广东地区两种兰花病毒病害的分子鉴定及检测

根据已报道的建兰花叶病毒(CyMV)和齿兰环斑病毒(ORSV)基因组核苷酸序列，在其cp基因上下游设计PCR引物。CyMV预计扩增产物784bp，ORSV预计扩增产物604bp。以采集自广东省顺德的墨兰和文心兰表现病毒病症状的病株叶组织总RNA为模板，进行RT—PCR扩增。对预期大小的5个扩增产物进行克隆和测序，结果表明，来源于不同兰种或同一兰种不同兰场的病样CyMV引物扩增产物核苷酸序列存在少量差异，但均与世界各地的CyMV分离物cp基因高度同源；而来源于不同兰种的病样ORSV引物扩增产物核苷酸序列完全相同，与世界各地的ORSV分离物cp基因高度同源。因此可将侵染广东兰花的两种病毒鉴定为CyMV和ORSV。混合上述两种病毒的PCR引物，采用双重RT—PCR扩增，对采自广东顺德23个兰场共153份样品进行病毒检测，76份(49．7％)检出CyMV，52份(34．0％)检出ORSV，2份(1．3％)同时检出CyMV和ORSV。

Identification and Detection of Two Major Viruses Infecting Orchids by Molecular Technique

Based on reported Cymbidium mosaic virus(CyMV)and Odontoglossum ringspot virus(ORSV)genomic cDNA sequences, two pairs of PCR primers for viral coat protein gene with expected ampliffied sizes of 784bp and 604bp were designed respectively, and RT-PCR was performed with the total RNA as templates extracted from diseased orchid plants (Cymbidium sinense var. margicoloratum and Oncidium sp.) collected from Shunde, Guangdong province, China. Five amplified DNA fragments with expected sizes were cloned and sequenced. Although the nucleiotide sequences of DNA fragments amplified from different orchid species or different farms with CyMV primer showed slightly diversity, they had highly homology to cp gene of CyMV isolates around the world. Otherwise, the nucleiotide sequences of the two DNA fragments amplified from different orchid species with ORSV primer were completely identity and highly homologous to cp gene of ORSV isolates around the world. Therefore the two viruses infecting orchids in Guangdong were identified as CyMV and ORSV. Simultaneous detection of the two orchid viruses by dual RT-PCR was conducted with primers mixture. Out of the 153 samples collected from 23 farms, 76 tested positive for CyMV, 52 positive for ORSV and 2 positive for both CyMV and ORSV.