Detection of Tomato Leaf Curl New Delhi Virus-ES by Real-Time Quantitative PCR

The tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite, single-stranded DNA begomovirus (genus Begomovirus, family Geminiviridae) that was first identified in India in 1995 affecting solanaceous cucurbitaceous crops in India. In 2012 a new virus strain, denominated ToLCNDV-ES, was first detected in Europe affecting zucchini squash, melon, cucumber, and to a lesser degree, tomato, in Spain and other Mediterranean countries. The virus is currently listed as a quarantine pest by EPPO. ToLCNDV-ES is reported to be transmitted by Bemisia tabaci whiteflies. Symptoms that are produced by the virus in zucchini are curling, chlorosis and vein thickening of leaves, stunted growth, and fruit deformation and abortion However, viruses of cucurbits often cause severe symptoms and mixed infections are common. Therefore ToLCNDV needs to be confirmed through laboratory analysis, either through PCR or through ELISA. ELISA provides a good basis for virus detection and confirmation in virus surveys, while PCR-based tests overall have higher sensitivity. In this manuscript, we propose a protocol to detect and quantify the viral load of ToLCNDV in any of its horticultural hosts.