侵染木薯的两种单组分菜豆金色花叶病毒属病毒及卫星分子基因组结构特征分析

 为明确木薯(Manihot esculenta Crantz)植株叶片皱缩、畸形是否由菜豆金色花叶病毒属病毒侵染引起,从云南省红河州田间采集具有疑似感染症状的木薯植株叶片样品,应用菜豆金色花叶病毒属病毒简并引物、种专化性引物及卫星分子引物进行PCR扩增、克隆,通过测序分析其核苷酸序列特征并对其进行系统进化分析。结果显示,从采集疑似病叶中共克隆获得4条菜豆金色花叶病毒属病毒DNA-A全序列和6条beta卫星分子全序列,经全序列分析发现侵染木薯的2种菜豆金色花叶病毒属病毒分离物分别属于烟草曲茎病毒(tobacco curly shoot virus,TbCSV)和中国胜红蓟黄脉病毒(ageratum yellow vein China virus,AYVCNV)。TbCSV木薯分离物全基因组核苷酸序列与分离自云南的TbCSV-YN2247(KX290925)分离物亲缘关系最近,相似性最高达到96.67%;AYVCNV木薯分离物全基因组核苷酸序列与分离自云南的AYVCNV-YN4326(KU601622)分离物亲缘关系最近,相似性最高达到95.80%;侵染木薯的beta卫星分子分别为赛葵黄脉病毒beta卫星(malvastrum yellow vein betasatellite,MaYVB)和中国胜红蓟黄脉病毒beta卫星(ageratum yellow vein China virus betasatellite,AYVCNB),MaYVB-YN6332-12全基因组核苷酸序列与分离自云南的MaYVB-Y216(KX290925)分离物亲缘关系最近,相似性最高达到96.4%;AYVCNB-YN6338-17全基因组核苷酸序列与分离自海南的AYVCNB-Hn9(KU601622)分离物亲缘关系最近,相似性最高达到90.4%,表明木薯是这两种菜豆金色花叶病毒属病毒的新寄主。单组分菜豆金色花叶病毒属病毒及其伴随beta卫星分子可以复合侵染木薯植株为首次发现。     

野茼蒿黄脉病毒的田间寄主范围及其基因多样性特征

为明确野茼蒿黄脉病毒(Crassocephalum yellow vein virus,CraYVV)的田间寄主范围及其群体基因结构特征,分别采用克隆和测序技术对采自云南省西双版纳傣族州、红河哈尼族彝族自治州的7种杂草和2种作物进行CraYVV的分离和鉴定,并通过生物信息学软件对分离到的CraYVV进行基因组结构、重组及基因遗传结构分析。结果显示,在赛葵、龙葵、臭牡丹、水茄、豨莶、野茼蒿和一点红7种杂草以及茄子和草莓2种作物中均分离到CraYVV,共获得20条CraYVV全长序列;CraYVV所有分离物分属2个株系,将其命名为YJ株系和JH株系;JH株系具有地理隔离特征,与YJ株系存在一定的遗传距离;重组分析显示,CraYVV是由烟草曲茎病毒(tobacco curly shoot virus,TbCSV)和云南烟草曲叶病毒(tobacco leaf curl Yunnan virus,TbLCYnV)重组产生;遗传结构分析显示,CraYVV中C1基因变异最显著,其次是C4基因和基因间隔区。表明CraYVV能侵染5科9种杂草和作物,具有较广的寄主范围,且菜豆金色花叶病毒属病毒能够侵染草莓、CraYVV能够侵染茄子,显示来源于杂草的菜豆金色花叶病毒属病毒在自然条件下能够侵染作物。     

侵染青蒿的烟草曲茎病毒的分子鉴定及基因组结构特征分析

菜豆金色花叶病毒属病毒是一类在全球热带及亚热带地区造成严重经济损失的植物病毒,田间杂草是这类病毒重要的中间寄主。 本研究从云南省玉溪市采集了表现黄脉的青蒿植株,通过PCR扩增、克隆及测序从样品中获得两条菜豆金色花叶病毒属病毒DNA-A全基因组序列,分别为YN6393-23和YN6393-27,其全长均为2 739 bp,相似性为100%。序列分析发现,YN6393-23和YN6393-27的核苷酸序列与烟草曲茎病毒Tobacco curly shoot virus(TbCSV）的分离物YN4584的核苷酸序列相似性最高,为99.45%。根据国际病毒分类委员会对菜豆金色花叶病毒属病毒种的分类标准,全基因组序列相似性大于91%则为同种病毒,表明此病毒分离物为烟草曲茎病毒的一个分离物。这是菜豆金色花叶病毒属病毒侵染青蒿植株的首次报道。     

