侵染广东番茄的番茄褪绿病毒分子鉴定

在广东番茄上发现一种新病害,病株表现为叶片褪绿,叶脉颜色变深及叶片增厚等症状。利用番茄褪绿病毒Tomato chlorosis virus(ToCV)HSP70基因的两对特异引物对番茄病样进行RT-PCR检测,结果表明,从所采集的6份病样中均扩增到预期大小的DNA特异片段。对其中1份样品的扩增片段进行克隆与序列分析,结果表明,扩增片段包括1个长度为1 665个核苷酸的完整病毒基因,其核苷酸序列与已报道的ToCV HSP70基因有较高的同源性,表明广东番茄受到了ToCV的侵染。但ToCV广东番茄分离物的HSP70序列与国内外已报道的各分离物的同源性均低于82%,存在较大差异,其中与塞浦路斯tomato、约旦JU_20分离物的同源性最高,为81.8%。这是ToCV在广东发生的首次报道,也是该病毒HSP70基因序列存在显著差异分离物的首次发现。     

侵染番茄的番茄褪绿病毒山东泰安分离物的分子鉴定和序列分析

2012年秋季, 在山东泰安番茄主要种植区中采集到叶片褪绿, 叶脉颜色变深的疑似番茄褪绿病毒病和番茄侵染性褪绿病毒病的番茄样品。利用番茄褪绿病毒(Tomato chlorosis virus, ToCV)的特异引物 ToCV1/ToCV2和番茄侵染性褪绿病毒(Tomato infectious chlorosis virus, TICV)的特异引物TICV1/TICV2分别对样品进行扩增, 最后仅得到利用引物 ToCV1/ToCV2 扩增的101 bp 的核苷酸序列, 对该核苷酸序列克隆并测序。序列比对表明, 山东泰安地区分离物与已登录的番茄褪绿病毒(ToCV)分离物相似性都在99%以上。随后, 对山东泰安种植区ToCV番茄分离物进行外壳蛋白(CP)及热激蛋白(HSP70)序列的扩增、克隆和测序(GenBank登录号KC812620/KC812625), 经NCBI BLAST比对发现, 目的序列与番茄褪绿病毒日本番茄分离物ToCV Japan/Tochigi (GenBank登录号AB513442/AB513443)相似性最高为99%, 同属于毛型病毒属的番茄褪绿病毒, 这是首次明确山东地区番茄受到番茄褪绿病毒的侵染。     

北方四省区番茄褪绿病毒的分子鉴定

2015-2016年间,在山西省、内蒙古自治区、辽宁省、吉林省采集到疑似感染番茄褪绿病毒Tomato chlorosis virus(ToCV)的番茄植株。利用扩增ToCV外壳蛋白(coat protein,CP)和类热激蛋白(heat shock protein 70homolog,HSP70h)基因片段的两对特异性引物,通过RT-PCR对疑似样品进行分子检测,得到970bp和1 864bp的特异条带,经测序、比对确定为ToCV。序列分析表明,山西晋中分离物SXJZ(KX853540)的CP核苷酸序列与河南安阳分离物HNAYHX(KP264983)相似性为99.7%,内蒙古呼和浩特分离物NMHHHT(KU204709)和辽宁大连分离物LNDL(KU204707)的CP核苷酸序列与国内已报道的山东寿光分离物SDSG(KC709510)相似性分别为99.5%和99.7%,吉林长春分离物JLCC(KU306111)的CP核苷酸序列与山东聊城分离物SDLC(KC812622)的相似性为99.8%。而HSP70h核苷酸序列的相似性分析表明,山西晋中分离物SXJZ(KX853539)与日本分离物Tochigi(AB513442)相似性为99.4%,内蒙古呼和浩特分离物NMHHHT(KU204710)、辽宁大连分离物LNDL(KU204708)和吉林长春分离物JLCC(KX880384)与山东寿光分离物SDSG(KC709510)相似性分别为99.7%、99.8%和99.7%。试验结果明确了番茄褪绿病毒已蔓延传播到我国山西、内蒙古及东北地区。     

番茄褪绿病毒RT-PCR检测技术的优化及河南分离物的鉴定

为获得高效、灵敏和稳定的番茄褪绿病毒(Tomato chlorosis virus,ToCV)RT-PCR检测体系,选取文献报道的和重新设计的共9套ToCV的RT-PCR引物,经过一系列测试和比较,筛选出了灵敏度、特异性均较高的引物组合,并对反应条件、参数进行了优化,确定了最优反应条件。应用优化的ToCV RT-PCR检测体系对从河南设施蔬菜生产区采集的疑似感病番茄样品进行检测,扩增产物测序后经BLAST比对发现,河南分离物的CP和HSP70基因序列与GenBank上注册的ToCV典型分离物序列的相似性均达94%以上,通过对ToCV病毒粒子的电镜观察而进一步确定ToCV已在河南侵染设施番茄。     

