Molecular Characterization of Tobacco leaf rugose virus, a New Begomovirus Infecting Tobacco in Cuba

Tobacco (Nicotiana tabacum L.) is the main raw material for the cigar industry and one of the most important crops in Cuba comprising 49,654 ha. During the past 20 years, foliar rugosity and stunting symptoms have been observed in several tobacco producing areas. These symptoms were correlated with the presence of typical geminivirus nuclear inclusions and the transmission of the causal agent by whiteflies (Bemisia tabaci Genn) (1). To identify the suspect geminivirus, diseased leaf samples were collected in Havana province in 2000 and 2001. Sap extracts or leaf pieces were used to inoculate healthy tomato and tobacco plants by mechanical and graft inoculation procedures. Characteristic symptoms were reproduced in tobacco plants only by grafting (8 to 10 plants). DNA extracts from symptomatic plants were analyzed by Southern blot and polymerase chain reaction. The presence of a bipartite begomovirus was supported by the observation of hybridization signals (1.6 kb to 3 kb) at low stringency to probes derived from DNA-A and DNA-B of Taino tomato mottle virus. Furthermore, typical begomovirus amplicons of approximately 1.4 kb and 1.2 kb were amplified using the primer sets PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722 (2), respectively. Amplicons were cloned, and their nucleotide sequences (nt) obtained from two clones each. Sequence for component A was assembled, and some fragments were compared with those for other begomoviruses using CLUSTAL W. For the CP gene (756 nt) (GenBank Accession No. AJ488768), the comparison revealed the highest percentages of nt identity with Sida golden mosaic virus from Florida (SiGMV-F, GenBank Accession No. AF049336) (86%), Tomato mottle virus (GenBank Accession No. L14460) (83.5%), and the yellow vein strain of Sida golden mosaic virus from Honduras (GenBank Accession No. Y11099) (83.3%). In addition, the percentages of nt identity obtained using the core region (a 540-nt fragment located between positions 147 and 687) of the CP gene from the tobacco virus were calculated. The best scores were as follows: SiGMV-F, 87.8%; Jatropha mosaic virus (JMV) from Puerto Rico (GenBank Accession No. AF058025), 86.9%; and Tomato rugose mosaic virus (GenBank Accession No. AF291705), 86.3%. Finally, comparisons of the common region (CR, 144 nt) revealed the highest values with JMV from Jamaica (JMV-JM) DNA-A and DNA-B (GenBank Accession Nos. AF324410 and AF324411; 89% and 91.1%, respectively). Interestingly, the CR analysis revealed the presence of the Ori-associated iterative motif GGGGT, which is the same in the CR of JMV-JM. Although the data suggest that the tobacco begomovirus is related to the JMV-JM isolate, it is a new species, and the name of Tobacco leaf rugose virus (TbLRV) is proposed. References: (1) S. Quintero and J. Santiesteban, Agrotec. Cuba 11(1), 1979. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.