First report of Tomato leaf curl New Delhi virus affecting chilli pepper in Pakistan

Abstract

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus that infects tomato and watermelon crops in the Indian subcontinent (Padidam et al., 1995a; Mansoor et al., 2000). Chilli pepper is an important crop on the Indian subcontinent that often shows symptoms similar to tomato leaf curl, such as yellowing, leaf curling, a reduction in leaf size and stunting. Since chilli and tomato crops overlap in the field, it is likely that chilli peppers may become infected with tomato begomoviruses. To assess the presence of ToLCNDV in chilli pepper, plants with symptoms were collected from several locations in the Punjab province of Pakistan. To confirm begomovirus infection, total DNA was isolated from leaf samples, resolved in agarose gels and blotted onto nylon membranes and probed with a radioactively labelled probe of DNA A of ToLCNDV. The probe hybridized with bands in all the samples collected from affected plants, when the blot was washed at medium stringency. To confirm infection of ToLCNDV, a PCR procedure was carried out using degenerate begomovirus DNA A primers, and products were obtained from these samples. A nearly full-length clone of this DNA A product was partially sequenced and was found to share 95% sequence identity with DNA A of ToLCNDV. These results confirmed ToLCNDV infection in chilli pepper in Pakistan. The DNA A-positive samples were also tested by hybridization using ToLCNDV DNA B as a virus-specific probe (Padidam et al., 1995b). A positive signal was detected in six out of eight samples that were positive using the DNA A probe. The presence of ToLCNDV was further confirmed by PCR using specific primers for DNA B (Padidam et al., 1995a), based on the movement protein gene of ToLCNDV (BC1F 5′-CACCATGGCAATAGGAAATGATGGTATGGG-3′ and BC1R 5′-AAGGATCCTCTTAATTTTTTGAATAAATTTGGC-3′). The use of these primers produced products of the expected size from all samples positive by hybridization. These results prove the occurrence of ToLCNDV in chilli pepper crops in Pakistan. Previous reports have identified the presence of distinct begomoviruses in chilli crops (Shih et al., 2003), but this is the first confirmed finding of ToLCNDV.