两个马铃薯Y病毒黑龙江马铃薯分离物株系鉴定

为明确分离自黑龙江省克山县马铃薯上的2个病毒分离物KS4和KS7的分类地位,通过RTPCR扩增、克隆获得其基因组序列,利用重组分析程序包和最大似然法分别进行重组分析和系统发育分析。结果显示,分离物KS4和KS7的开放阅读框均有9 186个核苷酸,编码3 061个氨基酸,分离物KS4的核苷酸和氨基酸序列均与马铃薯Y病毒(potato virus Y,PVY)分离物Mb112一致率最高,分别为96.9%和98.4%;分离物KS7的核苷酸序列与PVY分离物12-94一致率最高,为97.4%,其氨基酸序列与PVY分离物SYR-Ⅱ-Be1一致率最高,为97.8%。重组分析表明,分离物KS4和KS7均为分离物N-605和Oz的重组体,其中KS4基因组5′-端的2 392个核苷酸来自分离物N-605,其余核苷酸来自分离物Oz;KS7基因组的第800～2 227个核苷酸和第5 637～8 950个核苷酸来自分离物N-605,其余核苷酸来自分离物Oz。系统发育分析发现,分离物KS4被聚类到N:O株系(PVY~(N:O)),分离物KS7被聚类到NTN株系(PVY~(NTN))b型。     

一株PVYNTN-NW黑龙江马铃薯分离物的检测鉴定

 马铃薯Y病毒(Potato virus Y,PVY)是马铃薯、烟草等茄科作物上的重要病毒,在与寄主共同进化过程中产生了许多株系。本文从黑龙江马铃薯样品中得到PVY分离物A12。ELISA结果表明A12被PVY^O的单克隆抗体特异性识别。A12开放阅读框为9 186个核苷酸,编码3 061个氨基酸,与SYR-II-Be1分离物的核苷酸和氨基酸序列一致率均最高,分别为98.3%和99.2%。系统发育分析发现A12与PVY^NTN-NW株系SYR-II型的分离物聚类到一起;重组分析表明,A12是N-605和O_z的重组体,重组类型与SYR-II-Be1相同。综合以上结果表明,A12属于PVY^NTN-NW株系SYR-II型。但与常见PVY^NTN-NW株系分离物在珊西烟引起叶脉坏死不同,A12产生花叶症状。A12辅助成分-蛋白酶在182位和245位的氨基酸均为精氨酸,而其它PVY^NTN-NW株系分离物为赖氨酸。本研究结果可为黑龙江马铃薯PVY的早期检测和有效防控提供理论指导。     

一株PVYNTN-NW黑龙江马铃薯分离物的检测鉴定

 马铃薯Y病毒(Potato virus Y,PVY)是马铃薯、烟草等茄科作物上的重要病毒,在与寄主共同进化过程中产生了许多株系。本文从黑龙江马铃薯样品中得到PVY分离物A12。ELISA结果表明A12被PVY^O的单克隆抗体特异性识别。A12开放阅读框为9 186个核苷酸,编码3 061个氨基酸,与SYR-II-Be1分离物的核苷酸和氨基酸序列一致率均最高,分别为98.3%和99.2%。系统发育分析发现A12与PVY^NTN-NW株系SYR-II型的分离物聚类到一起;重组分析表明,A12是N-605和O_z的重组体,重组类型与SYR-II-Be1相同。综合以上结果表明,A12属于PVY^NTN-NW株系SYR-II型。但与常见PVY^NTN-NW株系分离物在珊西烟引起叶脉坏死不同,A12产生花叶症状。A12辅助成分-蛋白酶在182位和245位的氨基酸均为精氨酸,而其它PVY^NTN-NW株系分离物为赖氨酸。本研究结果可为黑龙江马铃薯PVY的早期检测和有效防控提供理论指导。     

