广西部分烟区烟草曲叶病病原的分子鉴定

Molecular identification of the causal agents causing tobacco leaf curl disease in some regions of Guangxi

[目的]明确广西西部地区靖西(JX)、凌云(LY)、德保(DB)和乐业(LeY)等4个县市烟草曲叶病的病原。[方法]2010年5-6月分别从广西靖西、凌云、德保和乐业等县市采集具有典型曲叶症状的烟草叶片,用基于双生病毒DNA保守序列设计简并引物Bego-1和Bego-6对病叶组织总DNA抽提物进行PCR扩增和对PCR产物进行序列测定,用BLAST、Vector NTI、MEGA 4.0和Simplot program 3.2软件等进行病毒序列分析、系统进化树构建和病毒重组分析。[结果]从选取的9个表现典型曲叶症状的样品叶组织总DNA抽提物中均可扩增出约1500bp与预期大小相符的DNA片段。测序和序列比对分析显示,9个样品扩增产物核苷酸序列相似性为73.7%～99.2%,与已报道的双生病毒具较高的相似性。其中,JX-2与中国番茄曲叶病毒广西番茄分离物(G32)的相似性最高,达99.2%;JX-3和JX-5与云南胡椒曲叶病毒云南辣椒分离物(YN323)相似性最高,分别为92.5%和93.4%;LeY-1、LY-1、DB-1、JX-1、JX-4和JX-6则与中国番茄黄化曲叶病毒中国番茄分离物(CHI)和广西烟草分离物(G102)的相似性最高,均高于95.0%。基于PCR扩增产物及已报道的双生病毒属代表种相应核苷酸序列构建的系统进化树分析表明,9个广西烟草分离物分属3个簇群:中国番茄曲叶病毒簇、云南辣椒曲叶病毒簇和中国番茄黄化曲叶病毒簇。重组分析结果表明:JX-3是云南辣椒曲叶病毒和中国番茄曲叶病毒的重组病毒,JX-5是云南辣椒曲叶病毒和中国番茄黄化曲叶病毒的重组病毒。[结论]9个广西烟草分离物分属于4种双生病毒:中国番茄曲叶病毒和中国番茄黄化曲叶病毒,以及分别由上述两种病毒与云南辣椒曲叶病毒重组而来的2种重组病毒。其中,中国番茄曲叶病毒自然侵染烟草、云南辣椒曲叶病毒和中国番茄曲叶病毒及中国番茄黄化曲叶病毒的重组病毒等结果此前均未见报道。

[Objective] This study was carried out to determine the identity of the pathogens causing tobacco leaf curl disease in western regions of Guangxi.[Method] Nine tobacco samples with typical leaf-curled symptom were collected from Jingxi,Lingyun,Debao,and Leye counties of Guangxi Province in 2010.A pair of degenerate primers was designed based on the conserved sequence of DNA-A gene of geminiviruses and a 1500-bp DNA-A fragment was amplified by PCR.For bioinformatics analysis,the programs BLAST at NCBI,Vector NTI,MEGA 4.0,and Simplot program 3.2 were used for alignment,phylogenetic tree construction,and recombination analysis.[Result] A DNA fragment of about 1500bp could be amplified from all of these nine samples by PCR with the degenerate primers.Among these isolates,the sequence similarity was between 73.7% and 99.2%,of which the isolate JX-2 had the highest similarity（99.2%） with the isolate G32 of Tomato leaf curl China virus（ToLCCNV）,and the isolates JX-3 and JX-5 shared the highest similarity of 92.5% and 93.4%,respectively,with YN323 of Pepper leaf curl Yunnan virus（PepLCYNV）.A similarity of greater than 89% with CHI and G102 isolates of Tomato yellow leaf curl China virus（TYLCCNV） was found for isolates LeY-1,LY-1,DB-1,JX-1,JX-4 and JX-6.A phylogenetic tree was constructed using partial DNA-A sequences of the isolates,indicating that the nine isolates formed three clades： ToLCCNV（JX-2）,PepLCYNV（JX-3 and JX-5）,and TYLCCNV（the other six isolates）.[Conclusion] Tobacco leaf curl diseases in Guangxi Province was associated with four Begomovirus species,ToLCCNV,TYLCCNV,and two recombinant viruses derived from PepLCYNV and ToLCCNV（JX-3） and PepLCYNV and TYLCCNV（JX-5）.It was the first report that ToLCCNV infected tobacco in the field in China,as well as recombinant viruses were derived from PepLCYNV,ToLCCNV,and TYLCCNV.