侵染云南雾水葛的菜豆金色花叶病毒属病毒的全基因组结构特征

 为明确云南雾水葛表现叶片黄化、花叶等症状的植株是否被菜豆金色黄花叶病毒属病毒侵染,本研究通过PCR扩增、克隆测序及核苷酸序列特征分析,确定样品中病原种类及其系统进化关系。结果显示,该病样中检测到雾水葛金色花叶病毒(pouzolzia golden mosaic virus,PouGMV),该分离物全基因组具有典型的菜豆金色花叶病毒属单组分病毒结构特征,与来自越南的Vietam分离物(KC857508)亲缘关系最近,相似性最高达94.6%,与PouGMV其他分离物亲缘关系相对较远,相似性在88.9%~93.2%之间。病毒alpha卫星分子检测及分析显示,该样品中检测到的alpha卫星分子,其全长核苷酸序列与云南番茄黄化曲叶alpha卫星(tomato yellow leaf curl Yunnan alphasatellite,TYLCYnA)相似性最高,达到92.3%。研究结果表明云南雾水葛中分离到PouGMV,且异源伴随TYLCYnA卫星分子。这是雾水葛金色花叶病毒伴随alpha卫星分子侵染雾水葛的首次报道。     

一种侵染鳢肠的双生病毒基因组特征

菜豆金色花叶病毒属病毒是热带及亚热带地区经济作物的重要病原病毒,该类病毒在田间的杂草寄主范围较为广泛。本研究从云南红河流域采集到叶脉黄化的鳢肠植株,经克隆获得了菜豆金色花叶病毒属病毒分离物YN3306,该分离物核苷酸序列全长2749 nt,具有典型的双生病毒基因组结构特征。进一步分析发现,分离物属于金腰剑曲叶病毒(Synedrella leaf curl virus,SyLCV),RDP软件分析表明,该分离物是由烟草曲茎病毒(Tobacco curly shoot virus,Tb CSV)、云南烟草曲叶病毒(Tobacco leaf curl Yunnan virus,TbLCYnV)和金腰剑曲叶病毒重组产生的病毒。本研究首次报道了GenBank中在印度注册的SyLCV可以侵染鳢肠植株。     

侵染苣荬菜的中国胜红蓟黄脉病毒及伴随的卫星基因组结构特征

杂草是菜豆金色花叶病毒属病毒的重要中间寄主,常富集多种该属病毒及其卫星病毒。2014年,在云南红河常见杂草苣荬菜Sonchus arvensis上出现了疑似菜豆金色花叶病毒属病毒病症状。利用克隆、测序和生物信息学分析技术对其所含病毒进行分离鉴定,结果从1株病样中共获得了两条菜豆金色花叶病毒属病毒全序列、两条β卫星全序列和一条α卫星全序列。序列分析显示,两条菜豆金色花叶病毒属病毒全序列与中国胜红蓟黄脉病毒相似性最高,分别为99%和96%,确定为中国胜红蓟黄脉病毒的分离物。两条β卫星全序列与赛葵黄脉β卫星相似性最高,为97%,确定为赛葵黄脉β卫星的一个分离物。α卫星全序列与中国番茄黄化曲叶α卫星相似性最高,为86.3%,是中国番茄黄化曲叶α卫星的一个分离物。这是菜豆金色花叶病毒属病毒病害复合体在中国侵染苣荬菜的首次报道。     

安徽省太和县中国南瓜曲叶病毒自然侵染南瓜的检测分析

中国南瓜曲叶病毒(squash leaf curl China virus, SLCCNV)是一种植物单链DNA病毒, 属于双生病毒科Geminiviridae菜豆金色花叶病毒属Begomovirus?在自然条件下该病毒可以侵染多种葫芦科作物, 如南瓜?甜瓜等?该病毒由烟粉虱进行持久性传播, 是影响瓜类产量和品质的重要病害?2021年4月-5月, 安徽省太和县温室大棚中的南瓜叶片出现斑驳?皱缩?卷曲以及植株矮化等现象, 我们采集了具有典型病毒病症状的南瓜病叶样品以及烟粉虱虫体进行RT-PCR鉴定, 结合测序结果鉴定危害南瓜的病毒为SLCCNV?为了进一步明确安徽太和地区SLCCNV的系统进化特征, 我们测定了SLCCNV的外壳蛋白基因(coat protein, CP)序列并进行系统发育分析, 结果表明安徽太和县所检测到的SLCCNV分离物与中国广东和海南地区的分离物亲缘关系较近, 并且与越南?菲律宾?柬埔寨?泰国等一些国家的分离物处于同一个大分支, 存在较小的地域差异性, 而与印度和孟加拉国地区的分离物亲缘关系相对较远, 处于不同的大分支?本研究是安徽省SLCCNV侵染南瓜的首次报道, 期望为该病害的预警和防控提供理论依据?     

