测,证实CNa和SatC382在假重组株中能稳定共存。测定CNa和CNa-SatC382的14种寄主生物学反应,并统计两者接种昆诺藜、假酸浆、心叶烟、西葫芦7、14、21、28d的病情指数,结果显示:带卫星和不带卫星的CMV在各供试寄主上表现的症状差别不明显,病情指数也无显著差异。由此推测卫星RNASatC382没有改变辅助病毒CMV-CNa株系对寄主症状的影响。     

湖南首次检测到黄瓜绿斑驳花叶病毒

 黄瓜绿斑驳花叶病毒（Cucumber green mottle mosaic virus,CGMMV）自1935年首次报道^［1］以来,已广泛分布于亚洲、欧洲、南美洲的多个国家和地区,对葫芦科作物的生产造成了严重损失。CGMMV主要有两种重要的传播途径,一是带毒种子造成远距离传播,二是授粉、嫁接等农事操作造成的田间传播^［2］。     

A study of viral coat protein accumulation in lily chloroplasts from mixed virus infections of Lily mottle virus and Cucumber mosaic virus

Two viruses that frequently occur in many Lilium species are Lily mottle virus (LMoV) and Cucumber mosaic virus (CMV), which usually co-infect lilies causing severe disease symptoms. Recent reports have revealed that the viral coat protein (CP) affects chloroplast ultrastructure and symptom development. This study used western blot analysis to confirm that in leaves infected by mixed virus infections of LMoV and CMV, CPs of both viruses were accumulated in lily chloroplasts. Immunogold labelling further demonstrated that both the LMoV CP and CMV CP were localized in the stroma and the thylakoid membranes of the chloroplasts. In addition, it was found that CPs of both viruses were rapidly transported into isolated, intact chloroplasts (in vitro), and their transport efficiencies were positively related to CP concentrations. The lowest transmembrane concentration of CMV CP decreased from 38 μg mL⁻¹ recorded in the single CMV CP import system to 10 μg mL⁻¹ in the mixed import system of LMoV CP and CMV CP. CPs of both viruses exhibited species selection in their transmembrane transport into chloroplasts. This is the first report that the CPs from two viruses (LMoV and CMV) are simultaneously present in lily chloroplasts. Accumulation of high levels of LMoV CP and CMV CP inside the chloroplast appears to contribute to a synergistic interaction inducing the development of mosaic symptoms.     

应用MNP-RT-PCR方法检测黄瓜绿斑驳花叶病毒

 A novel RT-PCR method integrated with Magnetic Nano Particles (MNP), MNP-RT-PCR, was set up for detection of Cucumber green mottle mosaic virus (CGMMV). After the virus particles in crude sap were concentrated by MNP, viral RNAs were released and were detected by RT-PCR. CGMMV could be detected in as less as 10 ng watermelon leaf materials. Compared with normal RT-PCR, the method decreased the inhibitors of plant material and steps for extracting RNA, and also increased the sensitivity of RT-PCR detection in less time. The method is simple and suitable for quick detection of plant virus in a large number of samples.     

云南、福建、湖南烟区烟草花叶病主要病毒种类检测及黄瓜花叶病毒亚组鉴定

 采用抗原直接包被和双抗体夹心酶联免疫吸附测定法(ELISA)对采自云南、福建、湖南烟区烟草花叶病样品进行了病毒种类检测,利用三抗体夹心ELISA对黄瓜花叶病毒(Cucumber mosaic virus,CMV)的亚组类型进行了鉴定。在云南采集的520个花叶病样品中,烟草花叶病毒(Tobacco mosaic virus,TMV)、CMV和马铃薯Y病毒(Potato virus Y,PVY)总检出率分别为71.74%、55.01%和6.35%;在福建采集的150个花叶病样品中,TMV、CMV和PVY的总检出率分别为94%、24.66%和8.00%;在湖南采集的74个花叶病样品中,TMV、CMV和PVY的总检出率分别为58.11%、51.35%和2.70%。部分样品为2种以上病毒复合侵染。云南、福建和湖南采集的64个CMV阳性样品中,属亚组Ⅰ的样品为57个,占89.1%;属亚组Ⅱ的样品为10个,占15.6%;其中3个样品为亚组Ⅰ和亚组Ⅱ的复合侵染。     

