中国农业科学2017，Vol.50Issue(24)：4725-4734,10.DOI:10.3864/j.issn.0578-1752.2017.24.006
侵染西番莲的夜来香花叶病毒的分子鉴定及特异性检测
Molecular Identification and Specific Detection of Telosma mosaic virus Infecting Passion Fruit
摘要
Abstract
[Objective] The objective of this study is to identify Telosma mosaic virus (TeMV) on passion fruit in Fujian orchard, develop a molecular method for specific and rapid identification of TeMV, and to provide a reference for prevention and control of the virus.[Method]The Fujian passion fruit samples were detected by using the serology method, electron microscopic observation, universal degenerate primers RT-PCR and specific primers RT-PCR. The PCR products of the positive sample were cloned and sequenced. A set of specific primes amplified the total length of coat protein (CP) gene was designed according to the sequences of reported TeMV and sequence determination of this study, and then specific primers RT-PCR detection method was established after optimizing reaction conditions. The sequence determination results were analyzed with BLAST program and DNAMAN software, and the phylogenetic tree was constructed based on the CP gene sequences obtained in this study by using Bayesian inference (BI) method implemented in MrBayes. [Result]The serology results showed that one passion fruit sample exhibiting mosaic and crinkle symptom reacted with Potyvirus antiserum.The positive sample was found to have about 750 nm×12 nm linear virions by electron microscopic observation. The expected fragment was amplified from the positive sample by universal degenerate primers RT-PCR, and then cloned and sequenced. Sequence analysis showed that the obtained sequence from the positive sample was identical with the expected size (680 bp), and shared the highest nucleotide sequence identity (98.2%) with the reported TeMV gene sequence. The full-length sequence of CP gene obtained by specific primers RT-PCR was 816 bp (named BXGFJ-13 isolate), and the sequence of nucleotide and amino acid of BXGFJ-13 was 86.2%-98.4% and 88.2%-97.8% identity, respectively, with the reported TeMV isolates. The results of phylogenetic analysis showed that the 13 TeMV isolates could be divided into 3 groups, and the same area or host derived isolates preferentially clustered together, suggesting these isolates had a strong geographical and host specificity. BXGFJ-13 isolate obtained in this paper and Guangxi isolate of China (KJ789129) clustered into a branch with high posterior probability, and then clustered together with two Thailand isolates (AM409188, AM409187) into the 2nd group (Group Ⅱ), showing that BXGFJ-13 and Guangxi isolate had the closest phylogenetic relationship. The specific primers RT-PCR showed good specificity, which only amplify the expected fragment from TeMV-infected passion fruit sample, and no expected fragment was obtained from Cucumber mosaic virus (CMV), Beet mosaic virus (BtMV), Soybean mosaic virus (SMV), Ornithogalum mosaic virus(OrMV),Onion yellow dwarf virus(OYDV),East Asian Passiflora virus(EAPV)and healthy control. The sensitivity results showed that the target fragment could be amplified from diluted 102fold RNA.[Conclusion]According to the species demarcation criteria for the Potyvirus given by International Committee on Taxonomy(ICTV)and the results of serological detection and electron microscopic observation, TeMV on the passion fruit sample exhibiting mosaic and crinkle symptom in Fujian orchard was confirmed. The established assay of specific RT-PCR could be used for rapid detection of TeMV.关键词
西番莲/夜来香花叶病毒/分子鉴定/检测Key words
passion fruit/Telosma mosaic virus(TeMV)/molecular identification/detection引用本文复制引用
谢丽雪,张小艳,郑姗,张立杰,李韬..侵染西番莲的夜来香花叶病毒的分子鉴定及特异性检测[J].中国农业科学,2017,50(24):4725-4734,10.基金项目
国家质检公益性行业科研专项(201410076)、福建省现代农业水果产业技术体系岗位项目(闽农科教〔2017〕129)、福建省农业科学院新兴特色果类创新团队(STIT2017-2-4)、福建省农业科学院院管A类项目三农业一融合(A2017-15) （201410076）
