Comparing RT-PCR of individual samples with high throughput sequencing of pooled plant samples for field-level surveillance of viruses in blackberry and wild Rubus

Blackberry production is increasing in the Southeastern United States with the availability of new cultivars. In addition to high production costs, growers are challenged by virus diseases. Blackberry yellow vein disease (BYVD) significantly limits blackberry production. BYVD is associated with the crinivirus blackberry yellow vein–associated virus in mixed infections with other viruses. The specific disease etiology and ecological factors underlying BYVD are not well understood and rely on the effective diagnosis of several viruses involved in the complex. In 2021, we collected samples from blackberry plants showing BYVD symptoms, asymptomatic blackberry plants, and wild Rosaceae spp. from nine farms across South Carolina, for a total of 372 individual plant samples. RNA from individual samples was isolated and pooled into sample groups (i.e., symptomatic, asymptomatic, and wild) from each farm for a total of 24 pooled samples. We sequenced the pooled RNA using Illumina and analyzed sequence profiles using the Virtool bioinformatics application. We also tested each plant for six viruses by reverse transcriptase PCR or reverse transcriptase quantitative PCR and compared plant (PCR)-level and field (high-throughput sequencing [HTS])-level data. Virtool detected 17 known viruses in the pooled samples, including 11 blackberry viruses. PCR testing was mostly consistent with HTS, with some notable disagreements for specific viruses. Our study demonstrates that HTS could be used as an efficient tool to detect viruses in bulked samples in blackberry fields, although limitations to using HTS for field-level surveillance are also discussed here.