Energy Biosciences Institute, University of Illinois, 1206 West Gregory Drive, Urbana, IL 61801-3838, USA.
J Virol Methods. 2010 Oct;169(1):119-28. doi: 10.1016/j.jviromet.2010.07.008. Epub 2010 Jul 16.
Miscanthus x giganteus, energycane, and Panicum virgatum (switchgrass) are three potential biomass crops being evaluated for commercial cellulosic ethanol production. Viral diseases are potentially significant threats to these crops. Therefore, identification of viruses infecting these bioenergy crops is important for quarantine purposes, virus resistance breeding, and production of virus-free planting materials. The application is described of sequence-independent amplification, for the identification of RNA viruses in bioenergy crops. The method involves virus partial purification from a small amount of infected leaf tissue (miniprep), extraction of viral RNA, amplification of randomly primed cDNAs, cloning, sequencing, and BLAST searches for sequence homology in the GenBank. This method has distinct advantage over other virus characterization techniques in that it does not require reagent specific to target viruses. Using this method, a possible new species was identified in the genus Marafivirus in switchgrass related to Maize rayado fino virus, its closest relative currently in GenBank. Sugarcane mosaic virus (SCMV), genus Potyvirus, was identified in M.xgiganteus, energycane, corn (Zea mays), and switchgrass. Other viruses identified were: Maize dwarf mosaic virus (MDMV), genus Potyvirus, in johnsongrass (Sorghum halepense); Soil borne wheat mosaic virus (SBWMV), genus Furovirus, in wheat (Triticum aestivum); and Bean pod mottle virus (BPMV), genus Comovirus, in soybean (Glycine max). The method was as sensitive as conventional RT-PCR. This is the first report of a Marafivirus infecting switchgrass, and SCMV infecting both energycane and M. x giganteus.
芒属能源草、巨菌草和柳枝稷是三种正在评估用于商业纤维素乙醇生产的潜在生物质作物。病毒病是这些作物的潜在重大威胁。因此，鉴定感染这些生物能源作物的病毒对于检疫、抗病毒育种和生产无病毒种植材料非常重要。本文描述了一种用于鉴定生物能源作物中 RNA 病毒的无序列依赖性扩增方法。该方法涉及从小量感染叶片组织（迷你提取）中部分纯化病毒、提取病毒 RNA、随机引物 cDNA 扩增、克隆、测序以及在 GenBank 中进行序列同源性 BLAST 搜索。与其他病毒特征鉴定技术相比，该方法具有明显的优势，因为它不需要针对目标病毒的特定试剂。使用该方法，在柳枝稷中鉴定到了与玉米线条病毒亲缘关系最近的斐济病毒属中的一个可能的新种。在芒属能源草、巨菌草、玉米和柳枝稷中鉴定到了甘蔗花叶病毒（SCMV），属马铃薯 Y 病毒科。鉴定到的其他病毒还有：玉米矮花叶病毒（MDMV），属马铃薯 Y 病毒科，在约翰逊草中；土壤传播小麦花叶病毒（SBWMV），属斐济病毒属，在小麦中；菜豆荚斑驳病毒（BPMV），属黄瓜花叶病毒属，在大豆中。该方法与常规 RT-PCR 一样敏感。这是首次报道斐济病毒感染柳枝稷和 SCMV 感染能源草和芒属能源草。
