Isolation and Characterization of Begomovirus(es) from Cestrum nocturnum

Abstract

Night blooming jasmine (Cestrum nocturnum; family Solanacea) is an evergreen ornamental plant, grown widely across the tropical and subtropical regions of the world. Severe leaf curling and mild yellowing symptoms were observed on the leaves of night blooming jasmine. Leaves from symptomatic plants and non-symptomatic plants were collected from Shifa International hospital (Isolate CN-146) and National University of Sciences and Technology (NUST; Isolate CS-152), around Islamabad, Pakistan during 2013. Their total DNA was extracted using Cetyl Trimethyl Ammonium Bromide (CTAB) method. The presence of a begomovirus was confirmed with diagnostic PCR using diagnostic pair of primers: CPF/CPR (for the amplification of coat protein gene of begomovirus) and NSPF/NSPR (for the amplification of Nuclear Shuttle Protein gene). The products of expected sizes: (approx. 750bp) for CP and (approx… 900bp) for NSP was obtained from symptomatic samples of both isolates. There was no amplification from non-symptomatic samples. Full length amplification products were obtained using abutting primer pairs; SonAF/SonAR and KTBF/KTBR, for the amplification of DNA A and DNA B, respectively. The expected size products of (approx. 2800bp) were obtained from both the samples. The products were T/A cloned and potential clones were sequenced in both orientation. The complete nucleotide sequence for DNA A of isolate CN-146 and isolate CS-152 were determined to be 2755 bp and 2757bp, respectively in length. The isolate CS-152 and the isolate CN-146 showed 84.8 nucleotide sequence identity between them while highest nucleotide sequence identity (89% and 88.9, respectively) to Pedilanthus leaf curl virus (accession no. AM948961), indicating to be new species of begomovirus for which we proposed the names “Cestrum leaf curl virus (for isolate CN-146) and Cestrum leaf curl Islamabad virus (for isolates CS-152)”. The complete nucleotide sequences of DNA B, from isolate CS152 and the isolate CN-146 were determined to be of 2694 and 2692bp, respectively and showed highest nucleotide sequence identity (99%) with Tomato leaf curl New Delhi Virus DNA B (accession no.AM 849547). Since night blooming jasmine is widely grown as an ornamental plant in Pakistan, the presence of two different begomoviruses in the same host plants within short distance is a serious threat to other plants and to agriculture economy. This indicates that night blooming jasmine is a good host for begomoviruses. This is the first report of Cestrum leaf curl virus and Cestrum leaf curl Islamabad virus affecting night blooming jasmine.