海南岛番木瓜和扶桑上粉虱传双生病毒的检测及序列分析

粉虱传双生病毒(Whitefly-transmitted geminivirus,WTGV)是一类广泛发生在热带、亚热带地区植物上的具有孪生颗粒形态的单链DNA病毒,在分类上属双生病毒科(Geminiviridae)的菜豆金色花叶病毒属(Begomovirus),该属的大多数病毒都由2个组分(DNA-A和DNA-B)组成,为两条闭合环状ssDNA分子,长度相似,每条为2.5-2.8kb,少数病毒为单组分,仅有DNA-A组分[1].我国自1983年报道发现双生病毒以来,在云南、广西、广东和海南等省区的已相继发现多种双生病毒[2～6],表明这类病毒在我国的危害有蔓延和加重的趋势.本文对从海南番木瓜(Carica papaya)和扶桑(Hibiscus rosa-sinensis)上采集到的表现典型双生病毒症状的样品进行了分子检测,并对序列进行了分析.     

番茄烟粉虱传双生病毒PCR检测

烟粉虱传双生病毒(Whitefly-transmitted Gemini- viruses,WTGV)病是世界番茄生产上重要病害之一,已给美国、以色列、埃及、澳大利亚等国的番茄生产造成了严重损失,病原是一类具有孪生颗粒形态的单链环状DNA植物病毒,属双生病毒科(Geminiviridae)菜豆金色花叶病毒属(Begomovirus),其基因组由2个组分(DNA-A和DNA-B)组成,每个组分的大小为2.5～2.8kb,少数病毒为单组分;由烟粉虱(Bemisia tabaci)以持久方式传播[1].     

云南番茄曲叶病是由烟草曲茎病毒引起的

从云南省德宏田间表现曲叶症状的番茄植株上分离到病毒分离物Y41,采集的带病植株在实验室可经烟粉虱(Bemisia tabaci)传播到健康的番茄.用针对非洲木薯花叶病毒(ACMV)、印度木薯花叶病毒(ICMV)及秋葵曲叶病毒(OLCV)的15种单抗对病样进行TAS-ELISA检测,结果表明,番茄曲叶病是由菜豆金色花叶病毒属(Begomovirus)病毒引起的,但其抗原表位型与我国广西报道的中国番茄黄化曲叶病毒(TYLCCV)不同.对Y41进行DNA-A全序列测定和分析表明,Y41 DNA-A全长2743个核苷酸,共编码6个ORF,其中病毒链编码AV1和AV2两个ORF,互补链编码AC1、AC2、AC3和AC4 4个ORF.对Y41及其它双生病毒CP进行同源性比较及系统进化关系分析表明,Y41属于"旧世界"的粉虱传双生病毒,与我国报道的烟草曲茎病毒(TCSV)及印度报道的番茄曲叶Karnataka病毒(ToLCKV)同源性最高,达到98.8%.进一步比较基因组发现,Y41与TCSV AV1、AV2、AC1、AC2、AC3、AC4各ORF同源性分别为98.8%、96.6%、86.4%、93.3%、89.6%和89.7%,基因间隔区(IR)、DNA-A同源性分别为92.1%和93.4%,且在基因间隔区内含有相似的重复子序列及排列方式.这些结果表明:Y41是TCSV在自然条件下侵染番茄的一个分离物.     

