Detection, discrimination and discovery of a new Tobacco streak virus strain

Soybean plants that exhibited symptoms of virus infection were sampled from different counties of Oklahoma. These plants were tested serologically for 15 viruses known to infect soybean plants. Fifty-seven samples that exhibited typical virus-like symptoms did not test positive for any of the 15 viruses used in a dot-immunobinding assay (DIBA). Four samples were pooled and used for next generation sequencing using the 454-Roche protocol. Sequence and phylogenetic analysis of the sequences obtained revealed infection with a distinct strain of Tobacco streak virus (TSV). TSV was one of the 15 viruses initially tested for using DIBA and had tested negative. TSV belongs to the genus Ilarvirus and has been reported as a causal agent of bud blight in soybean crops in Brazil and the United States. Out of 10 reported primer pairs for TSV reverse transcription-polymerase chain reaction (RT-PCR), only two had the potential, based on sequence similarity, to amplify part of the genome of the distinct strain of TSV found in Oklahoma and only one was actually able to amplify the region. In this study, a new primer pair, specific to all known TSV and capable of amplifying the Oklahoma strain (TSV-OK), was designed from a highly conserved region of coat protein (CP) sequences and end-point PCR and quantitative RT-PCR detection methods were developed and their sensitivity assayed. This is the first report of specific primers designed from this highly conserved region in the CP of TSV for detection of TSV. Twenty-three of the 57 DIBA soybean samples that initially tested negative were retested with the new specific end-point PCR method and found positive for TSV infection.