Tobacco leaf curl Cuba virus, a new begomovirus infecting tobacco ( Nicotiana tabacum ) in Cuba

Tobacco (Nicotiana tabacum) is an important crop in Cuba, with 34 000 ha under cultivation. In 2002, a begomovirus Tobacco leaf rugose virus (TbLRV) was isolated from diseased tobacco plants showing symptoms of foliar rugosity and stunting (Domínguez et al., 2002). However, tobacco plants with foliar rugosity symptoms but without stunting and dwarfing have been also observed. To identify the causal agent(s), leaves from plants with symptoms were collected in the Sancti Spíritus province in 2004. The presence of a begomovirus in tobacco leaf samples was suggested by strong hybridization signals in Southern blots using DNA-A from TbLRV as a probe, under low stringency conditions. Healthy tobacco plants were inoculated using a bioballistic method (Finer et al., 1992) with total DNA extracts purified from infected leaves. Foliar rugosity symptoms similar to the source plants were observed in one out of three inoculated plants. Total DNA from the inoculated tobacco plant with symptoms was analysed by PCR, using two degenerate primer sets: PAL1v, 1978-PAR1c715 and PAL1c 1960-PAR1v722 (Rojas et al., 1993). Amplicons of approximately 1·4 and 1·2 kb were amplified, cloned and component A sequenced (GenBank accession no. AM050143). Sequences were compared with those from begomoviruses using the program CLUSTAL W. For the coat protein (753 nt) and rep genes (1083 nt), the highest percentages of identity (87·2 and 84·8%, respectively) were to Okra yellow mosaic Mexico virus (OkYMMeV; GenBank accession no. DQ022611). Overall, the DNA A sequence showed the closest nt sequence identity (83·6%) to OkYMMeV. Finally, comparisons of the common region (CR; 145 nt) revealed the closest identity (80·1%) to Tomato mottle Taino virus DNA-A (ToMoTV, GenBank accession no. AF012300). Interestingly, CR analysis revealed the presence of the Ori-associated iterative motif GGGGG, which is also found in ToMoTV. The data suggest that the begomovirus in tobacco is a new species and the name Tobacco leaf curl Cuba virus (TbLCCuV) is proposed.