粉虱传双生病毒的TAS-ELISA及PCR快速检测

 利用粉虱传双生病毒(WTGs)多克隆抗体及单克隆抗体,建立了三抗体夹心ELISA (TAS-ELISA)检测WTGs的方法,并发现了单克隆抗体SCR18可广泛用于我国WTGs的检测。利用根据WTGs基因组上基因间隔区及外壳蛋白基因保守序列设计的引物,建立了PCR特异检测WTGs的方法。对田间病样的TAS-ELISA和PCR检测表明,粉虱传双生病毒在云南省烟草、番茄和南瓜上均存在,2种方法的测定结果是一致的。     

烟粉虱中甘薯双生病毒(sweepoviruses)半巢式PCR快速检测技术的建立与应用

甘薯双生病毒(sweepoviruses)是侵染甘薯的一类重要病毒,通过烟粉虱以持久方式传播,我国甘薯上至少存在8种甘薯双生病毒.本研究根据我国已报道的8种甘薯双生病毒基因组保守区设计了一组引物,建立了单头烟粉虱中甘薯双生病毒的半巢式PCR快速检测方法.特异性和灵敏性分析结果表明,半巢式PCR具有较高的特异性和灵敏性,...     

北京地区番茄黄化曲叶病毒病的鉴定及防治对策

番茄黄化曲叶病毒病是一种由烟粉虱传播的病毒病,给番茄生产造成严重威胁。2009年在北京郊区调查时发现部分保护地种植的番茄植株表现典型黄化曲叶症状。通过提取典型症状样品总DNA利用粉虱传双生病毒检测简并引物PA/PB,进行PCR扩增到541bp的特异条带。通过测序和核苷酸序列比对表明该序列与番茄黄化曲叶病毒序列相似性最高为99%。分子检测结果表明北京郊区部分保护地种植的番茄已被烟粉虱传播的番茄黄化曲叶病毒侵染危害。     

南疆温室番茄黄化曲叶病病毒种类的分子鉴定

为明确南疆温室番茄黄化曲叶病的病毒种类,利用双生病毒的兼并引物通过PCR扩增,对采集的20个番茄病株进行了分子检测.从20个病株中均扩增到约500 bp的目标片段,对其中4株进行克隆和测序,其相互间序列同源性为97.1％ ～99.3％,与番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)的同源性较高,为98.6％ ～ 99.5％.随机选取莎车分离物KS2-5进行全基因组的克隆和测序,KS2-5 DNA全长为2781 nt(序列号:JQ807735),具有典型的双生病毒基因组特征,与TYLCV其它分离物同源性达到98.9％～99.5％,而与其它粉虱传双生病毒的序列同源性较低,为68.3％ ～75.5％,表明危害南疆温室番茄的病毒种类为番茄黄化曲叶病毒TYLCV.     

广西胜红蓟黄脉病样中发现多种菜豆金色花叶病毒属的病毒

 双生病毒科(Geminiviridae)病毒是一组植物单链DNA病毒,它的特点是病毒粒子形态为孪生颗粒状。双生病毒分为菜豆金色花叶病毒属(Begomovirus)、甜菜曲顶病毒属(Curtovirus)、玉米线条病毒属(Mastrevirus)和番茄伪曲顶病毒属(Topocuvirus)^[1]。     

中国番木瓜曲叶病毒南宁分离物的基因组结构特征

 从广西南宁田间表现曲叶症状的番木瓜植株上分离到病毒分离物G4,经三抗体夹心ELISA (TAS-ELISA)检测,G4与粉虱传双生病毒的抗体呈阳性反应。对G4 DNA-A全序列测定和分析表明,G4 DNA-A全长2 748个核苷酸,共编码6个ORFs。同源性比较及系统进化关系分析表明,G4 DNA-A与在亚洲发现的粉虱传双生病毒关系较近,其中与我国报道的中国番木瓜曲叶病毒(PaLCuCNV)同源性最高,达到98.0%。进一步比较发现,G4 DNA-A编码的AV1、AV2、AC1、AC2、AC3和AC4与PaLCuCNV相应ORFs的氨基酸同源性分别为98.4%、95.7%、97.5%、97.8%、94.1%和94.6%,表明G4应属于PaLCuCNV的一个分离物。G4编码的ORFs与中国胜红蓟黄脉病毒(AYVCNV)、辣椒曲叶病毒(PepLCV)及烟草曲茎病毒(TbCSV)有较高的氨基酸同源性,可能起源于共同的祖先。利用DNA-B及卫星DNAβ的保守引物均未能从G4分离物中扩增出相应的组分。     

