Design and validation of an infectious clone of ToLCNDV, an emergent virus in south-east Spain

Tomato leaf curl New Delhi virus (ToLCNDV) (family Geminiviridae, genus Begomovirus) is an important pathogen that severely affects tomato and cucurbits production. Original from the Indian Subcontinent, it has been recently introduced in Spain causing severe epidemics in cucurbit crops. ToLCNDV genome consists of two single-stranded circular DNA molecules, denominated DNA-A and DNA-B, of approximately 2.7 kb each. Partial dimeric DNA-A and DNA-B clones were constructed in a binary vector and used to agroinoculate zucchini (Cucurbita pepo 'Brillante') and pumpkin (Cucurbita moschata 'Avalon F1') plants. Symptoms were observed in plants of both species at 100 and 50% frequency for zucchini and pumpkin plants, respectively. First symptoms appeared 7 days post-inoculation, and they were clearly observed at 14 days post-inoculation. Visual observation was confirmed by molecular techniques, such as molecular hybridization and qPCR. This new ToLCNDV infectious clone, based on a Spanish isolate, constitutes a useful tool for basic and applied virology studies.