侵染甜椒的番茄褪绿病毒的分子鉴定

番茄褪绿病毒（Tomato chlorosis virus, ToCV）是一种由粉虱传播的RNA病毒,可以侵染番茄和辣椒等常见蔬菜,已在世界多地造成严重危害。2012年在北京市大兴区调查蔬菜病毒病时发现保护地栽培的甜椒植株表现脉间褪绿症状,且植株上烟粉虱数量多,初步推测为ToCV危害。通过提取显症样品总RNA,利用ToCV特异性引物进行反转录PCR,扩增得到463 bp的目标条带。通过测序和核苷酸序列比对分析,表明该序列与国外已报道的ToCV HSP70h（the heat shock protein 70 homolog,热激蛋白70家族）基因序列相似性为99%。这是对ToCV在我国侵染甜椒的首次报道。     

番茄褪绿病毒侵染黄瓜的首次报道

番茄褪绿病毒Tomato chlorosis virus(ToCV)是严重危害世界经济作物的一种病毒,寄主范围广泛。田间调查发现黄瓜Cucumis sativus表现出叶片黄化、脉间褪绿的疑似番茄褪绿病毒感病症状,同时叶片背面聚集了大量烟粉虱。采用RT-PCR方法对样品叶片和烟粉虱进行检测,ToCV感染率为65%,且发病叶片上烟粉虱携带ToCV。为进一步确定黄瓜是否为番茄褪绿病毒的新寄主,室内利用农杆菌侵染性克隆接种健康黄瓜,结果显示:接种30 d的黄瓜新生叶片出现褪绿症状。采用ToCV HSP70基因的引物对田间黄瓜叶片、烟粉虱和室内黄瓜新生叶片进行RT-PCR,扩增出约450 bp的条带,在NCBI上BLAST显示与KC887999.1的同源性最高,为99%。这些数据表明黄瓜是番茄褪绿病毒的寄主。这是ToCV感染黄瓜的首次报道。     

西瓜花叶病毒2号南瓜分离物的鉴定及其外壳蛋白基因序列分析

 利用电镜和酶联免疫吸附测定法(ELISA)在黑龙江省采集的南瓜病样中检测到西瓜花叶病毒2号(WMV-2)。再利用免疫PCR (IC-PCR)和反转录PCR (RT-PCR)方法,扩增获得其外壳蛋白(CP)基因片段,并克隆到pGEM-T载体中。核苷酸序列测定表明,该分离物CP基因全长为852个核苷酸,编码由284个氨基酸组成的31.8 kDa蛋白。与国外已报道的WMV-2 CP基因相比,其核苷酸序列同源性为92.2%~94.0%,由此推导的氨基酸序列同源性为94.5%~98.1%。与国内2个分离物相比,和山西分离物核苷酸和氨基酸的同源性都达到98.5%,和郑州分离物核苷酸和氨基酸的同源性分别为91.5%和95.0%。     

一种基于RPA的番茄褪绿病毒检测方法

番茄褪绿病毒Tomato chlorosis virus(ToCV)引起番茄褪绿病毒病,给番茄生产造成严重危害。开发快速准确的检测方法对该病害的防控具有重要意义。利用番茄褪绿病毒外壳蛋白(CP)基因序列,设计特异性引物,建立了ToCV的重组酶聚合酶等温扩增(recombinase polymerase amplification, RPA)检测方法,同时分析了该方法的灵敏度和特异性。结果表明,建立的ToCV-RPA方法在38℃恒温下40 min可从ToCV阳性的番茄样品中扩增出246 bp的特异性条带。扩增时间短,对设备要求低,且与番茄其他病毒无交叉反应,特异性好,灵敏度可达到PCR方法的10倍,适用于ToCV的快速检测。     

西瓜花叶病毒2号新疆昌吉分离物外壳蛋白基因核苷酸序列分析

 利用RT-PCR从新疆昌吉地区表现花叶、疱斑、扭曲等症状的南瓜病株上检测到西瓜花叶病毒2号新疆昌吉分离物(简称WMV-2-XJ-CJ),并测定了该分离物外壳蛋白(CP)基因序列。序列分析表明,新疆昌吉分离物CP基因全长850个核苷酸,编码197个氨基酸。与国内外报道的12个WMV-2CP基因相比,其核苷酸序列同源性为92.6%~98.3%,由此推导的氨基酸序列同源性为94.7%~99.3%。新疆昌吉分离物在CP N'端可变区明显不同于国内外报道的核苷酸序列。WMV-2新疆昌吉分离物与日本和郑州分离物较其它国家和地区的分离物多出6个核苷酸,但其核苷酸及其推导的氨基酸序列差异较大。新疆昌吉分离物外壳蛋白有2个氨基酸残基明显不同于其它分离物,其中蚜传株系的特征结构域DAG突变为DAE。     

