Viruses are a major biotic threat to cucurbit production in the southeastern United States. Many important cucurbit viruses are transmitted by whiteflies and aphids. Recent cucurbit virus research in Georgia is predominantly focused on whitefly-transmitted viruses (WTVs) and research is lacking on the aphid-transmitted viruses (ATVs) in cucurbit crops of Georgia. This study investigates the occurrence, prevalence, and emergence of ATVs in pumpkin and winter squash crops grown in a vegetable research farm at the University of Georgia-Tifton Campus during the fall season of 2022 and 2023. We screened plants for characteristic virus symptoms such as mosaic, mottling, yellowing, chlorotic spots, vein clearing, shoestring, upward curling, crumpling, blistering, and deformation of leaves. We collected symptomatic leaf samples of pumpkin and winter squash from the field. We processed the leaf samples for small RNA libraries for high-throughput sequencing (HTS) to identify ATVs present in the samples. We amplified viral genes for sanger sequencing and ran quantitative polymerase chain reactions (qPCR) for molecular validation of HTS results. HTS analysis revealed the presence of two ATVs, zucchini yellow mosaic virus (ZYMV) and papaya ringspot virus (PRSV). The qPCR results suggested a significant temporal shift in ATV’s abundance in these two crops. In 2022, PRSV and ZYMV incidence was observed in 56.25% and 31.25% pumpkin leaf samples. In winter squash, PRSV and ZYMV incidence was found in 50% and 32.14% leaf samples. Mixed infection of both viruses was at 28.12% for pumpkin and 25% for winter squash. In 2023, PRSV was not detected in pumpkins, and it was detected at a negligible level (0.62%) in winter squash. ZYMV was predominant in pumpkin (61.25%) and winter squash (42.50%). Phylogenetic analysis of ZYMV-encoded coat protein (CP) and helper component-protease (HC-Pro) suggested a close relationship with the European isolates. However, PRSV-encoded CP and NIa-VPg showed a close relationship with isolates from Australia, Papua New Guinea, Spain, and the United States. To the best of our knowledge, this is the first study to suggest single or mixed infections of ZYMV- and PRSV-infected pumpkin and winter squash in Georgia, USA. The findings of this study will serve as an important foundation for future research to understand the complex interactions between insect-transmitted viruses in cucurbit crops, which is vital for developing resistant cultivars and effective virus management strategies for commercial cucurbit vegetable crop production.
