Tobacco mottle leaf curl virus, a new begomovirus infecting tobacco in Cuba

An increase in begomovirus-associated symptoms has been observed in cultivated tobacco plants (Nicotiana tabacum) in Cuban fields in the past two decades. Previously, the presence of two new geminiviruses, Tobacco leaf rugose virus (TbLRV) (Dominguez et al., 2002) and Tobacco leaf curl Cuba virus (TbLCuCUV) (Morán et al., 2005) was shown. Further screening during the period 2002-2003 in Sancti Spiritus, a locality in the central region of the Cuba Island, showed the presence of a novel begomovirus. Symptomatic plants (approximately 1% of plants were affected) were spread over 39 ha and DNA extracts from 31 of them were analyzed by Southern blot hybridization and polymerase chain reaction. The presence of a begomovirus was detected in 26 of the samples by hybridization (hybridizing bands of 1.6 to 3 kb) at low washing stringency to probes derived from the DNA-A of TbLCuCUV. Furthermore, typical begomovirus amplicons of approximately 1.4 kb and 1.2 kb were amplified from 26 samples using the primer sets PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722 (Rojas et al., 1993), respectively. Amplicons were cloned, and their nucleotide sequences determined for two clones obtained with each primer pair. The complete sequence of the DNA A component was assembled from these fragments and deposited in the GenBank (Accession No. FM160943). DNA sequences were compared with those from other begomoviruses using PASC (http://www.ncbi.nlm.nih.gov/sutils/pasc) and CLUSTAL W. The comparison of the full-length nucleotide sequence of the DNA A component (2634 nt) revealed the highest percentage identities with TbLCuCUV (85.2%) and Wissadula golden mosaic St Thomas virus (84.6%) (DQ395343). The same analysis with the other previously identified Cuban tobacco-infecting begomovirus, TbLRV, showed 83% identity. For the CP gene (765 nt), the comparison revealed the highest percentages nucleotide identity (87%) with Sida golden mosaic virus (SiGMV, AF049336). Comparisons with the common region (CR) sequence (146 nt) revealed the highest nucleotide sequence identity (86%) with Sida golden yellow vein virus (SiGYVV-[Cu:Hav], AJ577395). Interestingly, the CR analysis revealed the presence of the ori-associated iteron motif AATTGGAGW, which is distinct from that in SiGYVV-(Cu:Hav). These data indicate that this virus is a new begomovirus for which the name Tobacco mottle leaf curl virus (TbMoLCV) is proposed.