北京地区辣椒上黄瓜花叶病毒和甜菜西方黄化病毒复合侵染的分子鉴定

辣椒是我国重要的蔬菜和经济作物,受多种病毒危害。2014年在北京市顺义区调查时发现部分种植的辣椒植株上叶片大面积黄化,边缘症状明显,个别植株叶片轻微上卷。提取典型症状样品的总RNA,反转录得到cDNA,分别用黄瓜花叶病毒(Cucumber mosaic virus,CMV)特异引物和马铃薯卷叶病毒属(Polerovirus)通用引物进行PCR检测,CMV特异引物和马铃薯卷叶病毒属通用引物分别扩增得到约650bp和1 400bp的特异条带。测序和核苷酸序列比对表明,其分别与CMV和甜菜西方黄化病毒(Beet western yellows virus,BWYV)序列同源性最高为99%和96%。这是对我国种植的辣椒上发生的CMV和BWYV复合侵染的首次报道。     

A Variant of Cucumber mosaic virus Is Restricted to Local Lesions in Inoculated Tobacco Leaves with a Hypersensitive Response

A variant of Cucumber mosaic virus, CMV(Y/GM2), was isolated from a tobacco plant with mild green mosaic symptoms that was regenerated in vitro from a yellow strain of CMV [CMV(Y)]-infected tobacco leaves by tissue culture. CMV(Y/GM2) has two amino acid substitutions at 36 and 111 positions in the coat protein encoded on RNA3. CMV, assembled by mixing in vitro transcribed CMV(Y) RNA1 and RNA2 plus infectious RNA3 transcribed in vitro from cDNA to RNA3 of CMV(Y/GM2), was prepared and designated as CMV(Y/GM2)tr. When tobacco (Nicotiana tabacum cv. Xanthi nc) plants were inoculated with CMV(Y/GM2)tr, large necrotic local lesions in which the virus was localized, developed on the inoculated leaves. This host response unique to CMV(Y/GM2)tr was similar to the hypersensitive response (HR), which is a common resistance response to avirulent pathogens and was observed in five cultivars of Nicotiana tabacum and eight Nicotiana species. The revertant virus, however, accumulated to quite different levels in the various hosts. CMV(Y/GM2)tr induced pathogenesis-related 1 (PR-1) protein accumulation and systemic acquired resistance (SAR) which were generally observed in the HR. However, when tobaccos were inoculated with CMV(S36P)tr and CMV(V111I)tr, which have an amino acid substitution at either the 36 or 111 position in the coat protein of CMV(Y), respectively, CMV(S36P)tr was restricted to the primary infection site without necrotic local lesion formation and PR-1 protein and SAR induction. CMV(V111I)tr, however, systemically spread and induced mild green mosaic symptoms, while the host had the HR to CMV(Y/GM2)tr. The localization of CMV(Y/GM2)tr at the primary infection site may not only be caused by the HR, but also by the restriction of virus systemic movement resulting from the amino acid substitution at position 36 in the coat protein of CMV(Y). Received 15 December 1999/ Accepted in revised form 18 April 2000     

Defective Long-distance Transport of <Emphasis Type="Italic">Cucumber mosaic virus </Emphasis>in Radish Is Efficiently Complemented by <Emphasis Type="Italic">Turnip mosaic virus</Emphasis>

A strain of Cucumber mosaic virus (CMV-D8) systemically infects Japanese radish (Raphanus sativus), but the Y strain of CMV (CMV-Y) only infects the inoculated leaves. Both of these strains cause severe systemic mosaic on the plants after dual infection with Turnip mosaic virus (TuMV). Synergistic interactions on long-distance transport of CMV-Y and CMV-D8 with TuMV were analyzed using an immunobinding assay. Direct tissue blots probed with either anti-CMV-Y or anti-TuMV antiserum clearly showed that CMV-Y efficiently spread and accumulated in the tissues of noninoculated upper leaves and roots when co-inoculated with TuMV, and that long-distance movement of CMV-D8 was enhanced by the presence of TuMV. Received 16 September 1999/ Accepted in revised form 5 February 2000     

基于外壳蛋白基因的湖南烟草黄瓜花叶病毒遗传多样性分析

为探明湖南烟草上发生的黄瓜花叶病毒Cucumber mosaic virus(CMV)的遗传多样性及分子进化特征,对来自湖南烟区的303份疑似感染病毒的烟草样品进行检测,分析CMV系统发育、遗传变异和群体结构等特征。结果表明：部分分离物的外壳蛋白(coat protein, CP)基因与NCBI上登录的CMV分离物的一致性为86.34%～98.42%;系统发育分析发现湖南烟草CMV分离物属ⅠB组,不同组间的分离物地理特征不明显,无重组现象,进化的主要驱动力是负选择;组间遗传变异比较明显,基因交流频率较低,受到遗传漂变影响,遗传多样性高,群体趋于扩张。研究结果为烟草抗CMV育种提供了理论依据,对病害防治具有重要意义。