马铃薯Y病毒HC-Pro第182位赖氨酸残基参与引起烟草叶脉坏死

 马铃薯Y病毒(potato virus Y,PVY)是侵染烟草的最重要病毒之一。不同PVY株系侵染烟草可引起不同症状,有些PVY株系可引起烟草叶脉坏死,严重影响烟草的产量和品质。PVY A12分离物属于NTN-NW株系,但侵染珊西烟(Nicotiana tabacum cv. Xanthi)不能引起叶脉坏死。分析发现,PVY分离物A12 HC-Pro第182和245位的氨基酸均为精氨酸(R),而能引起叶脉坏死的其他NTN-NW分离物HC-Pro的这2个位点均为赖氨酸(K)。PVY坏死株系N605的HC-Pro第182位和245位氨基酸也均为K。本研究通过定点突变,将N605侵染性克隆PVY^N605-GFP HC-Pro第245位残基K突变为R,突变体仍然能够引起叶脉坏死,而将其HC-Pro第182位残基K突变为R,突变体不能引起叶脉坏死。Western blot检测发现,2个突变体与野生型病毒CP蛋白在珊西烟中的表达水平没有明显差异。沉默抑制实验结果显示,2个突变体和野生型的HC-Pro抑制RNA沉默能力没有发生变化。初步确定PVY^N605-GFP HC-Pro第182残基K是引起叶脉坏死的关键氨基酸,推测PVY A12分离物不能引起烟草叶脉坏死的原因是其HC-Pro第182残基R引起的。     

基于全基因序列的中国北方3省(区)马铃薯Y病毒遗传多样性分析

本研究以马铃薯Y病毒(PVY)全基因组为基础,分析吉林、黑龙江和内蒙古3省(区)PVY群体遗传多样性和群体分化,并评估突变、重组、选择等遗传力所起的作用。根据已报道的PVY全基因序列保守区设计4对引物,采用片段重叠法对来自内蒙古和吉林的24个PVY分离物全基因序列进行测定,并联合NCBI中已登录的9个黑龙江分离物全基因组序列进行遗传多样性参数评估、群体分化检验和分子变异等分析。结果显示,我国北方3省(区)PVY群体遗传多样性高,其中内蒙古和黑龙江PVY群体遗传多样性高于吉林群体,并且3个群体之间呈现一定程度的遗传分化。分子变异分析发现在PVY基因组中存在1 786个变异位点,表明我国北方3省(区)PVY群体变异程度较高,并且这种高变异度有85.54%来自各个马铃薯种植区内PVY个体的遗传变异。重组分析和系统发育分析发现,我国北方3省(区)PVY群体中重组株系占比高达90.3%,并具有明显的株系多样性,表明PVY重组株系已成为我国北方3省(区)马铃薯种植区的流行株系。选择压力分析显示,使用FEL和IFEL法分别检测出501个和315个净化压力选择位点,这表明3省(区)PVY群体受净化选择压力为主。以上结果表明,中国北方3省(区)PVY群体遗传多样性高,突变、重组和自然选择都对遗传多样性和群体分化存在一定影响。     

马铃薯Y病毒N-Wi株系广西分离物的鉴定

 马铃薯Y病毒 (potato virus Y,PVY) 是一种重要的农作物病毒,可造成产量损失和产品质量下降。其宿主范围广泛,包括马铃薯、烟草、番茄和辣椒等经济作物。在广西从叶片表现斑驳褪绿症状的马铃薯上分离到一株PVY分离物DX,其基因组包含一个大的开放阅读框 (open reading frame,ORF),由9 186 nt组成,编码3 061个氨基酸。系统发育进化分析显示,分离物DX与PVY^N-Wi株系分离物IUNG-12、SGS-AG、MAF-VOY聚类成一个分支。重组分析表明分离物DX基因组在496 nt和2 388 nt存在重组位点,分别位于P1和HC-Pro/P3结合区,是分离物Oz和N605的重组体。通过机械摩擦接种,分离物DX可侵染茄科9种作物,引起本生烟叶片花叶、皱缩和泡状突起等症状;引起普通烟草和番茄的轻微花叶症状;引起马铃薯叶片花叶症状,接种辣椒没有发病。序列比对分析显示,分离物DX缺乏引起烟草叶脉坏死相关的氨基酸位点N₂₀₅、K₄₀₀和E₄₁₉。本研究比较了分离物DX对茄科共12种作物的侵染能力和病害症状差异,结果表明分离物DX可用于探索PVY在不同寄主中的致病机理。     

马铃薯Y病毒在种薯中检出率及其株系鉴定

 马铃薯是我国重要粮食和经济作物。马铃薯Y病毒(potato virus Y,PVY)是危害马铃薯生产的重要病害。种植脱毒种薯是防治PVY最有效的途径。马铃薯种薯携带PVY问题严重,但种薯中PVY株系还不清楚。本研究利用PVY特异性抗体检测了7个马铃薯品种362个种薯,发现不同品种种薯带毒率差异较大,最高达12%。通过RT-PCR方法扩增获得了7个PVY分离物编码区全序列。重组分析发现7个分离物基因组均为重组型,根据重组位点的差异可以分为PVY^NTN-NW(SYR-II型)、Rec-1、Rec-2和Rec-3等4种重组类型,后3种为新重组类型。系统进化分析发现,分离物HQH18G3-10与PVY^NTN-NW(SYR-II型)处于同一个大的分支,但与中国PVY大田分离物聚集在一起形成一个相对独立的组,命名为PVY^NTN-NW(CN型);其余6个分离物与数据库中的中国分离物聚集在PVY^N-Wi组。这暗示PVY中国分离物具有相对独立的进化过程,PVY马铃薯大田分离物和种薯分离物进化上相近。所有分离物均能在珊西烟上引起典型叶脉坏死症状,HQH18G3-10引起的坏死症状最为严重。本研究首次报道了我国种薯内PVY发生情况,对分析病毒发生发展规律和防控具有借鉴作用。     

