Isolation and Cloning of Begomovirus(es) from Islamabad

Abstract

Begomoviruses (family Geminiviridae) are a major threat to agricultural crops and horticulture plants in tropical and sub-tropical regions of the World. Ornamental and weed plants are thought to be the reservoir hosts of begomoviruses. Leaf samples of an ornamental plant Alcea rosea (Hollyhock; 2YI) showing crinkled leaf symptoms was collected from areas around Islamabad in 2014. Total genomic DNA was isolated from non-symptomatic and symptomatic samples by CTAB method. Full length amplification product (approx. 2800bp), for DNA-A component was obtained from isolate 2YI using a set of abutting primer pair SonAF/SonAR and was T/A cloned. No amplification was seen in non-symptomatic samples. A single colony of potential clone was sequenced in both orientations. The complete nucleotide sequence for DNA-A component of isolate 2YI was determined to be 2741bp in length. Comparison of the nucleotide sequence of isolate 2YI with sequences available in the GenBank database showed highest identity (86.6%) to Euphorbia leaf curl virus. Based on present species demarcation criteria, isolate 2YI was found to be a new species of begomovirus for which the name Hollyhock yellow vein mosaic Islamabad virus (HYVMIV) is proposed. Sequence comparison and recombination analysis showed that HYVMIV is generated as a result of recombination; with a major parent Pedilanthus leaf curl virus (PedLCV) (fragment between 31 to 2386bp) and minor parent Cotton leaf curl Multan virus (CLCuMV) (fragment between 2387 to 30bp). Attempts to amplify the second component (DNA-B or/and DNA betasatellite) in the infected sample failed to yield any results. As Hollyhock belongs to family Malvaceae and is infected by a virus recombinant of CLCuMV, it can be a new challenge for cotton crop and scientists.