芜菁花叶病毒不同分离物3'-cDNA片段的克隆及序列分析

 从河北、北京、山东泰安和潍坊等地区的萝卜、大白菜、甘蓝、油菜上得到8个芜菁花叶病毒(Turnip mosaic virus,TuMV)分离物,测定了它们3'-末端cDNA片段的序列。根据衣壳蛋白基因(cp)核苷酸序列可以将这8个分离物与另外2个TuMV分离物分为3组:泰安旧镇大白菜(JZBC)、甘蓝(JZGL)和萝卜(JZLB)分离物为一组;北京(BJILB)、潍坊萝卜分离物(WFLB)与引起萝卜红心病的TuMV分离物(WFLB99)为一组;泰安范镇、河北、潍坊(WFLB04)萝卜和泰安旧镇油菜(JZYC)上的TuMV分离物为一组。农壳蛋白氨基酸序列比较结果与此基本一致,所不同的是分离物WFLB99与所有分离物的差异均较大,形成单独一个分支。有必要对TuMV的变异情况和致病机理进行深入研究。     

重庆市萝卜芜菁花叶病毒的检测与序列分析

为明确重庆市萝卜感染芜菁花叶病毒(Turnip mosaic virus,TuMV)的情况,在重庆市巴南区、九龙坡区、北碚区等14个区县共采集了146份萝卜病毒病样品,采用酶联免疫吸附试验法(enzymelinked immunosorbent assay,ELISA)对病样进行TuMV检测,并通过PCR、克隆和测序等技术获得13个TuMV分离物CP核苷酸序列,利用软件分析重庆市TuMV CP基因序列的相似性,利用系统进化树分析CP基因遗传变异与系统进化。结果表明,共有105份样品的TuMV检测为阳性,检出率为71.9%;所得13个分离物CP基因均为864 bp,测序的13个分离物CP核苷酸相似性为89.1%~99.2%,与国内已报道的TuMV各分离物CP核苷酸相似性在88.3%~99.4%之间;所得的13个分离物均分布在basal-BR和world-B组中,在Asian-BR和basal-B组中均无分布,进一步分析发现有9个分离物属于word-B组,仅4个分离物属于basal-BR组。研究表明,TuMV在重庆市萝卜各种植区普遍发生,且word-B组为重庆市萝卜的优势毒源。     

芜菁花叶病毒长春分离物p3基因的克隆、序列分析及P3蛋白抗血清的制备

 用RT-PCR方法从长春感染芜菁花叶病毒( Turnip mosaic virus,TuMV)的十字花科蔬菜中扩增获得该病毒的p3基因,并对其序列进行了比较分析。结果表明,本研究所获得的10个TuMV分离物p3基因含1 065个核苷酸,其序列一致率为98.8%~99.6%,与GenBank中其他15个TuMV分离物核苷酸一致率为80.9%~99.4%。根据p3基因核苷酸序列构建的系统进化树显示:25个TuMV分离物可分为4个组,本研究得到的10个TuMV分离物均属于basal-BR组。将TuMV JCR06分离物p3基因N端663 bp片段克隆至原核表达载体pET-28a(+),并在大肠杆菌BL21(DE3) pLysS中表达出分子量约为28 kDa的融合蛋白。以纯化的融合蛋白为抗原免疫家兔,制备了P3蛋白的特异性抗血清。以TuMV侵染的萝卜为抗原,间接ELISA测定抗血清的效价为1∶2 048。Western blotting分析表明,制备的抗血清能与诱导表达的融合蛋白发生特异性反应。     

芜菁花叶病毒对油菜致病力差异及壳蛋白基因序列分析

 2004年春季参试的湖北、安徽2省11个芜菁花叶病毒(Turnip mosaic virus,TuMV)分离物感染4个油菜品种,病情指数幅度为26.2~76.0;秋季参试5个TuMV分离物感染14个油菜品种,病情指数幅度为38.3~55.9,均值方差分析表明,致病力差异分别达到极显著和显著水平。壳蛋白(CP)基因序列分析表明,来源于2省油菜、白菜、红菜薹、芝麻和萝卜的17个TuMV分离物与浙江分离物ZJB3序列同源性在97%以上,同属于MB类群;而另一个萝卜分离物WRS1与ZJR1、CH1和CH2分离物序列同源性在95.4%~98.7%之间,属于MR类群。类群间分离物序列同源性仅为88.0%~92.2%。遗传进化树分析表明,萝卜分离物WRS2在MB类群中单独构成一个分支,可能是MR类群和MB类群发生重组的后代。     

