Taino Tomato Mottle Virus, a New Bipartite Geminivirus from Cuba.

Abstract

Geminiviruses have become the most important virus group affecting tomatoes (Lycopersicon lycopersicum (L.) Karsten) in Cuba since they have been detected in all tomato-producing areas, causing serious losses. Recently, a whitefly-transmitted, monopartite geminivirus was detected in Cuba and identified as tomato yellow leaf curl virus-Israel (TYLCV-Is) (1). Samples collected from the main tomato-producing areas during the period 1995 to1996 were further analyzed by polymerase chain reaction (PCR) with degenerate primers (PAL1v1978 and PAR1c496) (2). Whereas in samples from most areas only TYLCV was detected, in some samples from the Havana area, two DNA fragments (approximately 1.4 and 1.1 kb) were amplified by PCR. The larger fragment was identified as part of the TYLCV-Is genome, confirming the previous report (1). The 1.1-kb fragment was cloned and its nucleotide sequence suggested that a new bipartite geminivirus was also present in those tomato samples. To clone the entire genome, tomato plants were inoculated by biolistics with DNA extract from field samples. After symptom expression, a viral DNA-enriched preparation from the inoculated tomatoes was independently digested with several restriction enzymes and the products were ligated into pZero plasmid (Invitrogen, San Diego, CA). Several clones in the 2.6-kb size range were characterized by restriction mapping and hybridization against component A and B heterologous probes. Two clones were selected as containing putative A and B components and their infectivity was tested by biolistic inoculation of tomato and pepper plants. The inoculated tomatoes developed a mild mottle in the younger leaves, whereas no symptoms were visible on the inoculated pepper plants. However, the presence of viral DNA was confirmed in both tomatoes and peppers by Southern blot hybridization analysis with A- and B-specific probes. Partial sequences of both components were obtained and their analysis showed that both components shared a 170-bases common region with a 95% identity. In addition, the nucleotide sequences of two open reading frames, one in each component (AC1 and BC1), were determined and compared with geminivirus sequences deposited in Gen-Bank. A dendogram generated with the CLUSTAL program and obtained with the AC1 and BC1 amino acid sequences, placed the new geminivirus in a cluster with tomato mottle virus (ToMoV; accession nos. L14460, L14461), Abutilon mosaic virus (AbMV; X15983, X15984), potato yellow mosaic virus (PYMV; D00940, D00941), and bean dwarf mosaic virus (BDMV; M88179, M88180). The percentages of identity obtained with the amino acid sequences were as follows. For AC1: ToMoV, 87%; PYMV, 79.5%; BDMV, 78.7%; and AbMV, 78%. For BC1 protein: BDMV, 92.8%; ToMoV, 89.1%; PYMV, 88.1%; and AbMV, 67.5%. In addition, the sequences were compared with partial nucleotide sequences (AC1, coat protein [CP], and common region) of a bipartite geminivirus affecting tomatoes in Jamaica (accession nos. U83855, U83854, and U83850). Interestingly, the common regions showed a higher percentage of identity (88%) than the CP and AC1 partial nucleotide sequences (86 and 74%, respectively). These data suggest that the virus reported here is a new geminivirus and the first bipartite geminivirus reported in Cuba. Thus, the name of Taino tomato mottle virus is proposed. (Taino refers to the name of the inhabitants of Cuba at the time of Columbus's arrival in the Caribbean).