Occurrence of Tomato Leaf Curl New Delhi Virus in Three Cucurbits Crops in Northern China and Its Fast and Accurate Detection through Heat-Labile Uracil-DNA Glycosylase Based LAMP Elucidated with Lateral Flow Dipstick.

Abstract

Tomato leaf curl New Delhi virus (ToLCNDV) poses a significant threat to cucurbit crops, causing severe yield losses. While being well documented in southern China, it has been found recently on melon, cucumber, and smooth luffa crops in Shandong province of northern China for past two years. Field surveys on symptomatic plants further confirmed its widespread occurrence and significant impact on these economically important crops. Whole-genome sequencing of the DNA-A and DNA-B components revealed over 99% sequence homology among the strains isolated from all three hosts. The phylogenetic analysis of sequencing data further confirmed that cucumber and smooth luffa strains clustered most closely with other Chinese ones, whereas the strain isolated from melons exhibited a closer evolutionary relationship to Indian isolates. A set of loop-mediated isothermal amplification (LAMP) primers targeting at the conserved regions of DNA-A was designed and an assay by integrating the heat-labile uracil-DNA glycosylase (UDG) and lateral flow dipstick (LFD) was developed for a rapid and visualized ToLCNDV detection. The optimal reaction conditions consisted of a 5 min incubation at 30°C, followed by an incubation at 62°C for 50 min. The assay was 10-fold more sensitive than conventional PCR with no cross-reactions detected among other cucurbit viruses. Therefore, the UDG-LAMP-LFD assay developed in this study provides a dependable diagnostic tool for the surveillance of ToLCNDV and the early detection of the virus on cucurbit seedlings.