长蒴母草黄脉病毒——一个双生病毒的新种

 从采集于海南儋州地区表现黄脉症状的长蒴母草(Lindernia anagallis)上分离到病毒分离物L2, DNA-A全序列分析结果表明, 全长2739个核苷酸(nt)(GenBank登录号:AY795900), 共编码6个ORF, 其中病毒链编码AV1(CP)、AV2, 互补链编码AC1、AC2、AC3、AC4。利用BLAST程序对DNA-A进行分析表明, 与L2 DNA-A有同源关系的病毒均为双生病毒科(Geminiviridae)菜豆金色黄花叶病毒属(Begomovirus)成员。进一步比较发现, L2 DNA-A与我国广东报道的广东番茄曲叶病毒(Tomato leaf curl Guangdong virus, ToLCGuV)(AY602165)全基因组核苷酸序列的同源性最近, 仅为77.0%, 说明L2为Begomovirus中的一个新种, 命名为长蒴母草黄脉病毒(Lindernia anagallis yellow vein virus, LAYVV)。与L2的IR区及各基因编码的氨基酸序列有最高同源性的病毒均来源于亚洲。利用DNA-B特异引物和DNA-β的特异引物, 均未检测到DNA-B和卫星DNA-β的存在。     

侵染新疆加工番茄的中国番茄黄化曲叶病毒 DNA-A的基因组特征

 从新疆加工番茄上分离到病毒分离物XJ26-4,对其基因组DNA-A 全序列测定表明,XJ26-4 DNA-A 全长2 737 个核苷酸(GenBank 登录号:FN985163),具有典型的双生病毒基因组特征。进一步序列比较发现,XJ26-4 DNA-A 与中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus, TYLCCNV)各分离物的同源性最高,达到91. 2% ~ 99. 5% ,而与其他双生病毒的序列相似性均在79. 5% 以下,表明XJ26-4 是TYLCCNV 的一个分离物。这是首次明确新疆加工番茄受到粉虱传双生病毒的侵染。     

云南省烟草上双生病毒的发生与分布

 已报道云南省烟草曲叶病(Tobacco leaf curl disease, TLCD)由3种菜豆金色花叶病毒属(Begomovirus)的双生病毒引起。它们是烟草曲茎病毒(Tobacco curly shoot virus, TbCSV)、云南烟草曲叶病毒(Tobacco leaf curl Yunnan virus, TbLCYNV)和中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus, TYLCCNV)。为了了解这3种病毒的分布, 2000~2005年, 从云南省4个市(州)采集了109个烟草曲叶病样品, 利用3种病毒的特异引物对采集样品进行了PCR检测。从保山市的烟草曲叶病样品中检测到3种病毒, 并且存在TbCSV+TbLCYNV、TbCSV+TYLCCNV和TbCSV+TbLYNV+TYLCCNV的复合侵染, 从大理州、文山州和红河州的烟草曲叶病样品中仅检测到TYLCCNV。对39个分离物部分序列的系统进化分析结果表明, 病毒分离物依据病毒种类和地理位置聚成几簇。对烟草曲叶病的流行和病害控制策略进行了讨论。     

广西粉虱传染类植物双生病毒调查

广西粉虱传染类植物双生病毒调查植物双生病毒（Ｇｅｍｉｎｉｖｉｒｕｓ）具有独特的球状对生病毒形态和单链ＤＮＡ，由于其经济和理论研究上的重要性，近年成为病毒学研究的热点之一，但是，国内有关此组病毒的报道极少。笔者自１９９１年以来在广西对粉虱传染类植物双生...     

A geminivirus causing vein yellowing of Ageratum conyzoides in Singapore

A vein-yellowing disease of Ageratum conyzoides in Singapore was shown to be caused by a geminivirus, here named ageratum yellow vein virus (AYVV), which was transmitted by the whitefly Bemisia tabaci but not by inoculation with sap or through seed. AYVV particles (30 × 20 nm) are serologically related to those of other whitefly-transmitted geminiviruses, and reacted with some monoclonal antibodies elicited by particles of African cassava mosaic or Indian cassava mosaic geminiviruses. However, the epitope profile of AYVV differed from the profiles of these viruses, and from those of geminiviruses from vein yellowing-affected A. conyzoides from India and from yellow leaf curl-affected tomato from either Singapore or India. The results provide further evidence of antigenic differences among geminiviruses that cause similar diseases in the same plant species in different geographical regions.     

