马铃薯Y病毒中国分离物的分子株系组成

 马铃薯Y病毒(potato virus Y,PVY)主要侵染马铃薯和烟草等茄科作物,给世界农业造成巨大经济损失。本文对测定的23个及GenBank中注册的52个中国PVY分离物ORF序列进行了系统发育、重组和选择压等分析。系统发育分析表明,根据ORF序列可把我国75个PVY分离物和国外30个参比分离物分成O、C、E、NTN-NW(SYR-I型)、NTN-NW(SYR-II型)、NTN(NTN-a型)、NTN(NTN-b型)、NA-N/NTN、Eu-N、N-Wi(N:O型)和N-Wi(N-Wi型)等11个分子株系,其中中国PVY分离物属于除E和C株系外的9个分子株系。除ME162、guiyang、PVYzu、SD-G、WA-13和CN:JL-1:17等 6个分离物基因组中未检测到重组,其余69个分离物均存在明显重组。根据重组位点的不同,中国PVY可分为11种重组类型,其中5种为新的重组类型。选择压分析表明,中国PVY分离物的11个基因均处于负选择,其中核内含体b基因受到的选择压最大,PIPO受到的选择压最小。基因流分析表明,黑龙江、河南和山东PVY分离物间基因交流频繁,马铃薯与烟草PVY分离物之间基因交流频繁。本研究的结果明确了中国PVY分离物的分子株系组成,对指导PVY的检测和防控具有积极作用。     

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.     

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.     

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.     

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.     

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。     

A novel type of Potato virus Y recombinant genome,determined for the genetic strain PVYE

Two Potato virus Y (PVY) isolates collected in Brazil, PVY‐AGA and PVY‐MON, were identified as recombinants between two parent genomes, PVY^NTN and PVY‐NE‐11, with a novel type of genomic pattern. The new recombinants had an ordinary PVY^NTN genome structure for approximately 6·7‐kb from the 5′‐end of the genome whereas the 3′‐terminal 3·0‐kb segment had two fragments of NE‐11‐like sequence separated by another small PVY^NTN‐like fragment. PVY strains are defined based on the hypersensitive resistance (HR) response in potato indicators. Both PVY‐AGA and PVY‐MON isolates did not induce the HR in potato cultivars carrying Ny, Nc, or (putative) Nz genes and thus were able to overcome all known resistance genes to PVY. Only one of the two isolates, PVY‐AGA, induced a vein necrosis reaction in tobacco. The biological responses of the potato indicators and tobacco defined PVY‐MON as an isolate of the PVY^E strain. To distinguish PVY‐AGA and PVY‐MON from other PVY^NTN isolates, an RT‐PCR test was developed utilizing new specific primers from the capsid protein gene area and producing a characteristic 955‐bp band. Serological profiling of these PVY isolates with three monoclonal antibodies revealed an unusual reactivity, where one of the two commercial PVY^N‐specific monoclonal antibodies did not recognize PVY‐AGA. The ability of these new PVY recombinants to overcome resistance genes in potato producing mild or no symptoms, combined with the lack of serological reactivity towards at least one PVY^N‐specific antibody may present a significant threat posed by these isolates to seed potato production areas.     

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。     

Serological Analysis and Coat Protein Sequence Determination of Potato Virus Y (PVY) Pepper Pathotypes and Differentiation from Other PVY Strains

The serological relationships of Potato Virus Y (PVY) isolates belonging to the pepper pathotypes 0, 1 and 1-2 were established by enzyme-linked immunosorbent assay (ELISA). PVY pepper pathotypes did not react with monoclonal antibodies which typically recognize non-pepper strains within the PVY group, leading to discrimination between these two groups of strains. No serological differences were found between the three PVY pepper pathotypes. The coat protein (CP) nucleotide and predicted amino acid sequences of the three different PVY pepper pathotypes were determined. The highest sequence similarity was found between pathotypes 0 and 1 (99.2%), while the lowest occurred between these two and pathotype 1–2 (98.1%). PVY strains from potato and tobacco appeared more distantly related. Phenetic analysis of the CP amino acid sequences showed that the PVY pepper pathotypes formed a tightly clustered group separate from other PVY strains.     

