采用多基因联合方法鉴定福建长乐和福清产区马铃薯Y病毒株系组成

【目的】马铃薯Y病毒(Potato virus Y,PVY)是马铃薯生产上危害较为严重的病毒,也是制约马铃薯可持续发展的主要病毒之一。论文旨在开发一套简便、准确、快速的PVY分子鉴定技术,并采用该技术及时查明福建省部分产区PVY病害的发生、分布及PVY株系组成。【方法】采用ELISA方法对采自福建省长乐市、福清市马铃薯种植区疑似受PVY感染的样品进行检测,并根据文献报道的PVY P1、VPg和CP基因保守区设计3对简并引物,对ELISA检测后的阳性样品进行基因扩增、克隆,并将获得的序列进行核苷酸序列一致性、重组位点、基因型分布和系统发育分析。【结果】ELISA检测结果表明,17份样品中有13个样品与PVY抗体呈阳性反应,其他呈阴性反应。13个阳性样品均能成功扩增出3个与P1、VPg和CP基因预期大小一致的特异片段。BLAST比对分析显示P1、VPg和CP基因与文献报道的已知PVY分离物的核苷酸序列一致性分别为72%—99%、85%—99%和88%—99%。P1、VPg和CP 3个基因联合序列分析显示,FQ01分离物与PVY~(N-Wi)株系的核苷酸序列一致性最高,FQ08分离物与PVYE株系的核苷酸序列一致性最高,CL01、CL02、CL05和CL13 4个分离物与PVY~(NTN-NW)株系SYR-I型的核苷酸序列一致性最高,CL03、CL04、FQ02、FQ06、FQ09、FQ11和CL12 7个分离物与PVY~(NTN-NW)株系SYR-II型的核苷酸序列一致性最高。重组分析显示,除CP基因外,长乐市和福清市两个产区的PVY分离物的P1和VPg基因中均检测到显著的重组信号。基因型统计分析结果显示,长乐产区的P1基因为N型(60%)和N×O重组型(40%),VPg基因均为N×O重组型,而CP基因均为O型,而福清产区的P1基因为N型(25%)和N×O重组型(75%),VPg基因为N×O重组型(87.5%)和O型(12.5%),而CP基因除了一个分离物为N型外,其他均为O型。系统发育分析显示分离物FQ01与PVYN-Wi株系聚为一簇,FQ08与PVYE株系聚为一簇,CL01、CL02、CL05和CL13与PVY~(NTN-NW)株系SYR-I型聚为一簇,CL03、CL04、FQ02、FQ06、FQ09、FQ11和CL12与PVY~(NTN-NW)株系SYR-II型聚为一簇,表明在系统发育关系上,分离物FQ01与PVYN-Wi株系最近,FQ08与PVYE株系最近,CL01、CL02、CL05和CL13与PVY~(NTN-NW)株系SYR-I型最近,CL03、CL04、FQ02、FQ06、FQ09、FQ11和CL12与PVY~(NTN-NW)株系SYR-II型最近。【结论】PVY在福建省长乐市、福清市马铃薯种植区普遍存在,重组株系已成为田间的主流株系,且PVY~(NTN-NW)已成为优势重组株系。

Strain Composition of Potato virus Y in Fujian Province Detected with the Concatenated Sequence Approach

【Objective】Potato virus Y (PVY) is one of the most destructive pathogens constraining sustainable development of potato industry. The objective of this study is to develop a fast, easy-to use and accurate approach to timely detect PVY strains and apply the approach to investigate the occurrence, distribution and composition of PVY strains in Fujian province. 【Method】ELISA method was used to confirm the presence of virus in the PVY-alike leaf samples randomly collected from Changle and Fuqing cities in Fujian province. Three pairs of degenerate primers designed from the conserved regions of P1, VPg and CP genes in the reference PVY sequences downloaded from GenBank were used to amplify the positive samples by ELISA test. Nucleotide identity and recombination events between isolates from Fujian and strains downloaded from GenBank were evaluated and phylogenetic tree was reconstructed using concatenated sequences of the three genes. 【Result】ELISA confirmed that 13 out of the 17 samples collected from the two potato producing areas were infected with PVY. RT-PCR amplifications of all 13 ELISA-positive samples generated the expected sizes of fragments corresponding to the three genes. Individual gene analyses showed that P1, VPg and CP sequences in Fujian isolates shared 72%-99%, 85%-99% and 88%-99% nucleotide identity with the reference strains, respectively. Concatenated sequence analysis showed that FQ01 and FQ08 isolates shared the highest sequence identity with PVY^N-Wi and PVY^E, respectively; CL01, CL02, CL05 and CL13 isolates shared the highest sequence identity with PVYN^TN-NW (SYR-I), whereas CL03, CL04, FQ02, FQ06, FQ09, FQ11 and CL12 shared the highest sequence identity with PVY^NTN-NW (SYR-II). Recombination signals were identified in P1 and VPg genes but not in CP of PVY, which isolated from Changle and Fuqing cities in Fujian province. Further analyses showed that in Changle City, P1 gene was composed of two lineages, with 60% being N lineage and 40% being N×O lineage. All VPg genes were N×O lineage and all CP genes were classified as O lineage. Similarly in Fuqing city, P1 gene was consisted of two lineages, with 25% being N lineage and 75% being N×O lineage; 87.5% VPg genes were N×O lineage and 12.5% was O lineage; and all except one (which was classified as N lineage) CP genes were O lineage. Phylogenetic tree revealed that isolates CL01, CL02, CL05 and CL13 were grouped with PVY^NTN-NW (SYR-I) and isolates CL03, CL04, FQ02, FQ06, FQ09, FQ11 and CL12 were clustered with the PVY^NTN-NW (SYR-II), whereas isolate FQ01 was grouped with PV^YN-Wi and isolate FQ08 was clustered with the PVY^E, respectively. These results indicated that evolutionarily, CL01, CL02, CL05 and CL13 are closer to PVY^NTN-NW (SYR-I); CL03, CL04, FQ02, FQ06, FQ09, FQ11 and CL12 are closer to PVY^NTN-NW (SYR-II), FQ01 is closer to PVY^N-Wi and FQ08 is closer to PVY^E, respectively. 【Conclusion】PVY occurs frequently in Changle and Fuqing cities in Fujian province and recombinant strains, particularly PVY^NTN-NW are dominant in the PVY isolates from the province.