马铃薯Y病毒在种薯中检出率及其株系鉴定

 马铃薯是我国重要粮食和经济作物。马铃薯Y病毒(potato virus Y,PVY)是危害马铃薯生产的重要病害。种植脱毒种薯是防治PVY最有效的途径。马铃薯种薯携带PVY问题严重,但种薯中PVY株系还不清楚。本研究利用PVY特异性抗体检测了7个马铃薯品种362个种薯,发现不同品种种薯带毒率差异较大,最高达12%。通过RT-PCR方法扩增获得了7个PVY分离物编码区全序列。重组分析发现7个分离物基因组均为重组型,根据重组位点的差异可以分为PVY^NTN-NW(SYR-II型)、Rec-1、Rec-2和Rec-3等4种重组类型,后3种为新重组类型。系统进化分析发现,分离物HQH18G3-10与PVY^NTN-NW(SYR-II型)处于同一个大的分支,但与中国PVY大田分离物聚集在一起形成一个相对独立的组,命名为PVY^NTN-NW(CN型);其余6个分离物与数据库中的中国分离物聚集在PVY^N-Wi组。这暗示PVY中国分离物具有相对独立的进化过程,PVY马铃薯大田分离物和种薯分离物进化上相近。所有分离物均能在珊西烟上引起典型叶脉坏死症状,HQH18G3-10引起的坏死症状最为严重。本研究首次报道了我国种薯内PVY发生情况,对分析病毒发生发展规律和防控具有借鉴作用。     

两个马铃薯Y病毒不同株系分离物在三种寄主上的致病性分析

马铃薯是重要的粮食和经济作物。马铃薯Y病毒(potato virus Y,PVY)是危害马铃薯安全生产的重要病毒。近年来,危害我国马铃薯的PVY株系组成发生了显著变化。PVY重组型株系尤其是PVY^NTN-NW SYRI和SYRII型成为优势株系,但与传统株系分离物相比,优势株系分离物在不同寄主上的侵染性及其致病力还不清楚。本研究分析了PVY^N株系代表性分离物PVY^N605和PVY^NTN-NWSYRI型分离物GZ在本氏烟、普通烟和辣椒上的侵染性,比较了二者在本氏烟和普通烟上的致病力。结果表明,PVY^N605和PVY^NTN-NWSYRI-GZ分离物均能侵染本氏烟和普通烟,并在普通烟上引起叶脉坏死;PVY^N605不能系统侵染辣椒品种‘特大牛角王’,而PVY^NTN-NWSYRI-GZ可系统侵染辣椒品种特大牛角王。PVY^NTN-NWSYRI-GZ在本氏烟细胞间的移动速度明显慢于PVY^N     

一个马铃薯Y病毒山东分离物的分离与鉴定

 从具有典型花叶症状的马铃薯叶片中分离到马铃薯Y病毒(Potato virus Y,PVY)(本文称PVY-SD-TA分离物),扩繁后,提纯病毒,电镜下可观察到700~900 nm×11 nm的病毒粒体,病组织超薄切片观察可见风轮状的内含体结构,寄主反应特性研究表明其能侵染2科13种植物。SDS-PAGE电泳检测病毒编码的外壳蛋白亚基的分子量为33 kDa。以PVY-SD-TA基因组RNA为模板,应用RT-PCR方法和特异引物合成了外壳蛋白基因。对cDNA全序列分析表明,PVY-SD-TA CP基因核苷酸序列与N株系的同源性为96%,与GenBank中登录序列号为AJ390306的O株系分离物的同源性最高,为99%;与国内不同学者报道的PVY中国流行株的同源性分别为96%,97%和98%。通过以上生物学特性和分子水平的研究将PVY-SD-TA鉴定为普通株系(PVY^O株系)。     