河南省地黄病毒病初步鉴定

 利用血清学、RT-PCR并结合核苷酸序列测定等方法,对河南省地黄病毒病进行了初步鉴定。结果表明,烟草花叶病毒(TMV)为侵染地黄的主要病毒;对TMV地黄分离物(TMV-RH) CP基因的序列分析结果表明,TMV-RH与TMV-U1株系CP基因的核苷酸同源性为86.5%,氨基酸同源性为94.3%;与已发表的TMV其它株系CP基因的核苷酸同源性在76.3%~88.5%之间,氨基酸同源性在79.3%~95.0%之间,同源性较低。根据不同株系CP的氨基酸序列进化树分析,推测该分离物可能为TMV的一个新株系。     

黄瓜绿斑驳花叶病毒山西西瓜分离物外壳蛋白基因序列分析

通过病毒dsRNA分离技术、非序列依赖性PCR扩增(sequence-independent amplification,SIA)技术和RTPCR等手段对采自山西太谷的西瓜病样进行了检测与鉴定,确定其为黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)所侵染。为明确CGMMV西瓜分离物(CGMMV-SXTG)的分类地位,本研究进一步克隆了CGMMV-SXTG的外壳蛋白基因(coat protein,CP)全序列并进行序列分析,系统进化树分析显示该分离物与亚洲分离物亲缘关系较近,而与欧洲分离物亲缘关系较远,表明该病毒的不同分离物在分组上可能与地域有一定的关系;接着对不同分离物的CP基因核苷酸序列和氨基酸序列进行同源性比对分析,结果表明该分离物与中国山东分离物(TANG)同源性最高,分别达到99.8%和99.6%。     

一个马铃薯X病毒分离物的外壳蛋白基因序列分析与株系鉴定

对山东省侵染马铃薯的一个马铃薯X病毒(PVX)分离物PVX—SD1的外壳蛋白(CP)基因进行了克隆和序列分析。以提纯的病毒RNA为模板，应用RT-PCR扩增目的基因，通过常规的基因克隆法将扩增的CP基因导入pUC19载体，测序。结果表明，PVX—SD1的CP基因长719bp，可编码248个氨基酸；与Gen—Bank中报道的15个有代表性的株系或分离物相比较，核苷酸同源性在80．1％-99．7％，氨基酸同源性在89．8％-100％；与欧洲株系UK3仅1个核苷酸不同，同源性为99．7％，氨基酸同源性达100％，表明它们可能为同一株系，属于X^3组．     

2种类型的烟草扭脉病毒外壳蛋白基因的序列和结构分析

 烟草丛顶病主要由烟草丛顶病毒(Tobacco bushy top virus ,TBTV)和烟草扭脉病毒(Tobacco vein distorting virus ,TVDV)复合侵染引起。其中,TVDV的CP蛋白可分别包装TBTV和TVDV形成病毒粒子;但也存在TVDV单独侵染不携带TBTV的病样。本文利用RT-PCR对自然状态下携带TBTV的TVDV(采自云南弥渡)和不携带TBTV的TVDV (采自云南保山)的cp基因进行序列克隆,发现TVDV弥渡分离物的cp基因为618个核苷酸,而TVDV保山分离物的cp基因为621个核苷酸,二者的核苷酸序列一致性为90.02 %,氨基酸序列一致性为89.81 %;而长度相同的TVDV cp基因序列一致性为98.5 % ~ 100 %。表明这2个cp基因属于2种不同类型。系统发育树分析也表明这2个cp基因属于进化距离较远的2个群。利用SWISS-MODEL进行同源建模,发现2种类型的TVDV CP蛋白的主要空间结构域基本一致,存在细小的结构域及结构域之间无规则卷曲角度的差异,整体空间结构没有发生明显改变。推断TVDV保山分离物CP蛋白中关键氨基酸的序列突变是导致CP无法包装TBTV,从而在病害传播过程中丢失TBTV的主要因素。     

一种侵染滇重楼的Potexvirus病毒分离物分子鉴定及其3′末端序列分析

从云南武定的滇重楼上得到一个病毒分离物Paris-YN,病毒粒体为弯曲线状。利用RT-PCR扩增获得一条1074bp的片段,序列比较分析发现其与马铃薯X病毒属(Potexvirus)病毒3′末端的结构最为相似,且与属内的白三叶草花叶病毒等20个不同分离物3′末端有36.7%～58.9%的同源性;该病毒cp基因长639个核苷酸,编码212个氨基酸(22.8kDa),与20个Potexvirus病毒分离物的CP氨基酸序列比较发现,Paris-YN与白三叶草花叶病毒的CP氨基酸同源性最高(60.1%)。证据表明,该分离物可能为Potexvirus的新成员,暂命名为重楼X病毒(Paris polyphylla virus X)。     

