S. Iram¹, L. Amrao¹, M.S. Mansoor¹*, A.H. Malik¹, R.W. Briddon² and Y. Zafar¹

¹ National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan
² Department of Disease and Stress Biology, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK

*smansoor@nibge.org

Accepted for publication: 21/09/04

Golden dewdrop (Duranta erecta) is a common ornamental plant of the family Verbenaceae that is often grown as garden hedge in Pakistan. During a recent survey for begomoviruses, symptoms typical of begomoviruses, including reduced leaf size, upward leaf curling and chlorosis, were observed on D. erecta throughout the Punjab and North West Frontier Province (NWFP). To detect the presence of a begomovirus nucleic acid extracts were prepared from leaf samples showing symptoms, collected from ten distinct locations in the Punjab and NWFP. The DNA was resolved in agarose gels, transferred to nylon membranes and hybridized with an [α32P]dCTP-labeled full-length clone of Cotton leaf curl Multan virus (pCLCUV003; Mansoor et al., 2003). The pattern of hybridizing single- and double-stranded DNA bands typical of the replication of geminiviruses was observed for all samples with symptoms. None were observed with samples without symptoms, confirming the association of a begomovirus with the disease.

Figure 1: Typical symptoms of leaf curl disease of D. erecta in Pakistan.	Figure 2: Symptoms of Duranta leaf curl on a shoot showing symptoms (left) compared to a shoot without symptoms from the same plant. Flowers are reduced in size and flowering is delayed in symptomatic shoots.

The presence of a begomovirus was further confirmed by PCR amplification using primers CLCV1 (virion-sense primer 5’-CCGTGCTGCTGCCCCCATTGTCCGCGTCAC-3’; annealing within the coat protein gene) and CLCV2 (complementary-sense primer 5’-CTGCCACAACCATGGATTCACGCACAG GG-3’; annealing in the C2 gene), designed to sequences conserved among begomoviruses from the Indian subcontinent (Mansoor et al., unpublished). An amplification product of the expected size (approx. 1700 bp) was produced from all symptomatic samples but not from non-symptomatic samples. For a sample originating from Faisalabad, the PCR product was cloned and end sequenced. The sequences obtained showed the highest levels of sequence identity to Croton yellow vein virus (91% over a stretch of 407 nucleotides in the virion-sense; Croton yellow vein virus is a provisional species in the genus Begomovirus) and Papaya leaf curl virus (93% identity over a stretch of 462 nucleotides in the complementary-sense). These findings suggest that the virus of Duranta is either a recombinant virus or a distinct begomovirus for which we suggest the name Duranta leaf curl virus.

We have recently shown that many begomoviruses of the Old World are associated with a single-stranded DNA satellite (DNA β; Briddon et al., 2003). Efforts to identify the presence of a DNA β in infected Duranta, using universal DNA β primers (Briddon et al., 2002), were uniformly negative. Efforts are now underway to identify the second genomic component (if any) and produce full-length clones of the virus. To the best of our knowledge this the first report of a begomovirus associated with leaf curl disease of Duranta.

Briddon RW, Bull SE, Amin I, Idris AM, Mansoor S, Bedford ID, Dhawan P, Rishi N, Siwatch SS, Abdel-Salam AM, Brown JK, Zafar Y, Markham PG, 2003. Diversity of DNA β; a satellite molecule associated with some monopartite begomoviruses. Virology 312, 106-121.

Briddon RW, Bull SE, Mansoor S, Amin, I, Markham PG, 2002. Universal primers for the PCR-mediated amplification of DNA ß; a molecule associated with some monopartite begomoviruses. Molecular Biotechnology 20, 315-318.

Mansoor S, Briddon, RW, Bull SE, Bedford ID, Bashir A, Hussain M, Saeed M, Zafar Y, Malik KA, Fauquet CM, Markham PG, 2003. Cotton leaf curl disease is associated with multiple monopartite begomoviruses supported by single DNA β. Archives of Virology 148, 1969-1986.

The British Society for Plant Pathology