来源于桃和苹果的苹果褪绿叶斑病毒的部分分子生物学特性和cp基因的原核表达

 从桃和苹果上分离得到苹果褪绿叶斑病毒ACLSV-HBP和ACLSV-C2个分离物,采用RT-PCR法进行扩增,所获扩增片段经序列测定,其全长分别为1768nt(ACLSV-HBP)和1751nt(ACLSV-C)。这2个分离物扩增片段全长的同源性为83%,mp基因片段核苷酸和推导编码氨基酸序列同源性分别为82.6%和87.1%;cp基因均由582nt组成,其核苷酸和推导编码氨基酸序列同源性分别为87.8%和95.9%。将2个分离物的cp基因与已报道ACLSV分离物进行序列同源性比较,结果显示ACLSV-HBP与SX/2的cp基因核苷酸序列及推导编码氨基酸序列同源性最高,分别为94.0%和96.4%。将ACLSV-HBP分离物的cp基因克隆到原核表达载体pGEX-KG,在大肠杆菌BL21(DE3)中诱导表达,SDS-PAGE分析表明,融合蛋白大小约为46kDa。Western-blot分析表明,该基因在大肠杆菌内得到高效表达,融合蛋白具有抗原性。     

Differential reaction of sweet pepper to infection with the crinivirus tomato chlorosis virus probably depends on the viral variant

The tomato chlorosis virus (ToCV), transmitted by whitefly species of the genera Bemisia and Trialeurodes in a semipersistent manner, causes significant losses in solanaceous crops including tomato (Solanum lycopersicum) and sweet pepper (Capsicum annuum). Worldwide reports of natural and experimental infection of sweet pepper plants with ToCV are contradictory, raising the question of whether the critical factor determining infection is related to the susceptibility of sweet pepper cultivars or the genetics of virus isolates. In this work, ToCV isolates obtained from different hosts and geographical origins were biologically and molecularly analysed, transmitted by B. tabaci MEAM1 and MED, and the reaction of different sweet pepper cultivars was evaluated under different environmental conditions. Brazilian ToCV isolates from tomato, potato (S. tuberosum), S. americanum, and Physalis angulata did not infect plants of five sweet pepper cultivars when transmitted by B. tabaci MEAM1. Temperatures did not affect the sweet pepper susceptibility to tomato-ToCV isolates from São Paulo, Brazil, and Florida, USA. However, sweet pepper-ToCV isolates from Spain and São Paulo, Brazil, were transmitted efficiently to sweet pepper plants by B. tabaci MEAM1 and MED. Although the results indicated that ToCV isolates from naturally infected sweet pepper plants seem to be better adapted to plants of C. annuum, phylogenetic analyses based on the complete nucleotide sequences of RNA1 and RNA2 as well as the p22 gene did not reveal significant nucleotide differences among them. Additional studies are needed to identify intrinsic characteristics of ToCV isolates that favour infection of sweet pepper plants.     

Tomato chlorosis virus: a new whitefly-transmitted, Phloem-limited, bipartite closterovirus of tomato

ABSTRACT Tomato chlorosis virus (ToCV) is the second whitefly-transmitted, phloem-limited, bipartite closterovirus described infecting tomato. ToCV is distinct from tomato infectious chlorosis virus (TICV), based on lack of serological and nucleic acid cross-reactions and differences in vector specificity. TICV is transmitted only by the greenhouse whitefly (Trialeurodes vaporariorum), whereas ToCV is transmitted by the greenhouse whitefly, the banded-wing whitefly (T. abutilonea), and Bemisia tabaci biotypes A and B (B. argentifolii). Double-stranded (ds) RNA analyses of ToCV show two prominent dsRNAs of approximately 7,800 and 8,200 bp, with several small dsRNAs. Digoxigenin-11-UTP-labeled riboprobes derived from cDNA clones representing portions of RNAs 1 and 2 were used in Northern blot hybridizations to detect two large nonhomologous dsRNAs and a subset of smaller dsRNAs. These probes were used in dot blot hybridizations to detect ToCV in infected tomato. Inclusion bodies and cytoplasmic vesicles were consistently observed in phloem tissues of ToCV-infected Nicotiana clevelandii. Computer-assisted sequence analysis showed significant homology between ToCV clones that hybridize specifically with RNAs 1 and 2 and the lettuce infectious yellows virus methyltransferase of RNA 1 and the HSP70 heat shock protein homolog of RNA 2, respectively. Thus, ToCV is another member of the growing subgroup of bipartite closteroviruses transmitted by whiteflies.     

