侵染云南早春西瓜的西瓜银色斑驳病毒和黄瓜花叶病毒分离鉴定

Isolation and identification of watermelon silver mottle orthotospovirus and cucumber mosaic virus infecting early spring watermelon in Yunnan Province

早春西瓜是云南热区的特色水果,近年来病毒病发生危害日益严重。为明确侵染早春西瓜的主要病毒,本研究采集了云南省西双版纳州勐海县的早春西瓜植株与果实病毒病样品,应用透射电子显微镜制样观察、间接酶联免疫吸附测定(ID-ELISA)和RT-PCR扩增克隆与测序分析进行检测鉴定。结果表明:侵染早春西瓜的病毒为西瓜银色斑驳病毒(WSMoV)和黄瓜花叶病毒(CMV);ID-ELISA对叶部样品WSMoV和CMV的检出率分别为70%和20%,复合侵染率为15%,RT-PCR对这两种病毒的检出率分别为100%和65%,复合侵染率为65%;并且,实验对发病果实种子进行RT-PCR扩增克隆和测序分析发现上述2种病毒复合侵染率达100%;系统发育树分析表明,云南西瓜WSMoV分离株21YV-40(GenBank登录号:OP617563)、21YV-43(GenBank登录号:OP867047)与云南分离株Banna-2011(GenBank登录号:KM242056)相似性最高,达到99.00%,云南西瓜CMV分离株CMVYN40(GenBank登录号:OP617565)、CMVYN46(GenBank登录号:OP617566)与泰国分离株A27(GenBank登录号:FN552545)相似性最高,达到98.00%。本研究明确了勐海县早春西瓜主要受到WSMoV和CMV的侵染且存在复合侵染,首次发现WSMoV和CMV复合侵染西瓜发病果实种子,为早春西瓜病毒病的防控提供了依据。

Watermelon planted in early spring is a characteristic fruit in hot area of Yunnan Province.Its viral diseases are more and more serious in recent years.To detect and identify the main viruses,the plants and fruits samples of watermelon were collected from Menghai county,Xishuangbanna state,and transmission electron microscope(TEM)observation,indirect enzyme-linked immunosorbent assay(ID-ELISA),RT-PCR amplification and analysis of viral genome sequences were carried out.The results showed that the viruses infecting watermelon were watermelon silver mottle virus(WSMoV)and cucumber mosaic virus(CMV).The detection rates of WSMoV and CMV using ID-ELISA were 70%and 20%,respectively,in leaf samples,and of complex infection was 15%.The detection rates of these two viruses by RT-PCR were 100%and 65%,respectively,and of complex infection was 65%.However,the complex infection rate of these two viruses detected by RT-PCR amplification could reach up to 100%in seeds.Phylogenetic analysis demonstrated that the genome sequences of WSMoV[21YV-40(GenBank accession no.:OP617563),21YV-43(GenBank accession no.:OP867047)]isolated in this study were similar to the squence of WSMoV[Banna-2011(GenBank accession no.:KM242056)]isolated from Yunnan watermelon in 2016,and the similarity was 99%.The genome sequences of CMV[CMVYN40(GenBank accession no.:OP617565),CMVYN46(GenBank accession no.:OP617566)]isolated in this study were similar to the sequence of CMV[A27(GenBank accession no.:FN552545)]isolated from watermelon in Thailand,and the similarity was 98%.This study demonstrated that the early spring watermelon was mainly infected by WSMoV and CMV,and complex infection was also common.The complex infection of WSMoV and CMV to both the plant and the seed of watermelon was reported for the first time.This study supplied a basis for virus prevention in the early spring watermelon.