侵染白附子的芋花叶病毒分子变异研究

为明确侵染白附子的芋花叶病毒(dasheen mosaic virus, DsMV)的分子变异情况,对51个DsMV白附子分离物(DsMV-BF)的外壳蛋白(Coat Protein,CP)基因和3个分离物的近全长基因组序列进行了克隆和测定,DsMV-BF的CP基因大小有855个和942个核苷酸两种类型,51个白附子分离物之间CP基因的核苷酸和氨基酸一致率分别为88.3%～100%和91.9%～100%,BF8、BF30和BF38分离物之间多聚蛋白的核苷酸和氨基酸序列一致率分别为82.9%～95.9%和90.7%～95.9%,与GenBank中其他分离物之间多聚蛋白的核苷酸和氨基酸序列一致率分别为76.9%～99.4%和85.6%～99.0%;P1基因的分子变异较大,P1基因大小有987个和990个核苷酸两种类型;CP基因核苷酸序列系统进化树分析结果表明,侵染白附子的DsMV分离物可分为两个亚组;重组分析结果表明BF8和BF30分离物各检测到1个重组事件,BF38检测到2个重组事件。     

侵染番红花的芜菁花叶病毒

 从表现花叶症状的德国进口番红花上获得一线状病毒分离物SA,线状病毒粒子长度为700~800nm。其在人工接种的11科39种植物中能侵染6科14种植物,在克里芙兰烟上产生系统坏死,在小白菜、花椰菜等十字花科植物上产生系统花叶或为隐症,在昆诺藜等藜科植物上为局部侵染。在番红花病叶、球茎及克里芙兰烟病叶组织中观察到卷轴状和片层状聚集的风轮状内含体。免疫吸附和免疫修饰电镜观察结果显示,SA与芜菁花叶病毒((TuMV)具有紧密的血清学关系。根据这些特征将SA鉴定为TuMV的一个分离物。这是TuMV侵染番红花的首次报道。     

Serotypic variation in turnip mosaic virus

A panel of 30 monoclonal antibodies (MAbs) was produced against four isolates of turnip mosaic virus (TuMV). The panel was tested in plate-trapped antigen ELISA tests against 41 TuMV isolates (with different host and geographical origins and of differing pathotypes). The antibodies were also tested against four other potyviruses (bean common mosaic virus, bean common mosaic necrosis virus, lettuce mosaic virus and zucchini yellow mosaic virus). The reactions were assessed quantitatively (using multivariate analysis) and qualitatively (using the standard deviation obtained against healthy leaf material). The MAbs recognized 16–17 TuMV epitopes that were not present in the other potyviruses and a further two potyvirus epitopes. The isolates were grouped into three serotypes. Only one isolate did not fit this grouping. The classification of seven isolates in coat protein amino acid sequence homology groups correlated with serotypes. There was no correlation between serotype and pathotype, or between reactions to individual MAbs and single lines. There was therefore no evidence that the epitopes recognized by the MAbs are elicitors for the resistance genes present in the Brassica napus lines. However, the sensitivity and specificity of the MAbs will be useful for both routine detection of TuMV and fundamental studies on plant–virus interactions.     

Identification of Isolates that Cause a Leaf Spot Disease of Brassicas as Xanthomonas campestris pv. raphani and Pathogenic and Genetic Comparison with Related Pathovars

ABSTRACT Twenty-five Xanthomonas isolates, including some isolates received as either X. campestris pv. armoraciae or pv. raphani, caused discrete leaf spot symptoms when spray-inoculated onto at least one Brassica oleracea cultivar. Twelve of these isolates and four other Xanthomonas isolates were spray- and pin-inoculated onto 21 different plant species/cultivars including horseradish (Armoracia rusticana), radish (Raphanus sativus), and tomato (Lycopersicon esculentum). The remaining 13 leaf spot isolates were spray-inoculated onto a subset of 10 plant species/cultivars. The leaf spot isolates were very aggressive on several Brassica spp., radish, and tomato causing leaf spots and dark sunken lesions on the middle vein, petiole, and stem. Based on the differential reactions of several Brassica spp. and radish cultivars, the leaf spot isolates were divided into three races, with races 1 and 3 predominating. A differential series was established to determine the race-type of isolates and a gene-for-gene model based on the interaction of two avirulence genes in the pathogen races and two matching resistance genes in the differential hosts is proposed. Repetitive-DNA polymerase chain reaction-based fingerprinting was used to assess the genetic diversity of the leaf spot isolates and isolates of closely related Xanthomonas pathovars. Although there was variability within each race, the leaf spot isolates were clustered separately from the X. campestris pv. campestris isolates. We propose that X. campestris isolates that cause a nonvascular leaf spot disease on Brassica spp. should be identified as pv. raphani and not pv. armoraciae. Race-type strains and a neopathotype strain for X. campestris pv. raphani are proposed.     

