Three Previously Unidentified Begomoviral Genotypes from Tomato Exhibiting Leaf Curl Disease Symptoms from Central Sudan.

Abstract

Field tomato plants exhibiting upward curling of leaflets, chlorosis, and stunting symptoms described for tomato leaf curl disease in Sudan (2) were collected in 1996 from Gezira (GZ) and Shambat (SH), Sudan. Disease symptoms were reproduced following experimental transmission of the causal agent(s) by the whitefly Bemisia tabaci from field tomato to virus-free tomato seedlings in a glasshouse at Gezira Research Station, Wad Medani, Sudan. Total nucleic acids were extracted from symptomatic tomato test plants. An ≈1.3-kbp fragment, diagnostic for begomovirus, was obtained from extracts by polymerase chain reaction using degenerate primers that amplify the coat protein gene (CP) and the respective flanking sequences for most begomoviuses (1). A second pair of degenerate primers was used to amplify a 2.3-kbp begomoviral fragment that overlaps both ends of the (CP) amplicon by >200 nt (1). At least 10 amplicons for each were cloned, and their sequences were determined, revealing three unique, tomato-infecting begomoviruses genotypes, two from GZ and one from SH. No B component was detected using degenerate primers that direct the amplification of a diagnostic fragment of the B component (1.4 kbp) for most bipartite begomoviruses. The organization of the three, apparently full-length viral genomes, was typical of other monopartite begomoviruses. A GenBank search revealed that the three viruses were previously undescribed. The GZ and SH tomato isolates are herein provisionally named ToLCV-GZ1 (GenBank Accession No. AY044137), ToLCV-GZ2 (GenBank Accession No. AY044138), and ToLCV-SH (GenBank Accession No. AY044139), respectively. All three tomato-infecting begomoviruses have identical stem-loop structures containing the conserved nonanucleotide motif characteristic of all members of the family Geminiviridae; however, the predicted Rep binding element located in the common region is unique for each virus. Phylogenetic analysis of the three viral sequences placed them in a large clade containing all other Old World begomoviruses. Distance comparisons among these and other well-studied begomoviruses indicated that ToLCV-GZ1 and ToLCV-SH shared an overall 90% nucleotide sequence identity, with ˜83% nucleotide sequence identity to ToLCV-GZ2. ToLCV-GZ1 and ToLCV-SH were 83% identical, with their closest relative, Tomato yellow leaf curl virus (TYLCV), while ToLCV-GZ2 shared 93% identity with TYLCV. The genomes of all three Sudan viruses contained regions of homologous nucleotide sequences, suggesting intermolecular exchange among these viruses. Exclusion of the homologous sequences (>800 nt) from the phylogenetic analysis indicated even lower shared nucleotide identities (<90%, the arbitrary cut-off for distinct species), which may warrant their classification as separate species. These three newly described begomoviruses are indigenous to central Sudan, and comprise a unique Old World lineage distinct from previously described begomoviruses associated with leaf curl disease of tomato in Africa and the Mediterranean Region.