侵染垂花悬铃花的木尔坦棉花曲叶病毒分子特征研究

 垂花悬铃花曲叶病是近期在广东发现的一种新病害,病株表现为叶片向上卷曲,叶脉肿大,叶脉变深绿色等症状。PCR检测结果显示, 该病样中均存在菜豆金色花叶病毒属病毒。基因克隆及序列分析结果表明,该病毒分离物（GD11）DNA-A全长为2 737 nt,具有菜豆金色花叶病毒属病毒基因组典型特征,为闭合环状单链DNA,编码6个ORFs;该序列与木尔坦棉花曲叶病毒(CLCuMV)各分离物序列的相似性均大于89.0%,其中与G6、Okra06及GX1等分离物序列的相似性大于99.0%。该病毒分离物也伴有卫星DNA β分子,其全长为1 348 nt,与CLCuMV各分离物的DNA β序列相似性大于85.0%,其中与G6、Okra06及GX1等DNA β的序列相似性均大于99.0%。因此,侵染广东垂花悬铃花的病毒分离物属于CLCuMV,且与入侵我国的朱槿分离物G6、黄秋葵分离物Okra06及棉花分离物GX1亲缘关系很近。本文首次报道了CLCuMV及其卫星β复合侵染垂花悬铃花。     

侵染假酸浆的泰国番茄黄化曲叶病毒全基因组结构特征

为明确假酸浆Nicandra physalodes叶片黄化、皱缩症状是否由菜豆金色花叶病毒属病毒侵染引起,本研究利用分子检测方法和生物信息学技术鉴定了假酸浆样品中的病毒种类。从采集的病样中克隆并获得了2条菜豆金色花叶病毒属病毒DNA-A全序列和1条beta卫星全序列,经全序列分析发现,该双生病毒的两条DNA-A全序列与泰国番茄黄化曲叶病毒(tomato yellow leaf curl Thailand virus, TYLCTHV)云南分离物TYLCTHV-YN1732一致性最高,达99.3%,亲缘关系较近;beta卫星的全序列与云南番茄曲叶beta卫星(tomato leaf curl Yunnan betasatellite, TLCYnB)的分离物YN5230一致性最高,达99.3%,亲缘关系较近。重组分析显示,假酸浆上分离的TYLCTHV-YN5735-12是一个重组病毒,有两个重组事件,一个主要发生在AV1的编码区,由中国番茄黄化曲叶病毒(tomato yellow leaf curl China virus, TYLCCNV)和广西大戟曲叶病毒(euphorbia lea...     

侵染中国陕西南瓜的中国南瓜曲叶病毒的鉴定和全基因组分析

为明确南瓜叶片上卷、黄化的症状是否由病毒侵染引起,本研究采用小RNA深度测序对采集自陕西地区的南瓜叶片样品进行了鉴定。结果显示,侵染南瓜样品的病毒可能是中国南瓜曲叶病毒(squash leaf curl China virus, SLCCNV)。经PCR扩增并且克隆测序获得了病毒的DNA-A和DNA-B组分的全基因组序列。序列比对发现,所克隆的DNA-A组分与SLCCNV海南分离物(SLCCNV-Hn61)DNA-A的一致性最高,为99.1%;DNA-B组分与SLCCNV-Hn61和三亚分离物SLCCNV-SY的DNA-B组分一致性最高,为96.8%。系统进化树分析发现所克隆的DNA-A和DNA-B组分分别与SLCCNV-Hn61和SLCCNV-SY的亲缘关系最近。以上研究结果表明侵染陕西南瓜叶片的病毒是SLCCNV的分离物。这是首次报道SLCCNV在陕西地区的危害,研究结果为当地经济作物南瓜的病害防控提供参考。     

Characterization of Tomato curly stunt virus: a new tomato‐infecting begomovirus from South Africa