Sequence analysis and classification of apparent recombinant begomoviruses infecting tomato in the nile and mediterranean basins

ABSTRACT Numerous whitefly-transmitted viral diseases of tomato have emerged in countries around the Nile and Mediterranean Basins the last 20 years. These diseases are caused by monopartite geminiviruses (family Gemini viridae) belonging to the genus Begomovirus that probably resulted from numerous recombination events. The molecular biodiversity of these viruses was investigated to better appreciate the role and importance of recombination and to better clarify the phylogenetic relationships and classification of these viruses. The analysis partitioned the tomato-infecting begomoviruses from this region into two major clades, Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus. Phylogenetic and pairwise analyses together with an evaluation for gene conversion were performed from which taxonomic classification and virus biodiversity conclusions were drawn. Six recombination hotspots and three homogeneous zones within the genome were identified among the tomatoinfecting isolates and species examined here, suggesting that the recombination events identified were not random occurrences.     

广东黄秋葵黄脉曲叶病样中检测到烟粉虱传双生病毒

黄秋葵是近几年来从日本和我国台湾引进的一种蔬菜作物。近期,广东的黄秋葵上发生了黄脉曲叶病。病株的典型症状表现为叶脉黄化,在叶片正面形成网络状,在叶背面叶脉肿大突起明显,病株幼叶小且向下卷曲,甚至整片幼叶黄化。植株早期被感染表现矮化。在发生黄脉曲叶病的黄秋葵田间,其病株率高达60%以上。用烟粉虱传双生病毒简并引物对随机采集的病样进行PCR检测,从这些病样中均能扩增出1条预期大小为570 bp的特异片段;基因克隆及测序分析结果表明,与该特异片段同源的均属双生病毒科菜豆金色花叶病毒属病毒DNA,其中与木尔坦棉花曲叶病毒（Cotton leaf curl Multan virus, CLCuMV）分离物G6相似性最高,为99%。这些研究结果表明,广东黄秋葵黄脉曲叶病中存在烟粉虱传双生病毒,该病害可能也是由CLCuMV侵染引起的。     

The transmission of geminiviruses by Bemisia tabaci

The cosmopolitan whitefly species, Bemisia tabaci (Gennadius) and Trialeurodes taporariorum (Westwood) have always been regarded as pests to a large range of worldwide crops. Both species are capable of transmitting plant viruses, with T. vaporariorum being the vector of only a few ‘clostero’-like viruses and B. tabaci the vector of viruses in several groups. The largest group of viruses transmitted by B. tabaci are the geminiviruses and B. tabaci is known to transmit around 60 members. Until recently, B. tabaci had been associated with only a limited range of host plants within any one region, although its total potential host range was large. Virus transmission was confined within the plant host range of each regional population of B. tabaci. The emergence of the polyphagous ‘B’ biotype of B. tabaci with its increased host range of more than 600 plant species, has resulted in geminiviruses infecting previously unaffected crops. As the ‘B’ biotype spreads further into Europe, European field and glasshouse crops have been shown to be susceptible to whitefly-transmitted viruses already endemic to other parts of the world. More than 20 colonies of B. tabaci, including both ‘B’ and non-‘B’ biotypes from disparate global locations have been compared for their ability to transmit more than 20 geminiviruses. All but two highly host-specific colonies were capable of transmitting most geminiviruses tested. However, some viruses were transmitted more efficiently than others. The virus coat protein or capsid is essential for vector recognition and transmission. By comparing transmissible viruses at the molecular level to viruses that are no longer whitefly-transmissible, the active epitope on the virus coat protein could be identified for designing future virus control strategies.     