Genetic diversity of Potato virus Y infecting tobacco crops in China

Genetic variability of Potato virus Y (PVY) isolates infecting potato has been characterized but little is known about genetic diversity of PVY isolates infecting tobacco crops. In this study, PVY isolates were collected from major tobacco-growing areas in China and single-lesion isolates were produced by serial inoculation on Chenopodium amaranticolor. Most isolates (88%) caused systemic veinal necrosis symptoms in tobacco. Of these, 16 isolates contained a PVY(O)-like coat protein (CP) and PVY(N)-like helper component proteinase (HC-pro) and, in this respect, were similar to the PVY(N-Wi), PVY(N:O), and PVY-HN2 isolates characterized from potato in Europe, the United States, and China, respectively; two isolates contained a PVY(O)-like HC-pro and a PVY(N)-like CP; another two isolates had recombination junctions in the CP-encoding region. Both the HC-pro and CP of PVY were under negative selection as a whole; however, seven amino acids in HC-pro and six amino acids in CP were under positive selection. Selection pressures differed between the subpopulations of PVY distinguished by phylogenetic analysis of HC-pro and CP sequences. When PVY isolates from potato were included, no host-specific clustering of the PVY isolates was observed in phylogenetic and nucleotide diversity analyses, suggesting frequent spread of PVY isolates between potato and tobacco crops in the field.     

Genetic diversity of the ordinary strain of Potato virus Y (PVY) and origin of recombinant PVY strains

The ordinary strain of Potato virus Y (PVY), PVY(O), causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. A novel substrain of PVY(O) was recently reported, PVY(O)-O5, which is spreading in the United States and is distinguished from other PVY(O) isolates serologically (i.e., reacting to the otherwise PVY(N)-specific monoclonal antibody 1F5). To characterize this new PVY(O)-O5 subgroup and address possible reasons for its continued spread, we conducted a molecular study of PVY(O) and PVY(O)-O5 isolates from a North American collection of PVY through whole-genome sequencing and phylogenetic analysis. In all, 44 PVY(O) isolates were sequenced, including 31 from the previously defined PVY(O)-O5 group, and subjected to whole-genome analysis. PVY(O)-O5 isolates formed a separate lineage within the PVY(O) genome cluster in the whole-genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVY(O) sequences separated into at least two distinct lineages on the whole-genome phylogenetic tree. To shed light on the origin of the three most common PVY recombinants, a more detailed phylogenetic analysis of a sequence fragment, nucleotides 2,406 to 5,821, that is present in all recombinant and nonrecombinant PVY(O) genomes was conducted. The analysis revealed that PVY(N:O) and PVY(N-Wi) recombinants acquired their PVY(O) segments from two separate PVY(O) lineages, whereas the PVY(NTN) recombinant acquired its PVY(O) segment from the same lineage as PVY(N:O). These data suggest that PVY(N:O) and PVY(N-Wi) recombinants originated from two separate recombination events involving two different PVY(O) parental genomes, whereas the PVY(NTN) recombinants likely originated from the PVY(N:O) genome via additional recombination events.     

小RNA深度测序鉴定昭通市烟草脉斑病病毒

烟草脉斑病在云南昭通市的烟草上发生危害严重,为明确昭通市烟草脉斑病的病毒种类,本研究采集昭通市的4个烟草种植区症状表现为疑似烟草脉斑病的烟草样品,采用小RNA深度测序技术对不同来源的混合样品进行小RNA测序分析。结果显示混合样品中的病毒分别属于烟草花叶病毒属Tobamovirus、马铃薯Y病毒属Potyvirus和马铃薯卷叶病毒属Polerovirus。根据不同病毒属设计通用引物,分别对不同地区的烟草样品进行RT-PCR验证,结果表明,烟草样品中病毒种类有烟草花叶病毒、烟草脉带花叶病毒、烟草脉扭病毒和马铃薯Y病毒的PVYN、PVYN-Wi和PVYNTN株系。     

Identification of the molecular make-up of the Potato virus Y strain PVY(Z): genetic typing of PVY(Z)-NTN