An unusual isolate of turnip mosaic potyvirus fromAbutilon theophrasti in Piedmont, Italy

An isolate of the poty virus turnip mosaic virus (TuMV-Ab) showing severe mosaic symptoms inAbutilon theophrasti from Piedmont (northwestern Italy) in 1993, has been found to be of an unusual pathotype and serotype. The isolate was easily transmitted byAphyis gossiypii and Myzus persicae and was not seed-transmitted inA. Theophrasti. The host range of TuMV-Ab was different from that of another Piedmont isolate of TuMV fromAlliaria officinalis and from a TuMV isolate fromBrassica napus. TuMV-Ab was characterized using the reactions on the fourB. Napus lines S4, R4, 165 and S1 as the rare pathotype 7, found only once previously in Europe. Tests with polyclonal antisera indicated that TuMV-Ab was only distantly related to the two other TuMV isolates. Serological characterization with a panel of 30 monoclonal antibodies showed that TuMV-Ab belonged to one of the less common serotypes (JPN).     

Variation in the pathogenicity of two Turnip mosaic virus isolates in wild UK Brassica rapa provenances

Brassica rapa can be infected with Turnip mosaic virus (TuMV) as a result of manual inoculation or aphid transmission, but infected plants have not been found in the field. In this study, B. rapa plants grown from seed collected from two field sites in southern England were mechanically inoculated with one of two distinct isolates (pathotypes) of TuMV under glasshouse conditions. These had either been isolated from Brassica oleracea growing wild in Wales, (GBR 83, pathotype 3) or Dorset (GBR 98, pathotype 1). Use of ELISA as an index of infection in manually inoculated B. rapa showed that although seed provenance had a small effect on the proportion of plants infected, the biggest factor was the virus isolate. Both virus isolates infected both lines of B. rapa , but invaded at different rates, although both resulted in easily discernible symptoms. The severity of symptoms was not related to amounts of virus in the infected plants. A significantly greater proportion of plants were infected with GBR 83 at 45 days post-inoculation (d.p.i.) than GBR 98. but GBR 98 caused significantly more severe and obvious symptoms as well as greater mortality at 119 d.p.i., in plants from both sites than GBR 83.     

Programmed cell death pathways induced by early plant‐virus infection are determined by isolate virulence and stage of infection

Programmed cell death (PCD) pathways caused by Turnip mosaic virus (TuMV) infection before symptom appearance were studied by light microscopy and electrolyte leakage following sap inoculation of Brassica carinata (Ethiopian mustard) TZ‐SMN‐44‐6 plants. Leaf responses to inoculation with avirulent (TuMV‐avir) and virulent (TuMV‐vir) isolates, and mock‐inoculation, were compared at 2, 20 and 52 h after inoculation (hai). The phenotypes induced were localized resistance (TuMV‐avir) and systemic susceptibility (TuMV‐vir). No visible TuMV symptoms were recorded in any inoculated plants during the 2–52 hai sampling period, but appeared as chlorotic spots in inoculated leaves at 5 days after inoculation. With TuMV‐vir alone, they were followed by systemic infection (mosaic). Dead cell number, deformation, percentage area and percentage integrated intensity, and conductivity of electrolyte leakage data, were analysed to examine their possible roles in stimulating cell death pathways. At 2 hai, dead cell number and percentage area were significantly greater for TuMV‐avir than TuMV‐vir infection or mock‐inoculation. Overall, isolate TuMV‐vir caused significantly greater cell deformation than TuMV‐avir, whereas wounding by mock‐inoculation had negligible effects. By 52 hai, isolate TuMV‐avir caused significantly greater electrolyte leakage than isolate TuMV‐vir or mock‐inoculation. This suggests both isolates triggered morphological changes consistent with apoptotic‐like PCD and necrosis‐like PCD that depended upon isolate virulence and stage of infection, respectively. These findings highlight how quantification of dead cell deformation and electrolyte leakage offer a new understanding of compatible and incompatible plant responses to early virus infection in plants.