Revealing hidden viruses inducing similar yellowing symptoms or remaining asymptomatic in cucurbit crops

Abstract Mixed viral infections occur frequently in plants, leading to diseases that can be difficult to diagnose, especially when an unknown virus is hidden behind expression of symptoms common to other viruses. We monitored aphid‐ and whitefly‐transmitted viruses in two cucurbit crops (melon and watermelon) for three consecutive seasons (2021–2023) across three production regions in Spain, focusing on the common yellowing and mosaic symptoms. A total of 984 symptomatic leaf samples from 246 field plots were tested for 10 frequently found cucurbit viruses. Cucurbit aphid‐borne yellows virus (CABYV) was the most prevalent virus infecting both cucurbit crops over the three seasons. Additionally, occurrence of watermelon mosaic virus (WMV) in samples with mosaic symptoms was significant. However, an important proportion of yellowing symptomatic samples tested negative for known viruses. Using a sequence‐independent approach, we identified a novel polerovirus, Pepo aphid‐borne yellows virus (PABYV), which was widespread, infecting both crops. Analysis of our long‐term cucurbit frozen‐sample collection revealed that PABYV had emerged in Spain in 2018, possibly unnoticed as its yellowing symptoms were similar to CABYV, with which it was frequently associated. Additionally, we found the cryptic Cucumis melo endornavirus (CmEV) in all tested melon samples from 2011 and, for the first time, in pumpkin. Genetic characterization of CABYV, PABYV and CmEV populations revealed a replacement of ancient CABYV isolates by contemporary ones, while PABYV and CmEV isolates were genetically homogenous among their populations. This study underlines the need for continuous surveillance and further investigation into common symptoms of mixed viral infections.