从烟草上分离的中国番茄曲叶病毒及其卫星DNA全基因组结构特征

本研究从具有典型曲叶病症状的广西靖西烟草病植株上分离到病毒分离物JX-2,全基因组序列测定结果表明,JX-2 DNA-A 全长2 738个核苷酸,共编码6个开放阅读框架(open reading frames,ORFs),其中病毒链编码AV1 (CP)和AV2两个ORFs,互补链编码AC1、AC2、AC3和AC4 共4个ORFs.BLAST结果表明,JX-2 DNA-A与中国番茄曲叶病毒(Tomato leaf curl China virus,ToLCCNV)各分离物的相似性在93.0%~99.7%之间,其中与ToLCCNV广西番茄分离物ToLCCNV-G32的相似性最高,达99.7%,而与其它双生病毒的同源性均在88.0%以下,表明JX-2是ToLCCNV的一个分离物.基于JX-2和已报道的双生病毒属代表种DNA-A全基因组核苷酸序列构建的系统进化树显示,JX-2与ToLCCNV-G32分离物的亲缘关系最近,并与ToLCCNV其它分离物形成一个分支,而与其它10种双生病毒的亲缘关系均相对较远.利用双生病毒卫星DNAβ的特异性引物β01/β02在JX-2样品中扩增到DNAβ分子(JX-2β),全长为1 341个核苷酸,其互补链编码1个ORF (即βC1),并包含一个富含A序列和一个卫星病毒保守序列.序列分析表明,JX-2β与ToLCCNV伴随的DNAβ的相似性在91.0%~96.1%之间,其中与ToLCCNV-G61DNAβ和ToLCCNV-G18 DNAβ的相似性最高(96.1%),与其它卫星DNAβ的相似性均低于61.8%.基于JX-2β全基因组核苷酸序列构建的系统进化关系树显示,JX-2β与ToLCCNV G61分离物伴随的DNAβ亲缘关系最近,并形成一个独立的分支,再与ToLCCNV 其余两个分离物伴随的DNAβ形成一个较大的分支.这是首次报道从烟草中分离到的中国番茄曲叶病毒及其伴随卫星DNA分子的全基因组结构特征.     

入侵我国的木尔坦棉花曲叶病毒及其为害

棉花曲叶病是世界棉花生产上最具毁灭性的病毒病害,已在巴基斯坦、印度、苏丹、埃及和南非等国棉花产区广泛流行,造成巨大经济损失。目前,已克隆了与该病害相关的植物病毒8种,木尔坦棉花曲叶病毒（CLCuMV）即是其中之一,这些病毒均属双生病毒科菜豆金色花叶病毒属。CLCuMV是引起巴基斯坦、印度棉花曲叶病大流行的主要病原之一。该病毒由烟粉虱以持久方式传播,也可以嫁接传播,但不能通过机械摩擦接种传播和种子带毒传播;其基因组仅含有DNA-A组分,并伴随卫星β分子。自2006年首次在我国广东朱槿上检测与鉴定到该病毒以来,目前已在我国广东、广西和海南等多个地理区域发现该病毒引起的病害,受侵染寄主植物包括朱槿、黄秋葵、棉花和垂花悬铃花;同时,已入侵我国的CLCuMV及其卫星β分子的各地理区域和不同寄主来源的分离物DNA序列相似性均大于99％,遗传较稳定。基于文献报道及作者近年的研究,本文对棉花曲叶病的分布、病原、CLCuMV特性、已入侵我国的CLCuMV现状进行了较全面的综述,同时对入侵我国的CLCuMV来源及其威胁我国棉花生产的风险进行了讨论。CLCuMV＂对我国棉花等作物的威胁日益加剧,本研究可为该病毒的防控提供参考。     

侵染朱槿的木尔坦棉花曲叶病毒及其卫星DNA全基因组结构特征

从广州朱槿上分离到病毒分离物G6,全序列测定结果表明,G6 DNA-A全长为2 737个核苷酸.序列比较显示,G6 DNA-A与木尔坦棉花曲叶病毒(CLCuMV)各分离物的同源率均大于89%,其中与CLCuMV-[62]的同源率最高(96.1%),与拉贾斯坦棉花曲叶病毒(CLCuRV)的同源率87.1%～89.8%,而与其他菜豆金色花叶病毒属病毒同源率均在87%以下.DNA-A系统进化关系分析显示,G6与CLCuMV各分离物的亲缘关系最近,聚在一起形成一个分支,而与其他几种双生病毒的亲缘关系相对较远.利用DNAβ特异引物β01和β02,从G6中扩增到卫星DNA分子(DNAβ).序列分析结果表明,G6 DNAβ全长1 346个核苷酸,推导其互补链上编码一个ORF(C1).序列比较结果表明,G6 DNAβ与CLCuMV DNAβ的同源率最高(92.1%),与CLCuRV DNAβ的同源率为88.7%,而与其他已报道的DNAβ的同源率均在80%以下.DNAβ系统进化关系分析显示,G6 DNAβ与CLCuMV DNAβ形成一个独立的分支,再与CLCuRV及MYVV-[Y47]的DNAβ形成一个较大分支.从上述研究结果可以得出,侵染广东朱槿的病毒分离物G6应该是CLCuMV一个分离物.     

