Development of a SYBR Green-based real-time RT-PCR method using a newly designed tomato mottle mosaic virus-specific primer set and the assessment of its seed transmission properties in tomato seeds.

Tomato mottle mosaic virus (ToMMV), first reported in Mexico in 2009, is an emerging virus that globally threatens the yield of crops belonging to the family Solanaceae. The international trade of ToMMV-contaminated seeds is potentially associated with its global spread; however, the epidemiological features of virus-contaminated seeds remain unclear, and the availability of effective seed assays to detect ToMMV is limited. Therefore, we have developed a SYBR Green-based real-time RT-PCR (SYBR-RT-qPCR) for ToMMV detection, which supports the detection of ToMMV with 10–100 times improved sensitivity compared to available methods; up to 1.54 copies were detected while the common Solanaceae-infecting tobamoviruses were not amplified. Additionally, virus-contaminated seeds were obtained from two cultivars of ToMMV-infected tomato plants. A direct immunostaining assay revealed the presence of ToMMV in the seed coat and hair of both cultivars. Although ToMMV was detected in all tested seeds, seed-to-seedling ToMMV transmission rates were only 0.42% and 0.93% in tomato cvs. Carol Passion and Rejina, respectively. Using these seed lots harboring seed-to-seedling-transmissible ToMMV, we confirmed the efficacy of this SYBR-RT-qPCR method for detecting ToMMV in a bulk sample (n = 400, including one ToMMV-contaminated seed). Therefore, our detection method is effective in actual seed inspection. This is the first report of a practical ToMMV-specific seed inspection assay based on SYBR-RT-qPCR; moreover, crucial properties related to the seed transmission of ToMMV have been revealed. The insights and testing methods reported in this study could help prevent the spread of ToMMV through virus-contaminated seeds.