Use of High Throughput Sequencing for Improved Virus Detection in Strawberry Certification

To protect global strawberry production, clean plant programs rely on rigorous certification and quarantine testing to prevent the spread of disease. For decades, biological graft indexing has been the "gold standard" for virus detection; however, it often fails to identify viruses that remain latent in single infections or viruses requiring extended periods to induce symptoms. High throughput sequencing (HTS) offers an alternative that identifies all known and emerging viruses independent of the nucleotide sequences of their genomes including those undetected by traditional bioassays. This study compared HTS with graft indexing using 73 virus-infected donor plants, 584 indicator clones, and two replicates to evaluate HTS as a replacement for conventional graft bioassay testing. Donor material was analyzed via HTS and simultaneously grafted onto two replicates of four Fragaria indicator clones (UC-4, UC-5, UC-10, UC-11). Indicator symptoms were monitored weekly over eight weeks and again following natural dormancy. The RT-qPCR/RT-PCR testing for virus transmission was also conducted at eight weeks and after dormancy. HTS identified viral infections in all donor plants, detecting 17 different viruses across eight families, and revealing mixed infections in 75% of plants. In contrast, 83% of indicators developed symptoms within the current standard of eight weeks post graft. Only 61% of the indicator plants displayed symptoms both before and after dormancy, while 7% remained asymptomatic throughout despite receiving grafts from HTS-positive donors. Given the limitations of biological indexing, this research strongly supports the integration of HTS as a frontline detection tool to enhance the reliability and efficiency of certification programs.