广东省国兰病毒病害调查及CymMV和ORSV基于cp基因的系统进化分析

Investigation of Chinese Cymbidium viral disease,and phylogenetic analysis of cp gene of Cymbidium mosaic virus and Odontolossum ring spot virus in Guangdong Province

为调查广东省国兰病毒病发生情况,采用双抗体夹心-酶联免疫吸附测定(double sandwich enzyme-linked immunosorbent assay,DAS-ELISA)法对采集自广州市、汕头市和韶关市兰花主产区的53份具有典型病毒病症状的国兰叶片进行检测,选取35份病叶应用RT-PCR技术对检测结果进行验证,并基于cp基因序列对检测到的病毒进行系统进化分析。DAS-ELISA检测结果显示,53份病叶中有44份检出病毒,总检出率为83.02%;其中,有14份仅检出建兰花叶病毒(Cymbidium mosaic virus,CymMV),有19份仅检出齿兰环斑病毒(Odontolossum ring spot virus,ORSV),有11份检出CymMV和ORSV复合侵染,检出率分别为26.42%、35.85%和20.75%。RT-PCR检测结果显示,35份病叶中有30份检出病毒,总检出率为85.71%;其中,有21份仅检出CymMV,有6份仅检出ORSV,有4份检出CymMV和ORSV复合侵染,检出率分别为60.00%、17.14%和11.43%,但未检测到其它病毒。表明广东省国兰病毒病害主要由CymMV和ORSV侵染引起。序列和系统进化分析显示,来源于不同地区和寄主的CymMV和ORSV分离物的cp基因序列极为保守,CP氨基酸序列相似性分别大于97.31%和93.67%,且CymMV和ORSV多样性种群的分化与寄主种属和地理隔离关系不大。

To investigate the viral disease of Chinese Cymbidium in Guangdong Province,the double sandwich enzyme-linked immunosorbent assay(DAS-ELISA)was used to detect the virus in 53 samples of Chinese Cymbidium leaves with typical viral-symptoms,which were collected from the main orchid producing regions in Guangzhou,Shantou and Shaoguan cities.Thirty-five out of the 53 samples were selected to verify the DAS-ELISA results by reverse transcription polymerase chain reaction(RTPCR).Additionally,sequence and phylogenetic analysis of these viral isolates detected in this study were constructed based on the deduced amino acid sequences of cp gene.DAS-ELISA results revealed that 44 out of the 53 samples were virus infected,with a detection rate of 83.02%.Thereinto,14,19 and11 samples were detected to be infected with Cymbidium mosaic virus(CymMV),Odontolossum ring spot virus(ORSV),and CymMV combined with ORSV,with detection rates of 26.42%,35.85%and20.75%,respectively.RT-PCR results showed that 30 out of the 35 samples were infected with virus(detection rate was 85.71%).Among them,there were 21,6 and 4 samples were detected to be infected with CymMV(60.00%),ORSV(17.14%),and CymMV combined with ORSV(11.43%)respectively,while no other virus was detected.The above results indicated that CymMV and ORSV were still main viral pathogens of Chinese Cymbidium in Guangdong Province.Sequence and phylogenetic analysis showed that cp genes of CymMV and ORSV isolates collected from different regions and various hosts were highly conserved,and the sequence similarities of the deduced amino acid were greater than97.31%and 93.67%,respectively.The differentiations of diverse viral populations had little relationship with host species and geographical isolation.