黑皮果蔗花叶病病原鉴定

Identification of mosaic disease pathogen in chewing cane badila

在南宁市郊采集到1份表现褪绿条纹、花叶的黑皮果蔗样品(NN-Ba2),经差速离心和PEG二次沉淀法提取病毒粗汁液,在电镜下观察到略弯曲的线状病毒粒子;DAC-ELISA检测显示NN-Ba2与甘蔗花叶病毒多克隆抗体和单克隆抗体呈阳性反应;根据SCMV和SrMV CP基因分别设计合成特异引物,利用RT-PCR对NN-Ba2进行分子鉴定,扩增出大小约900 bp的预期片段,健康对照未扩增到任何片段;NN-Ba2扩增片段经克隆后测序得到719个有效核苷酸,测序结果在DDBJ上用BLAST进行同源性分析,结果显示该序列与已报道的SCMV广东、云南、广西等地分离物对应的核苷酸序列同源性为97.36%～97.77%,根据马铃薯Y病毒科(Potyviridae)病毒种和株系的划分标准,NN-Ba2鉴定为SCMV。田间采集的另外10份表现花叶症状的果蔗样品经ELISA和RT-PCR检测,证明也感染了SCMV。

A sample of leaves from chewing cane badila （NN-Ba2） showing chlorotic stripe and mosaic symptoms was collected from Nanning suburb. Virus was purified from the infected leaves by the method of differential centrifugation and secondary PEG sedimentation. Slightly convex filamentous particles were observed under electron microscope. The results of DAC-ELISA test indicated that the virus isolate showed positive reaction with polyclonal antibody and monoclonal antibody of sugarcane mosaic virus （SCMV）. A 900 bp fragment was obtained by RT-PCR using specific primers based on the coat protein gene of SCMV and sorghum mosaic virus （SrMV）, respectively, but no fragment was amplified in the control. Nucleotide sequence analysis indicated that the fragment contained 719 nucleotides. Homology analysis using the BLAST showed that the isolate had 97.36%-97.77% nucleotide sequence homology compared with isolates separated from Guangdong, Yunnan and Guangxi. The isolate was identified as SCMV according to the classified standards of Potyviridae. Another ten samples of chewing cane badila leaves showing mosaic symptoms were identified as SCMV by the methods of ELISA and RT-PCR.