侵染苘麻的菜豆金色黄花叶病毒的鉴定及基因组结构分析

为明确引起我国山西晋中地区苘麻叶片表现皱缩和花叶症状的病原物及其基因组分子特征, 本研究利用双生病毒简并引物扩增获得病毒基因组部分序列,经测序、比对后设计特异性引物扩增病毒基因组序列, 进而通过生物信息学方法构建系统发育树并进行序列分析。结果表明:引起苘麻叶片皱缩、花叶的病原物为番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV), 将该分离物命名为TYLCV-Abu, GenBank登录号为OP293347, 但未扩增到β卫星。该病毒DNA-A基因组全长为2 782 bp, 含有6个开放阅读框。TYLCV-Abu分离物与TYLCV茄子分离物KSQ1-3(GenBank登录号KC428753)的核苷酸序列一致性最高, 为98.99%, 其中C4和V2编码的蛋白变异较大。重组结果分析显示,分离物TYLCV-Abu是由TYLCV-F(GenBank登录号KY971326)和TYLCV-KSQ1-3重组得到, 重组区域为其基因组2 617－2 782 nt区域。这是首次从苘麻样品中扩增到TYLCV全基因组序列并进行分析。     

Infectivity analysis of a soybean isolate of Mungbean yellow mosaic India virus by agroinoculation

Yellow mosaic disease (YMD) of legumes endemic to South Asia are caused by begomoviruses transmitted by whiteflies. Based on molecular characterization, two distinct viruses – Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) – were found previously to be the etiological agents of YMD in legumes. Here, host range studies with a soybean isolate of MYMIV (MYMIV-[Sb]) were carried out by both whitefly transmission and agroinoculation. MYMIV-[Sb] was similar to a cowpea isolate of MYMIV (MYMIV-[Cp]) in its ability to infect cowpea, thus differing from blackgram (MYMIV) and mungbean (MYMIV-[Mg]) isolates, which do not infect cowpea. Genomic analysis of DNA A and DNA B components of these MYMIV isolates show characteristic differences in complete DNA B nucleotide sequence correlating with host range differences.     

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。     

Malvastrum leaf curl Guangdong virus is a distinct monopartite begomovirus

Virus isolates GD6, GD7, GD8, GD9 and GD10 were obtained from Malvastrum coromandelianum showing leaf curl symptoms in Guangdong Province of China. A specific 500 bp product was consistently detected in total DNA extracts, amplified with universal primers specific for members of the genus Begomovirus. Analysis of their partial DNA sequences revealed that they are isolates of the same begomovirus species, sharing 92·8%–97·1% nucleotide sequence identity. The complete DNA sequences of both GD6 and GD9 were found to be 2767 nucleotides, with all the characteristic features of begomovirus genome organization. The two isolates have less than 85·2% nucleotide sequence identity with other reported begomoviruses. Consequently, GD6 and GD9 are considered to be isolates of a novel begomovirus species, for which the name Malvastrum leaf curl Guangdong virus (MLCuGdV) is proposed. Sequence analyses suggest that MLCuGdV may have arisen by recombination between viruses related to Papaya leaf curl China virus , Tomato leaf curl Philippines virus and other undiscovered virus ancestors. Neither the DNA-B component nor the DNAβ molecule associated with these begomovirus isolates was found. An infectious clone of GD6 was constructed. GD6 efficiently infected Nicotiana benthamiana , N. glutinosa and Petunia hybrida by agro-inoculation, and Malvastrum coromandelianum by whitefly transmission, inducing leaf curling, vein swelling and stunting symptoms. GD6 was also infectious in N. tabacum , but did not induce observable disease symptoms.     

