Plant Virus Identification and Detection Using High-Throughput Sequencing

Identification and detection of plant viruses is an important part of the testing and certification system provided by Naktuinbouw (Netherlands Inspection Service for the Horticulture) and BKD (Flower Bulb Inspection Service). Traditionally, ELISA and the more recently PCR based assays are applied to screen for known viruses or virus families. Furthermore, indicator plants are used as a safety net to detect the presence of unknown viruses or unusual combinations. Despite the wide acceptance, robustness and low costs of these methods, viruses can be missed using these targeted detection methods. The numbers of unidentified viruses in plants are estimated relatively high due to a fast mutation rate, host specificity or low abundance. They can cause novel disease symptoms, can be part of a virus complex or can be present without showing any symptoms. Using the non-targeted High-throughput sequencing of RNA from infected leaves, both known and unknown viruses can be detected. We generated a fast detection tool by sequencing the total RNA fraction (RNAseq) or the small RNAs in combination with an in house plant virus database. All tested viruses (including a viroid) could be detected with both the methods. However, a by far higher mapping percentage was reached at lower sequencing costs with small RNA sequencing. Preliminary results show that both RNAseq and small RNA sequencing are suitable to also identify unknown viruses de novo.