Differential seasonal prevalence of yellowing viruses infecting melon crops in southern California and Arizona determined by multiplex RT-PCR and RT-qPCR

Viruses transmitted by the whitefly ( ) are an increasing threat to cucurbit production in the southwestern United States as well as many other cucurbit production regions throughout the world. The crinivirus, cucurbit yellow stunting disorder virus (CYSDV), has severely impacted melon production in California and Arizona since its introduction to the region in 2006. Within the past few years, another crinivirus, cucurbit chlorotic yellows virus (CCYV), and the whitefly-transmitted ipomovirus, squash vein yellowing virus (SqVYV) were found infecting melon plants in California's Imperial Valley as well. Both CYSDV and CCYV, as well as an aphid transmitted polerovirus, cucurbit aphid-borne yellows virus (CABYV), occur together in the region and produce identical yellowing symptoms on cucurbit plants. Mixed infections of these four viruses both in the Sonoran Desert and other regions pose challenges for disease management and efforts to develop resistant varieties. A multiplex single-step RT-PCR method was developed that differentiates these viruses from one another, and this was used to determine the prevalence and distribution these viruses in melon samples collected from fields in the Sonoran Desert melon production region of California and Arizona, USA during spring and fall melon seasons from 2019 through 2021. TaqMan probes were also developed, optimized, and applied in a single-step multiplex RT-qPCR to quantify titers of these four viruses in plant samples, which frequently carry mixed infections. Results of the multiplex RT-PCR analysis demonstrated that CYSDV is the predominant virus during the fall season, whereas CCYV was by far the most prevalent virus during spring seasons each year. Multiplex RT-qPCR was used to evaluate differential accumulation and spatio-temporal distribution of viruses within plants and suggested differences in competitive accumulation of CCYV and CYSDV within melon. This study also provides the first official report of SqVYV in Arizona, USA, and offers an efficient method for virus detection and quantification for breeding and disease management in areas impacted by cucurbit yellowing viruses.