Development, validation, and application of reverse transcription real-time and droplet digital PCR assays for the detection of the potyviruses watermelon mosaic virus and zucchini yellow mosaic virus in cucurbits

Among the cucurbit-infecting viruses, watermelon mosaic virus (WMV) and zucchini yellow mosaic virus (ZYMV) (Potyvirus: Potyviridae) are responsible for severe symptoms on cucumber, melon, watermelon, and zucchini cultivations worldwide. In this study, reverse transcription real-time PCR (real-time RT-PCR) and droplet-digital PCR (RT-ddPCR) assays targeting the coat protein (CP) genes of WMV and ZYMV were developed and validated according to the international standards of plant pest diagnosis (EPPO PM 7/98 (5)). First, the diagnostic performance of WMV-CP and ZYMV-CP real-time RT-PCRs was evaluated, and the assays displayed an analytical sensitivity of 10−5 and 10−3, respectively. The tests also showed an optimal repeatability, reproducibility and analytical specificity, and were reliable for the virus detection in naturally infected samples and across a wide range of cucurbit hosts. Based on these results, the real-time RT-PCR reactions were adapted to set up RT-ddPCR assays. These were the first RT-ddPCR assays aiming at the detection and quantification of WMV and ZYMV and showed a high sensitivity, being able to detect until 9 and 8 copies/µL of WMV or ZYMV, respectively. The RT-ddPCRs allowed the direct estimation of the virus concentrations and opened to a broad range of applications in disease management, such as the evaluation of partial resistance in breeding processes, identification of antagonistic/synergistic events, and studies on the implementation of natural compounds in the integrated management strategies.