北京地区番茄黄化曲叶病病毒分离物测定及株系的初步鉴定

 番茄黄化曲叶病毒病是番茄生产中的一种毁灭性病毒病害,2009年传入北京。利用烟粉虱传双生病毒简并引物PA/PB对2010年~2011年采集自北京市5个区县的53个番茄样品进行检测,30个表现典型黄化曲叶病症状的样品均扩增得到约500 bp的特异条带,测定了其中7个样品的部分序列,经序列比对分析表明其为番茄黄化曲叶病毒（Tomato yellow leaf curl virus, TYLCV）。利用TYLCV特异引物TJ-F/TJ-R、TY-F/TY-R对样品BJDXXY、BJFS02、BJFS03、BJMY2231进行TYLCV基因组克隆和序列测定,经分析4个样品携带的TYLCV基因组长度均为2 781碱基,编码6个蛋白。基因组序列比较发现,这4个分离物与TYLCV-Israel株系同源性达到98%以上;通过建立系统发育树,发现BJDXXY、BJFS02、BJFS03与河北分离物（HBLF4）、山东分离物（SDSG）亲缘关系较近,BJMY2231与上海分离物（TYLCV-Israel）、江苏分离物（JSNJ1）亲缘关系较近。     

trans-Dominant Inhibition of Geminiviral DNA Replication by Bean Golden Mosaic Geminivirus rep Gene Mutants

ABSTRACT Geminiviruses are a group of single-stranded DNA viruses that cause major losses on a number of important crops throughout the world. Bean golden mosaic virus (BGMV) is a typical bipartite, whitefly-transmitted geminivirus that causes a severe disease on beans (Phaseolus vulgaris) in the Western Hemisphere. The lack of natural resistance to geminiviruses has led to attempts to engineer resistance, particularly through the use of pathogen-derived resistance strategies. The rep gene contains several conserved domains including nucleoside triphosphate (NTP)-binding and DNA-nicking domains and is the only geminiviral gene necessary for replication. Previous analysis by our group and others has demonstrated that the NTP-binding and DNA-nicking domains are necessary for geminiviral DNA replication. The ability of the rep gene and rep gene mutants to interfere with geminiviral DNA replication, when expressed in trans, was examined using a transient assay in a tobacco suspension cell culture system. Wild-type (wt) and mutant rep genes were cloned into plasmids under the control of the cauliflower mosaic virus 35S promoter for in planta expression and coinoculated into tobacco cells with infectious clones of various geminiviruses. The wt rep gene from BGMV-GA was able to support replication of BGMV-GA DNA-B. Several different rep gene mutants, with function-abolishing mutations in the NTP-binding or DNA-nicking domains, were potent trans-dominant inhibitors of geminiviral DNA replication.     

海南甘蔗病毒病的调查及其病原分子鉴定

 海南是我国重要的甘蔗生产省份之一,但其甘蔗主要种植区感染病毒的种类和数量尚不十分清楚,且海南甘蔗花叶病病原病毒缺乏分子水平的系统鉴定。为明确海南甘蔗病毒病的种类、数量、分布及危害情况,本研究拟建立较为完整的甘蔗病毒病检测技术体系,对海南甘蔗病毒病展开调查,为甘蔗抗病毒基因工程及健康种苗发展提供参考。     

Tomato dwarf leaf curl virus, a new bipartite geminivirus associated with tomatoes and peppers in Jamaica and mixed infection with tomato yellow leaf curl virus

Genomic characterization using nonradioactive probes, polymerase chain reaction with degenerate primers for whitefly transmitted geminiviruses and nucleotide sequencing were used to describe a new bipartite geminivirus, associated with dwarfing and leaf curling of tomatoes and peppers in Jamaica. Partial DNA-A and DNA-B clones were obtained. DNA sequence analysis showed that tomato and pepper samples have a similar geminivirus associated with them. Nucleotide sequence identity > 92% between the common regions of DNA-A and DNA-B confirmed the bipartite nature of the Jamaican geminivirus isolates. Nucleotide sequence comparisons of DNA-A and DNA-B with those of geminiviruses representing the major phylogenetic groups of Western Hemisphere geminiviruses showed the greatest similarity to potato yellow mosaic virus and members of the Abutilon mosaic virus cluster of geminiviruses. This new virus is given the name tomato dwarf leaf curl virus (TDLCV) because of the dwarfing and leaf curling symptoms associated with infected tomato plants. Polymerase chain reaction and Southern hybridization showed mixed infections of TDLCV with tomato yellow leaf curl virus from Israel in 16% of the field samples of tomatoes and peppers.