Detection by next generation sequencing of a multi-segmented viral genome from sugarcane associated with Ramu stunt disease.

Ramu stunt disease of sugarcane was first reported in Papua New Guinea in the mid 1980s. The disease can reduce sugarcane yields significantly and causes severe stunting and mortality in highly susceptible cultivars. The causal agent of Ramu stunt has been investigated but its characterization has not been completed. Sugarcane cv. Ragnar from Papua New Guinea with symptoms of Ramu stunt was analyzed by next generation sequencing. Total RNA was extracted and whole transcriptome shotgun sequencing was performed using an Illumina platform. Over thirty-seven million reads with an average length of 100 nucleotides were obtained. More than eighteen thousand contigs were assembled and subjected to BLASTX analysis. Twenty-one contigs were virus related and six were associated with plant viruses. The BLAST algorithms revealed sequence similarity to Tenuivirus and Phlebovirus, genera of viruses whose members contain genomes consisting of multiple RNA segments. The six contigs derived from the RNA sequencing data correspond to six RNAs that compose the Ramu stunt virus genome. Primers were designed for each of the six RNAs and RT-PCR amplicons were obtained only from the symptomatic sugarcane. There was concordance between the sequence data of the contigs obtained from the NGS and that of the amplicons obtained by RT-PCR. The NGS approach allowed us to determine the complete genomic sequence of Ramu stunt virus. It is likely that this virus is the causal agent of Ramu stunt disease.