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

Authors: MONDAL, SHAONPIUS - Oak Ridge Institute For Science And Education (ORISE); Hladky, Laura; Wintermantel, William - Bill

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/24/2023
Publication Date: 2/1/2023
Citation: Mondal, S., Hladky, L.L., Wintermantel, W.M. 2023. Differential seasonal prevalence of yellowing viruses infecting melon crops in southern California and Arizona determined by multiplex RT-PCR and RT-qPCR. Plant Disease. https://doi.org/10.1094/PDIS-06-22-1512-RE.
DOI: https://doi.org/10.1094/PDIS-06-22-1512-RE

Interpretive Summary: Viruses transmitted by the whitefly (Bemisia tabaci) 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 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. These three viruses are often found as mixed infections in melon and other cucurbit crop plants and also threaten cucurbit production in other parts of the world. In June and September of 2019, several leaf samples were collected from melon fields in Yuma County, Arizona and Imperial and Riverside Counties, California exhibiting foliar yellowing symptoms characteristic of crinivirus infections. A robust and rapid detection method was required to efficiently determine which viruses were present in field samples due to similarity in symptoms. A method was developed to efficiently detect individual and mixed virus infections of CYSDV, CCYV, and SqVYV, as well as the aphid-transmitted polerovirus Cucurbit aphid borne yellows virus (CABYV) which produces similar yellowing symptoms to those of CYSDV and CCYV in a single test (Multiplex RT-PCR). Field samples collected from the 2019 and 2020 spring and fall melon crops in the Sonoran Desert of California and Arizona. These samples as well as frozen archived RNA extracts from previous samplings from the region were evaluated and confirmed single, double, and triple virus infections. TaqMan probes, which allow measurement of the amount of virus present in a sample were also developed, optimized, and validated to quantify these four viruses from single and mixed infections. Results demonstrated differences in virus accumulation within melon plants as well as the presence of two and three-way mixed virus infections including CYSDV, CCYV, SqVYV, and/or CABYV in low desert melon production areas of the United States, and also provide the first report of CCYV and SqVYV in Arizona, USA. This approach provides valuable resources for both detection and quantification of emerging viruses infecting cucurbit crops, and a framework that can be adapted for regions throughout the world with different virus combinations.

Technical Abstract: Viruses transmitted by the whitefly (Bemisia tabaci) 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 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. These three viruses are often found as mixed infections in melon and other cucurbit crop plants and also threaten cucurbit production in other parts of the world. In June and September of 2019, several leaf samples were collected from melon fields in Yuma County, Arizona and Imperial and Riverside Counties, California exhibiting foliar yellowing symptoms characteristic of crinivirus infections. A robust and rapid detection method was required to efficiently determine virus mixtures in field samples. A multiplex two-step RT-PCR method was developed, and further validated on a single-step RT-PCR method to efficiently detect individual and mixed virus infections of CYSDV, CCYV, and SqVYV, as well as the aphid-transmitted polerovirus Cucurbit aphid borne yellows virus (CABYV) which produces similar yellowing symptoms to those of CYSDV and CCYV. Field samples collected from the 2019 spring and fall melon crops as well as frozen archived RNA extracts were evaluated and confirmed single, double, and triple virus infections. TaqMan probes were developed, optimized, and validated to quantify these four viruses in plant samples and to evaluate differential accumulation and spatio-temporal distribution within plants during mixed virus infections. Results demonstrated differences in virus accumulation within melon plants as well as the presence of two and three- way mixed virus infections including CYSDV, CCYV, SqVYV, and/or CABYV in low desert melon production areas of the United States, and also provide the first report of CCYV and SqVYV in Arizona, USA. This approach provides valuable resources for detection and quanitification of viruses infecting cucurbit crops, and a framework that can be adapted for regions throughout the world with different virus combinations.