The biological and molecular characterization of a virus recognized as a distinct begomovirus species, Tomato curly stunt virus (ToCSV), first observed in South Africa in 1997, is reported here. Whitefly‐transmission and host‐range studies were carried out using a Bemisia tabaci colony identified as the B‐biotype. The experimental host range of ToCSV spanned primarily species in the Solanaceae and Fabaceae. The complete ToCSV genome (2·766 kb) was amplified by PCR, cloned, and the DNA sequence determined. Phylogenetic analysis revealed that ToCSV was most closely related to Tobacco leaf curl Zimbabwe virus (TbLCZV), at 84% nucleotide identity, indicating that ToCSV is a new species in the genus Begomovirus that is probably endemic to southern Africa. The ToCSV genome sequence contained all of the hallmark coding and non‐coding features characteristic of other previously recognized monopartite begomoviruses. ToCSV is only the second begomovirus described from southern Africa that infects solanaceous species. Neither a begomoviral DNA‐B component nor a satellite‐like DNA molecule was detected by PCR in extracts of ToCSV‐infected plants.     

Agroinoculation Shows Tobacco leaf curl Yunnan virus is a Monopartite Begomovirus

We demonstrated that only 2 out of 15 isolates of Tobacco leaf curl Yunnan virus (TbLCYNV) were associated with the satellite DNAβ molecules. To investigate the infectivity of this virus, an infectious clone of TbLCYNV isolate Y143 (TbLCYNV-Y143) was agroinoculated or whitefly transmitted into Nicotiana benthamiana, N. glutinasa, Petunia hybrida and N. tabacum. TbLCYNV-Y143 alone was able to induce severe upward leaf curling, vein thickening or stunt symptoms in these plants. Co-inoculation of TbLCYNV-Y143 with DNAβ molecules associated with other begomoviruses induced similar symptom types on these plants. This indicates that TbLCYNV is a monopartite begomovirus. The relevance of results that only two isolates of TbLCYNV were associated with DNAβ molecules is discussed.     

Association of Tomato leaf curl Joydebpur virus and a betasatellite with leaf curl disease of eggplant

Leaf samples (five) from brinjal/eggplant fields showing upward leaf curling symptoms were collected from Varanasi, Uttar Pradesh state, India. The full length genome of begomovirus and associated betasatellite were amplified by PCR, cloned and sequenced. Sequences of homologous DNA-A and its betasatellite in all samples were the same. The samples failed to amplify DNA-B, suggesting that the begomovirus associated with leaf curl disease of eggplant was monopartite. The complete genome (homologous of DNA-A) consists of 2758 nts, whereas the betasatellite has 1352 nts and the genome organization is typical of Old World begomoviruses. The sequence analysis showed high levels of nucleotide sequence identity (79.8–91.7%) of virus with Tomato leaf curl Joydebpur virus (ToLCJoV) infecting chilli in India, suggesting it as a strain of ToLCJoV based on the current ICTV taxonomic criteria for begomovirus strain demarcation. However, the betasatellite associated was identified as a variant of Tomato leaf curl Bangladesh betasatellite (ToLCBDB), with which it shared highest sequence identity of 84.7–94.8%. Phylogenetic analyses of the genome further supported the above results. The recombination analyses of both genome and betasatellite showed that a major part of genome sequences are derived from begomoviruses (ToLCJoV, ChiLCuV, AEV) infecting chilli, tomato, ageratum and betasatellite from PaLCuB as the foremost parents in evolution, suggesting this as a new recombinant virus strain. This is the first report of a monopartite begomovirus and a betasatellite molecule associated with the leaf curl disease of eggplant.     

水蜡A病毒在呼和浩特市和哈尔滨市紫丁香上的发生及其基因组分子特征分析

为明确侵染紫丁香Syringa oblata并引起褪绿花叶症状的病毒种类及其基因组分子特征,利用透射电子显微镜对分离自呼和浩特市和哈尔滨市的紫丁香病样中的病毒粒子进行观察,并通过小RNA高通量测序和RT-PCR技术对其进行检测分析。结果表明,在紫丁香显症叶片的病毒粗提液中观察到长约600 nm、宽约13 nm的线状病毒粒子。利用小RNA高通量测序和RT-PCR技术从病样中检测到水蜡A病毒（Ligustrum virus A,LVA）,发病率为3.7%。呼和浩特市紫丁香分离物LVA-Sob的基因组序列全长8 525 nt,包含6个开放阅读框,分别编码Rep（1 968 aa）、TGB1（229 aa）、TGB2（107 aa）、TGB3（60 aa）、CP（294 aa）和NABP（119 aa）共6个蛋白。序列一致性分析表明,分离物LVA-Sob与韩国水蜡树分离物LVA-SK的基因组序列一致率高达97.9%,而与我国辽宁省暴马丁香分离物LVA-DX的基因组序列一致率仅为73.6%。在这3个LVA分离物基因组中没有检测到重组事件;基于基因组和cp基因序列的系统发育树显示这3个LVA分离物形成一个分支,并与瑞香S病毒（daphne virus S,DVS）有较近的亲缘关系。     