烟粉虱传播的番茄褪绿病毒和番茄黄化曲叶病毒对不同番茄品种的复合侵染

为明确烟粉虱传播的番茄褪绿病毒(Tomato chlorosis virus,ToCV)与番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)对不同番茄品种的复合侵染情况,于2015年11月在山东省寿光市温室内采集13个番茄品种共390份疑似发病植株叶片,对不同番茄品种的TYLCV抗性和2种病毒的复合侵染以及温室内发病番茄植株上烟粉虱成虫的带毒率进行检测。结果表明,采集的13个番茄品种经分子标记检测鉴定均为TYLCV杂合抗性;不同番茄品种ToCV与TYLCV的复合侵染率存在明显差异,大果番茄粉宴和贝瑞上复合侵染率最高可达73.3%,而樱桃番茄八喜上未检测到这2种病毒的复合侵染。此外,在发病番茄植株上采集的烟粉虱成虫体内可检测到2种病毒,其中烟粉虱ToCV带毒率为90.7%,TYLCV带毒率为80.0%,同时检测到ToCV与TYLCV的概率为71.3%。表明ToCV和TYLCV的复合侵染在山东省番茄生产中普遍发生,烟粉虱可同时携带这2种病毒并广泛传播。     

Screening tomato genotypes for resistance and tolerance to Tomato chlorosis virus

Tomato chlorosis virus (ToCV) is an emerging crinivirus in Brazil that causes an economically important disease in tomato (Solanum lycopersicum) and other solanaceous species. ToCV is transmitted predominantly by the whitefly Bemisia tabaci Middle East‐Asia Minor 1 (MEAM1, formerly biotype B), in a semipersistent manner. As all cultivated tomato varieties and hybrids are susceptible to this crinivirus, the main alternatives for the control of the disease are the use of healthy seedlings for transplanting and the chemical control of the insect vector. The objective of this work was to evaluate the responses of tomato genotypes to infection with this crinivirus and their tolerance to the disease in order to support the development of other alternatives for disease control. Resistance to infection was evaluated by ToCV inoculation with viruliferous B. tabaciMEAM1 followed by virus detection by RT‐PCR and RT‐qPCR. To measure tolerance to the disease, plant development and fruit yield of ToCV‐infected and healthy plants were compared. Among 56 genotypes, only the lineage IAC‐CN‐RT (S. lycopersicum ‘Angela Gigante’ × S. peruvianum ‘LA 444‐1’) was highly resistant to infection with ToCV. Tolerance to the disease over two trials with different genotypes showed variable results. The effect of ToCV on plant development varied from 2.9% to 71.9% reduction, while yield loss varied from 0.2% to 51.8%. The highly ToCV‐resistant lineage IAC‐CN‐RT, which is also resistant to a Spanish isolate of ToCV, might be useful for tomato breeding programmes.     

Lack of synergistic effects in tomato plants co-infected with tomato severe rugose virus and tomato chlorosis virus

The begomovirus Tomato severe rugose virus (ToSRV) and the crinivirus Tomato chlorosis virus (ToCV), in single and co-infections, are very common in tomato crops in Brazil. Both viruses are transmitted by the whitefly Bemisia tabaciMEAM1 (biotype B). The objective of this study was to analyse the interaction between ToSRV and ToCV in tomato plants of cultivars Santa Clara and Kada. Plants at 15, 30 and 45 days after emergence were inoculated with 30 viruliferous B. tabaci per plant. The following treatments were compared: plants inoculated with ToSRV, ToCV, ToSRV + ToCV, and healthy (control). The interaction between these viruses was analysed by measuring the virus titre by qPCR and the fresh and dry weights of the aerial parts of the tomato plants. Based on two independent assays, no significant effects for co-infection of ToSRV and ToCV on virus titres and plant development were observed compared to single infections. The dry weight of tomato plants of both cultivars infected with ToSRV, ToCV, or co-infected did not differ significantly. However, the dry weight of Santa Clara tomato plants infected with ToSRV, ToCV and ToSRV + ToCV showed mean reductions of 21.5%, 25.5% and 32%, respectively, compared to healthy plants, and mean reductions for Kada were 31.7%, 37.5% and 38%, respectively.