Sources of resistance to Watermelon Chlorotic Stunt Virus in melon

Watermelon Chlorotic Stunt Virus (WmCSV) is a bipartite geminivirus residing in the genus Begomovirus (family Geminiviridae) and is whitefly transmitted ( Bemisia tabaci ). WmCSV infects members of the Cucurbitaceae , and represents a considerable threat to melon and watermelon crops in Sudan, Yemen, and Iran. One hundred twenty-eight melon accessions, mostly collected from Sudan, were screened in the open-field in Sudan under severe whitefly inoculation pressure in order to identify sources of resistance to WmCSV. One WmCSV resistant line HSD 2445-005 selected from this first screening along with 22 new melon accessions were agro-inoculated with an infectious Sudanese clone of WmCSV. Ten accessions exhibited resistance to WmCSV infection upon agro-inoculation. Six of them were confirmed to have complete resistance to WmCSV through graft-inoculation as well as multiple field trials in Sudan. These lines include the Sudanese accession HSD 2445-005, the South African accession PI 282448, and three Indian accessions: 90625, PI 124112, and PI 414723. Resistant plants exhibited no disease symptoms, and virus DNA could not be detected by hybridization with a WmCSV-specific probe.     

番茄黄化曲叶病毒辽宁葫芦岛分离物的检测和序列分析

采用已报道的菜豆金色花叶病毒属的通用引物PA/PB,从辽宁葫芦岛地区的3份番茄样品中扩增到Begomoviruses的保守区域,获得3个长度约500 bp的克隆序列,同源性为99.58%.DNA-A全基因组序列分析结果显示,3份样品DNA-A全长均为2 781 bp(分别命名为LNhud1、LNhud2和LNhud3分离物),具有典型双生病毒结构,与番茄黄化曲叶病毒(TYLCV)山东分离物(TYLCV-[CN:SD:SDWF-L7:12],KC999850)的核苷酸同源性最高(99.6%).根据双生病毒分类标准,认为LNhud1、LNhud2和LNhud3是TYLCV的辽宁分离物.系统关系树表明,这3个分离物属于TYLCV-Isreal株系.     

Reduced incidence of tomato yellow leaf curl virus and leafminer in a tomato cultivar in northern Thailand

Tomato yellow leaf curl virus (TYLCV), a whitefly-vectored begomovirus, is a major limiting factor for tomato production worldwide, including Thailand. Field evaluation of commercial tomato cvs. for resistance to the virus endemic in northern Thailand has not been reported. Experiments conducted in northern Thailand in 2011–2013 compared the field performance of three tomato cvs. from the US with a commonly grown Thai cultivar. There were few significant differences observed among the cvs. in marketable yield, and the severity of root-knot and root rot. However there were significant differences noted in TYLCV incidence; virus incidence ranged from 38 to 100% and 8–41% in experiments one and two, respectively. In both experiments ‘Husky Cherry Red’ from the US had the lowest TYLCV incidence, leafminer damage (Liriomyza sp.), and progression of foliar necrosis primarily caused by thrips feeding and possibly exacerbated by air pollution. TYLCV infection was confirmed by a simplified method of nuclear inclusion visualization with light microscopy in experiment one, and by virus inclusion detection and PCR in experiment two.     

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。     

Tomato leaf curl geminivirus in Australia: occurrence, detection, sequence diversity and host range

The occurrence of whitefly transmitted geminiviruses in Australia was studied using a mixed DNA probe capable of detecting a range of distinct geminiviruses. The only geminivirus species detected was Tomato leaf curl virus (TLCV), which is spread across a vast geographical region of far-northern coastal Australia, an area inhabited by the Australasian-Oceania biotype of Bemisia tabaci . The newly introduced silverleaf whitefly, B. tabaci biotype B, forms high population densities in the eastern coastal region of Queensland and is currently located approximately 150 km from the nearest known TLCV-infected area. The viral host range appeared to be narrow and of 58 species of crop plants and weeds inoculated using the B biotype, only 11 became infected with the virus, including five that did not show foliar symptoms. A DNA fragment of 694 nt, including the complete C4 open reading frame (ORF), the overlapping N-terminal part of the C1 ORF and the viral iterons involved in replication, was amplified from 11 TLCV field isolates and sequenced. Sequence analysis revealed an overall sequence variation of up to 14% in this region, as well as the presence of distinct viral iterons.     

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.