广东番茄曲叶病毒G2分离物基因组DNA-A的分子特征

从采集于广东的番茄曲叶病病株上分离到病毒分离物G2 ,序列分析结果表明 ,其DNA_A为单链环状 ,全长2 74 4nt,共有 6个ORF ,其中病毒链上编码AV1(CP)、AV2 ,互补链上编码AC1、AC2、AC3和AC4。BLAST结果显示 ,与G2基因组有同源关系的病毒均属双生病毒科菜豆金色花叶病毒属。序列比较结果显示 ,G2与菜豆金色花叶病毒属病毒的DNA_A序列同源率均不超过 83% ,其中同源率最高的是PaLCuCNV_[G10 ](82 8% )。进一步比较发现 ,它们的基因间隔区 (IR)变异最大 (同源率为 30 9%～ 81 8% ) ;CP氨基酸序列的同源率较高 (77 6 %～ 99 2 % ) ,AC4蛋白氨基酸序列的同源率较低 (4 3 5 %～ 78 8% )。系统进化关系分析结果也显示 ,G2与已报道的菜豆金色花叶病毒属病毒的亲缘关系均较远。因此 ,G2可能是双生病毒科菜豆金色花叶病毒属中一个未报道的新种 ,命名为广东番茄曲叶病毒 (TomatoleafcurlGuangdongVirus ,ToLCGDV)     

烟草曲茎病毒Y128分离物及伴随卫星的分子鉴定*

从云南保山田间表现曲叶症状的烟草植株上分离到病毒分离物Y128,病株在实验室可经烟粉虱(Bemisiatabaci)传播到健康烟草上。分别用针对烟草曲茎病毒(TbCSV)、云南烟草曲叶病毒(TLCYNV)、中国番茄黄化曲叶病毒(TYLCCNV)及泰国番茄黄化曲叶病毒(TYLCTHV)等田间常复合侵染的云南粉虱传双生病毒的特异性引物对Y128DNA-A进行PCR扩增,结果表明Y128是烟草曲茎病毒(TbCSV)的1个分离物。利用DNAβ的特异性引物β01和β02,在Y128中扩增到卫星DNA分     

侵染广西烟草的中国番茄黄化曲叶病毒及其伴随的卫星DNA分子的基因组特征

从中国广西靖西的烟草病株上分离到病毒分离物G102和G103,用双生病毒特异性引物均扩增出约500bp的片段,两者序列同源性达99%。对G102基因组DNA-A全序列测定表明,其全长为2728个核苷酸,与中国番茄黄化曲叶病毒(TYLCCNV)同源性最高,达96.5%。进一步研究发现,G102和G103都伴随有长为1342个核苷酸的卫星DNA分子(DNAβ),这两个DNAβ分子的全序列与TYLCCNV的DNAβ同源性最高,分别为92.9%和93.4%。这是首次明确广西分离的TYLCCNV也伴随有卫星分子。     

中国烟草曲叶病毒广西株的初步研究

双生病毒(Geminivirus)是一种具有孪生颗粒形态的单链环状DNA植物病毒[1].根 据基因组结构特征及传播介体,双生病毒可分为三个亚组[1]:亚组Ⅰ双生病毒全 部为叶蝉传播的单组份基因组病毒,基因组大小在2.6～2.8kb之间,其代表病毒是玉米条纹病毒(MSV);亚组Ⅱ双生病毒是单组份基因组病毒,基因组大小在2.7～3.0kb之间;亚组Ⅲ双生病毒全部为粉虱(Bemisiatabaci)传播,基因组大小为2.5～2.8kb,除番茄曲叶病毒TLCV -AUS [2]等几个病毒为单组份基因组外,大多数亚组Ⅲ双生病病毒均为双组份基因组,稍大的组份叫DNA A,一般编码四个基因,稍小的组份叫DNA B,编码二个基因.烟草曲叶病 毒(TbLCV)和番茄黄化曲叶病毒(TYLCV)都属于双生病毒亚组Ⅲ[3,4].     

