Submitted to: Journal of Virological Methods
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/12/2010
Publication Date: 10/1/2010
Publication URL: hdl.handle.net/10113/49212
Citation: Agrindotana, B.O., Ahonsia, M.O., Domier, L.L., Gray, M.E., Bradley, C.A. 2010. Application of sequence-independent amplification (SIA) for the identification of RNA viruses in bioenergy crops. Journal of Virological Methods. 169(1):119-128. Interpretive Summary: Energy cane, Miscanthus, and switchgrass are three potential biomass crops being evaluated as feed stocks for commercial production of ethanol. Virus diseases can significantly reduce the amounts of biomass these plants produce each year and consequently are potentially significant threats to their value. Therefore, identification of viruses infecting these bioenergy crops is important for quarantine purposes, breeding virus-resistant varieties, and production of virus-free planting materials. Usually, we need to characterize and develop diagnostic reagents for a virus before it can be correctly identified. However, because these are new crops to North America, there is little information about which viruses will infect them. To circumvent this problem, we developed techniques to identify viruses with no prior information about the viruses. To test the method, virus-infected corn, soybean and wheat plants were analyzed. The new methods correctly identified all three test viruses. In the bioenergy crops, the method detected a new virus in switchgrass and a virus commonly found in corn in both Miscanthus and switchgrass. The method was very sensitive and will be of interest to scientists who are interested in identifying and characterizing new viruses or new virus diseases of plants.
Technical Abstract: Miscanthus x giganteus, Saccharum spp. (energy cane), 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. Here, we report application of sequence-independent amplification for the identification of RNA viruses from bioenergy crops. The method combines 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 GenBank. This method has a distinct advantage over other virus characterization techniques in that it does not require virus-specific reagents to identify viruses. Using this method, we identified a possible new species 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. x giganteus, energycane, sweet corn, and switchgrass. Other viruses identified were: Maize dwarf mosaic virus (MDMV), genus Potyvirus, in johnsongrass; Soilborne wheat mosaic virus (SBWMV), genus Furovirus, in wheat; and Bean pod mottle virus (BPMV), genus Comovirus, in soybean. 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.