Ageratum yellow vein China virus Is a Distinct Begomovirus Species Associated with a DNAbeta Molecule

ABSTRACT Ageratum conyzoides plants exhibiting yellow vein symptoms, collected near Haikou, Hainan Province, China, contained begomoviral DNA-A-like molecules. The complete sequences of the molecules from two samples, Hn2 and Hn2-19, were shown to consist of 2,768 and 2,748 nucelotides (nt), respectively. These sequences have more than 97% nucleotide sequence identity, but less than 86% identity with other reported begomovirus sequences. In line with the taxonomic convention for begomoviruses, Hn2 and Hn2-19 are therefore considered to represent isolates of a distinct begomovirus species, for which the name Ageratum yellow vein China virus (AYVCNV) is proposed. Sequence alignment shows AYVCNV has arisen by recombination among viruses related to Ageratum yellow vein virus, Papaya leaf curl China virus, and an unidentified begomovirus. Southern blot analyses revealed that all plants sampled contained molecules resembling DNAbeta. DNAbeta molecules from three samples were 1,323 or 1,324 nt long and had >98% sequence identity but <81% identity with previously reported DNAbeta sequences. Infectious clones of Hn2 and its associated DNAbeta were constructed and agroinoculated to plants. Hn2 alone caused sporadic asymptomatic systemic infection of Nicotiana benthamiana, N. glutinosa, Lycopersicon esculentum, Petunia hybrida, and A. conyzoides but its accumulation was much enhanced in plants co-inoculated with DNAbeta. The co-inoculated N. benthamiana, N. glutinosa, P. hybrida, and L. esculentum plants developed leaf curling or leaf crinkling symptom; those in A. conyzoides were typical of ageratum yellow vein disease. When the DNAbeta molecules associated with four other Chinese begomoviruses were coinoculated with Hn2 to N. benthamiana and N. glutinosa, the DNAbeta molecules were replicated, and the plants developed systemic symptoms of types that were specific for each DNAbeta. This illustrates that there is less specific interaction between monopartite begomovirus and DNAbeta than between the DNA-A and DNA-B of begomoviruses with bipartite genomes.     

Tomato leaf curl Guangxi virus is a distinct monopartite begomovirus species

Three begomovirus isolates were obtained from tomato plants showing leaf curl symptoms in Guangxi province of China. Typical begomovirus DNA components representing the three isolates (GX-1, GX-2 and GX-3) were cloned and their full-length sequences were determined to be 2752 nucleotides. Nucleotide identities among the three viral sequences were 98.9–99.7%, but all shared <86.7% nucleotide sequence identity with other reported begomoviruses. The sequence data indicated that GX-1, GX-2 and GX-3 are isolates of a distinct begomovirus species for which the name Tomato leaf curl Guangxi virus (ToLCGXV) is proposed. Further analysis indicated that ToLCGXV probably originated through recombination among viruses related to Ageratum yellow vein virus, Tomato leaf curl China virus and Euphorbia leaf curl virus. PCR and Southern blot analyses demonstrated that isolates GX-1 and GX-2 were associated with DNAβ components, but not isolate GX-3. Sequence comparisons revealed that GX-1 and GX-2 DNAβ components shared the highest sequence identity (86.2%) with that of Tomato yellow leaf curl China virus (TYLCCNV). An infectious construct of ToLCGXV isolate GX-1 (ToLCGXV-GX) was produced and determined to be highly infectious in Nicotiana benthamiana, N. glutinosa, tobacco cvs. Samsun and Xanthi, tomato and Petunia hybrida plants inducing leaf curl and stunting symptoms. Co-inoculation of tomato plants with ToLCGXV-GX and TYLCCNV DNAβ resulted in disease symptoms similar to that caused by ToLCGXV-GX alone or that observed in infected field tomato plants.