Cloning and sequencing of partial DNA-A of Cassava mosaic virus

Abstract

Polymerase chain reaction of Cassava mosaic virus revealed that out of the 50 samples analysed only two samples, one from Musiri (Trichy district) and other from Mallur (Salem district), were detected with ICMV infection as 904 bp fragment of DNA-A amplified. All the other samples from various districts of Tamil Nadu were detected invariably with SLCMV as they amplified 599 bp of DNA-A. A 599 bp fragment of DAN-A was cloned and sequenced from the sample collected from Mallur. The nucleotide sequence has been submitted to GenBank under the accession number DQ303479. The nucleotide sequence was compared with other cassava infecting geminiviruses and other geminiviruses in GenBank. Cluster dendrogram revealed that the cloned sequence was most closely related to ICMV, Maharastra strain rather than SLCMV, forming one cluster. Comparative sequence analyses showed that the cloned fragment shared a maximum sequence identity with ICMV at nucleotide levels (93%) than with SLCMV (88%).     

Cloning, restriction mapping and phylogenetic relationship of genomic components of MYMIV from Lablab purpureus

The present work describes cloning of genomic components of whitefly transmitted geminivirus infecting Lablab purpureus syn. Dolichos lablab (commonly known as Dolichos bean or Hyacinth bean). The genome characterization using PCR with geminiviral degenerate primers and DNA sequencing were used to describe the bipartite virus associated with yellow mosaic disease of Dolichos lablab. Full-length DNA-A and DNA-B clones were obtained. The DNA-A sequence analysis showed that the isolate was similar to other Mungbean yellow mosaic India virus (MYMIV) isolates reported earlier. The nucleotide sequence analysis of the full-length DNA-A of virus isolate revealed more than 97% homology with Mungbean yellow mosaic India virus-[Cowpea] (AF481865), while the DNA-B also showed >95% homology with MYMIV-[Cp] (AF503580) and MYMIV-[Sb] (AY049771). The phylogenetic analysis of present isolate showed close relationship to legume geminiviruses. The nucleotide sequence analysis showed presence of six open reading frames (ORFs) in DNA-A, with 2 ORFs aligned in sense and 4 ORFs in antisense orientation. Similarly, DNA-B contained two open reading frames (ORFs), one in sense and another in antisense orientation.     

Mixed Infection of Two Begomoviruses in Malvastrum coromandelianum in Fujian, China

Virus isolates were obtained from three Malvastrum coromandelianum plants showing vein thickening symptoms in Fujian Province, China. A fragment of approximately 500 bp was amplified from all the samples by PCR using the special degenerate primer pair PA/PB for begomoviruses. Sequence differences among the partial DNA-A fragments revealed that all three samples contained two virus isolates. Isolate I and isolate II share the highest nucleotide sequence identity (98–99%), respectively, with Malvastrum leaf curl Guangdong virus (MLCuGdV) and Ageratum yellow vein virus (AYVV). The complete nucleotide sequences of Fs1 and Fs2 isolates representing each virus were determined to be 2741 and 2756 nucleotides, respectively. Alignment and phylogenetic analysis showed that the complete DNA-A sequences of Fs1 and Fs2 were most closely to those of MLCuGdV (AM503104) and AYVV (AB100305), with 90.4% and 93.3% nucleotide sequence identity, respectively. Fs1 and Fs2 are considered therefore to be isolates of MLCuGdV and AYVV, respectively. This is the first report of AYVV in M. coromandelianum.     

