湖南省长沙市番茄黄化曲叶病毒的检测与系统发育分析

Detection and phylogenetic analysis of Tomato yellow leaf curl virus in Changsha,Hunan,China

【目的】对湖南省长沙市发生的疑似番茄黄化曲叶病毒(TYLCV)样本进行检测,明确TYLCV在湖南长沙的发生情况,为TYLCV的防控提供科学依据。【方法】2016年3和11月,在湖南长沙采集疑似TYLCV样本48份,采用已发表的TYLCV不同株系的全基因组序列,利用DNAMAN进行序列比对,根据保守序列设计特异性引物TY-T-F和TYT-R、TY-P-F和TY-P-R对番茄样本的DNA进行PCR、Southern印迹杂交(Sourthern blotting)检测和系统发育分析。对呈阳性条带的PCR产物进行切胶回收并送样测序,在NCBI上进行BLAST比对分析,利用系统发育分析TYLCV湖南长沙分离物与国内外株系的相似性。【结果】采集的48份样本中有42份样本能扩增出目的条带,阳性检出率为87.5%;将42个阳性克隆序列导入MEGA 7.0,得到27个分离物。对测序结果进行同源性分析、多重比对、聚类分析及进化分析,结果显示27个湖南长沙TYLCV分离物与我国其他省(市)分离物的相似性在98.5%~99.8%,与伊朗株系(AJ132711和GU076453)、墨西哥株系(FJ012358)、澳大利亚株系(GU178814)和葡萄牙株系(AF105975)的相似性分别介于90.7%~91.5%、95.4%~95.5%、98.6%~98.8%和99.3%~99.4%。【结论】TYLCV已在湖南长沙发生危害,需加强检疫工作,防止从疫区调运感病番茄,以控制病害扩散蔓延。

【Objective】In order to clarify the occurrence of Tomato yellow leaf curl virus(TYLCV)in Changsha,Hunan,the scientific basis for the prevention and control of TYLCV was provided,and the suspected TYLCV samples collected in Changsha,Hunan were tested.【Method】In March and November of 2016,48 samples of suspected TYLCV were collected in Changsha,Hunan.Sequences from the published whole genomes of different TYLCV strains were aligned by using DNAMAN,then the specific primers TY-T-F and TY-T-R,TY-P-F and TY-P-R were designed according to the conserved fragments,and the DNA of the tomato sample was detected by PCR,Southern bloting and phylogenetic analysis.The PCR products of some positive bands were excised for electrophorosis gel and sequenced.The sequencing result were performed by BLAST comparison analysis on NCBI,and the similarity among Changsha isolates,domestic and foreign strains were studied by phylogenetic analysis.【Result】Among the 48 samples collected,42 samples were dected the target band with a positive detection rate of 87.5%.The 42 positive clone sequences were introduced into MEGA 7.0,and 27 isolates were obtained.Homology analysis,multiple alignment,cluster analysis and evolution analysis were performed on the sequencing results.The results showed that the similarity between 27 Changsha TYLCV isolates and isolates from other provinces(municipalities)in China was 98.5%-99.8%,and the similarities between Changsha TYLCV isolates and Iranian strains(AJ132711 and GU076453),Mexican strain(FJ012358),Australian strain(GU178814)and Portuguese strain(AF105975)were 90.7%-91.5%,95.4%-95.5%,98.6%-98.8%and 99.3%-99.4%respectively.【Conclusion】TYLCV has outbroken in Changsha,Hunan.Quarantine work needs to be executed to prevent the transfer of susceptible tomatoes from the affected provinces in order to control the epidemic of the disease.