Abstract
Tomato samples with typical symptoms of tomato yellow leaf curl virus (TYLCV) infection were collected from six tomato growing regions in Tanzania and dot-blotted on nylon membranes. The membranes were hybridised with nucleic acid probes synthesized to detect TYLCV from Israel and Sardinia. Viral DNA was extracted from the samples by phenol-chloform procedure and amplified by polymerase chain reaction using a primer pair (OTY 2 and OTY 6) designed to amplify a 1.2kb fragment containing the coat protein, intergenic region and replication-associated protein. The amplified DNA was ligated to pBluescript II KS+ and transformed into Esherichia coli strain JM 83 cells by electroporation. Colonies containing the insert were sequenced using a Li-Cor DNA Semi-automatic Sequencer. The BLAST programme was used to search for viruses with similar sequences. Phylogenetic relationships with 20 geminiviruses were established using the CLUSTAL function of the Vector NT1.5 software. Amplified DNA was digested with Alu I and electrophoresed in polyacrylamide gel. Tomato yellow leaf curl virus samples from all the regions hybridised with both probes. Restriction analysis showed similar banding patterns for all the isolates. Both sequence comparison and phylogeny showed that TYLCV from Tanzania was closely related to TYLCV-Sar. The TYLCV isolates from the six regions were genetically the same.
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