The effects of co‐infection by different Potato virus Y (PVY) isolates on virus concentration in solanaceous hosts and efficiency of transmission

The influence of co‐infection on concentration and accumulation of genetically different isolates of Potato virus Y (PVY) in potato and tobacco plants and the efficiency of transmission by Myzus persicae of PVY isolates from doubly versus singly infected plants were evaluated. The vector ability to simultaneously transmit two virus isolates was examined. Eight PVY isolates represented three strain groups: PVY^O (pathotype and serotype O), PVY^NW (pathotype N and serotype O), and PVY^NTN (pathotype and serotype N). Different diagnostic methods, including DAS‐ELISA, multiplex RT‐PCR, aphid transmission tests and bioassays, were applied to detect the presence of PVY isolates in source and assay plants. Significant reductions in concentrations of certain PVY isolates during co‐infection with other isolates were found both in potato and tobacco plants. The observed effects were both isolate‐ and host‐dependent in form. The highest rates of virus transmission by single aphids were recorded with PVY^NTN isolates, and the lowest ones with PVY^O isolates. Individual aphids of M. persicae were able to simultaneously transmit two PVY isolates. The frequency of transmission was generally low, but it reached as high as 20% for one of the isolate combinations. The findings presented in the work provide proof for antagonistic within‐plant interactions between isolates of PVY, with some implications of these interactions for virus transmission by aphid vectors. Consequently, this research contributes to a better understanding of the epidemiology of the disease caused by PVY.     

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。     

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。     

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。     

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。     

Brazilian Potato virus Y isolates identified as members of a new clade facilitate the reconstruction of evolutionary traits within this species

Potato virus Y (PVY) is a plant virus distributed worldwide that causes damage to several species of the Solanaceae family. It was established long ago that groups of PVY isolates defined by phylogenetic analyses correlate strongly with those demarcated by differential biological properties. Consequently, life‐history traits of this viral species can be inferred by phylogenetic analysis. In this study, characteristics of PVY isolates sampled in different tobacco fields in Brazil were analysed and most of the tested Brazilian PVY isolates were assigned to the recently described unconventional serogroup Y^U. The analysis of molecular diversity of the coat protein (CP) cistron from some Y^U isolates made it possible (i) to identify specific amino acid residues in the N‐terminal of the CP protein and (ii) to assign some Y^U isolates to a new PVY clade. The symptoms caused by isolates belonging to this new PVY ‘Brazilian’ clade and their ability to infect selected susceptible hosts led to the conclusion that neither veinal necrosis symptoms expressed on infected tobacco plants nor adaptation to potato or pepper hosts are ancestral characteristics of PVY. These observations suggest that PVY has gained a remarkable new biological property and broadened its host range over time.     

利用RNA介导的抗病性获得高度抗马铃薯Y病毒的转基因烟草

 以马铃薯Y病毒坏死株系(PVY^N)的RNA为模板,应用反转录-聚合酶链式反应(RT-PCR)方法,扩增出长度为801 bp的非翻译的马铃薯Y病毒外壳蛋白基因。将扩增的片段克隆到pBSK的BamHI和KpnI之间并进行了序列测定。用BamHI和KpnI从重组克隆载体上切下该基因并插入到质粒pROKII内得到植物表达载体pPVYCP。通过根癌农杆菌(LBA4404)介导的方法转化烟草NC89,经卡那霉素抗性筛选、PCR和Southern blot检测,获得82株转基因植株。Northern blot和Western blot分析表明,转基因植株只在RNA水平上得到了表达。抗病性试验表明转基因植株之间抗性水平存在着差异,其中有7株是对PVY^N高度抗病性的植株。转基因植株抗病特异性试验初步表明,对PVY^N表现高度抗病的植株对PVY^O也具有高度抗病性。