Molecular identification of 16S rII phytoplasma group in commercial pistachio cultivars in Iran

Evaluation of phytoplasmas infection was conducted in the pistachio-growing areas of Iran (Rafsanjan in Kerman province) in early autumn of 2011. A total of 30 pistachio trees collected from a pistachio orchard in Rafsanjan showing Psylla damage symptoms and 10 samples with different abnormal symptoms from miscellaneous orchards were tested for the presence of phytoplasma. By using nested PCR with primer pairs P1/P7 and internal primer sets R16F2N/R16R2 and fU3-rU5, amplified fragment of expected size was observed in some trees with deformation and yellowing symptoms. On the basis of nucleotide sequence analysis of 16S?rDNA amplified by PCR, this phytoplasma was classified in group 16S?rII. In addition, we observed false positive reaction in three trees of Ahmadaghaei cultivar by using primer sets R16F2N/R16R2 and sequence analysis of ~1250bp PCR product indicated that amplified fragment was related to Schinus terebinthifolius; a species of flowering plant in the Anacardiaceae family.     

Increased pathogenicity in a pseudorecombinant bipartite geminivirus correlates with intermolecular recombination.

Most whitefly-transmitted geminiviruses possess bipartite DNA genomes, and this feature may facilitate viral evolution through pseudorecombination and/or recombination. To test this hypothesis, the DNA-A and DNA-B components of the geminiviruses bean dwarf mosaic virus (BDMV) and tomato mottle virus (ToMoV) were exchanged, and the resultant pseudorecombinants were serially passaged through plants. Both pseudorecombinants were infectious in Nicotiana benthamiana but induced attenuated symptoms and had reduced DNA-B levels. Serial passage experiments revealed that the BDMV DNA-A plus ToMoV DNA-B pseudorecombinant could not be maintained beyond three passages. In contrast, the ToMoV DNA-A plus BDMV DNA-B pseudorecombinant was maintained during serial passage through N. benthamiana and Phaseolus vulgaris and, after three to five passages, became highly pathogenic. Furthermore, the increased pathogenicity of this pseudorecombinant was consistently associated with an increased level of DNA-B, which eventuated in equivalent levels of both components. Sequence analysis of the DNA-B component of the more pathogenic pseudorecombinant revealed that intermolecular recombination had taken place in which most of the BDMV DNA-B common region was replaced with the ToMoV DNA-A common region. This recombinant DNA-B component, which contained the ToMoV origin of replication, was the predominant DNA-B component associated with the more pathogenic pseudorecombinant. These results provide the first demonstration of recombination between distinct bipartite geminiviruses and establish that the bipartite genome can facilitate viral evolution through pseudorecombination and intermolecular recombination.     

Chinese tomato yelIow Ieaf curl virus— a new species of geminivirus

Chine tomato yellow leaf curl virus (TYLCV-CHI) and other geminiviruses were analysed with 20 monoclonaI antibodies. It was shown that TYLCV-CHI is serclogicaIly close to Chinese tabacco Ieaf curl virus (TbLCV-CHI). The fragment of TYLCV-CHI DNA including the common region (CR), N-terminal of coat protein gene and AV1 gene was amplified by PCR and cloned, and its DNA sequence was determined. These raults showed that TYLCV-CHI is different from other known geminiviruses in the world, and is a new whitefly-transmitted gerninivirus.     

Detection and Molecular Identification of Aster Yellows Phytoplasma in Date Palm in Egypt

Phytoplasma‐like symptoms were detected in date palm trees (Phoenix dactylifera L.) in Al‐Giza Governorate in Egypt. Symptoms varied from leaf chlorotic streaks, stunting and marked reduction in fruit and stalk sizes. Direct and nested PCR of symptomatic samples using P1/P7 and R16F2n/R16R2n primers, respectively, of the 16S rRNA gene, resulted in a DNA amplification product of c. 1.3 kbp. Symptomless samples collected from the same location and the healthy control produced no product upon amplification. Products were cloned into TOPO TA vector for sequencing. Data generated were deposited in the GenBank (Accession KF826615 ). A BLAST search showed that the sequence of the 16SrRNA gene shared ‘Candidatus Phytoplasma asteris’ (16SrI group) with other isolates. Phylogenetic analysis revealed that the isolate clustered with the date palm phytoplasma causing Al‐Wijam disease in Saudi Arabia.