Potato virus Y (PVY) strains were originally defined by interactions with different resistance genes in standard potato cultivars. Five distinct strain groups are defined that cause local or systemic hypersensitive responses (HRs) in genetic background with a corresponding N gene: PVY(O), PVY(N), PVY(C), PVY(Z), and PVY(E). The nucleotide sequences of multiple isolates of PVY(O) and PVY(N) differ from each other by ≈8% along their genomes. Additionally, complete genome sequences of multiple recombinant isolates are composed of segments of parental PVY(O) and PVY(N) sequences. Here, we report that recombinant isolate PVY-L26 induces an HR in potato 'Maris Bard' carrying the putative Nz gene, and is not recognized by two other resistance genes, Nc and Ny(tbr). These genetic responses in potato, combined with the inability of PVY-L26 to induce vein necrosis in tobacco, clearly define it as an isolate from the PVY(Z) strain group and provide the first information on genome structure and sequence of PVY(Z). The genome of PVY-L26 displays typical features of European NTN-type isolates with three recombinant junctions (PVY(EU-NTN)), and the PVY-L26 is named PVY(Z)-NTN. Three typical PVY(NTN) isolates and two PVY(N) isolates, all inducing vein necrosis in tobacco, were compared with PVY-L26. One PVY(NTN) isolate elicited HR reactions in Maris Bard, similar to PVY-L26, while two induced a severe systemic HR-like reaction quite different from the quasi-symptomless reaction induced by two PVY(N) isolates. 'Yukon Gold' potato from North America produced HR against several PVY(NTN) isolates, including PVY-L26, but only late and limited systemic necrosis against one PVY(N) isolate. Consequently, according to symptoms in potato indicators, both PVY(Z) and PVY(NTN) isolates appeared biologically very close and clearly distinct from PVY(O) and PVY(N) strain groups.     

Diversity Among Potato virus Y Isolates Obtained from Potatoes Grown in the United States

ABSTRACT Potato field isolates (Solanum tuberosum) of Potato virus Y (PVY) collected from the midwestern and western United States were characterized using serological, molecular, and biological assays. PVY field isolates were grouped into the previously defined categories: PVY(O), European PVY(NTN), North American PVY(NTN), and PVY(N:O) recombinant and four previously undefined groups. Studies reported here agree with published reports from Europe and elsewhere in North America as PVY isolates capable of causing veinal necrosis in tobacco indicator plants appear in high frequency. In contrast to European experiences, PVY tuber necrosis isolates have a PVY(O) coat protein rather than that of PVY(N). Several PVY(N:O) recombinant isolates induced potato tuber necrotic ringspot disease (PTNRD) in the highly susceptible potato cv. Yukon Gold. The PTNRD symptoms produced by these PVY(N:O) recombinants were atypical compared with lesions found on the same cultivar infected with either the European or North American PVY(NTN) isolates. These PVY(N:O) isolates produced a roughly circular, sunken necrotic lesion on the surface of the tuber instead of the typical external sunken ring pattern displayed by PVY(NTN) isolates. This study establishes the complex nature of PVY populations within the U.S. potato industry and clearly demonstrates the diverse nature of PVY in the United States.     

马铃薯Y 病毒贵州黔西烟草分离物P1 基因序列分析

 Potato virus Y (PVY) is the type member of the genus Potyvirus and infects several important solanaceous crops, including tobacco. PVY is one of the most widespread and economically destructive viruses.The P1 region of PVY had been used to determine the diversity, evolution and the homology based on the
geographic location of the virus. The P1 region of PVY tobacco isolate from Qianxi in Guizhou Province was cloned and sequenced. Database searches and multiple sequence alignment showed subtle variation within the same strain, while apparent variation between O strain and the other three strains, N, NTN and N∶O. Moreover, variation in P1 region of Qianxi isolate derived from the gene mutation rather than the recombination among genomes. Phylogenic analysis revealed that the clustering only discriminated O strain from others. Consequently, sequence analysis in P1 region is unable to afford strain determination.     

来自安徽不同寄主PVY分离物的血清学鉴定及其cp基因分子进化分析

 Tobacco and potato samples showing symptoms of PVY were collected from different regions in Anhui Province, and the ELISA results of partial samples were positive. The total RNA was extracted from the positive samples by TRIZOL methods. Specific primer pair was designed to amplify cp gene of PVY by RT-PCR. Sequencing results indicated that the full length of cp gene of PVY from tobacco (PVY-CP-4) and pota-to (PVY-CP-7) is 801 nts, and each of them encodes 266 amino acids. A phylogenetic tree based on alignment of cp nucleotide sequences was constructed and the sequence comparing of cp gene was conducted. The results showed that PVY-CP-4 could be grouped into one branch with PVY^O and PVY^N:O and shared the highest sequence similarity (99.4%) with PVY^O (EF026074). It was suggested that PVY-CP-4 derived from tobacco in Hefei might belong to PVY^O. PVY-CP-7 clustered together with PVYN and PVYNTN and formed another branch. Furthermore, PVY-CP-7 shared the highest sequence similarity (98.3%) with PVY^NTN(AJ890347), PVY^NTN (EF026075) and PVY^NTN(AJ585342). It was supposed that PVY-CP-7 derived from potato in Wuhe probably belonged to PVY^NTN.