Molecular characterization of a Moroccan isolate of Tomato yellow leaf curl Sardinia virus and differentiation of the Tomato yellow leaf curl virus complex by the polymerase chain reaction

Abstract

The polymerase chain reaction (PCR) was used to identify an isolate of Tomato yellow leaf curl Sardinia virus (TYLCSV) from southwestern Morocco and to detect the members of the Tomato yellow leaf curl virus (TYLCV) complex. Thirty-five tomato samples with typical TYLCV symptoms were collected from infected tomato fields in the Souss-Massa region. PCR was performed with a general primer pair based on the coat protein (Cp) gene of the TYLCV complex, as well as with specific primer pairs for TYLCV and TYLCSV. Of the 35 samples tested, 29 generated a viral DNA product with the general primer pair, 29 samples gave a viral DNA product with the TYLCV-specific primers, and of these, 9 also gave a product with the TYLCSV primer pair; 6 samples did not give any PCR product with either primer pair. The full-length genome of TYLCSV was amplified with overlapping primers at the unique NcoI site in the TYLCSV genome (GenBank accession number X61153). The full-length genome of the TYLCSV isolate from Morocco is 2,777 nucleotides long (accession number AY702650) and is almost identical (97% nucleotide identity) to a TYLCSV isolate from Murcia, Spain (accession number Z25751). A PCR-based diagnostic method was developed to distinguish between TYLCV and TYLCSV in Morocco. To diagnose the TYLCV/TYLCSV complex a general primer pair was designed that anneals to a conserved region of the Cp gene. To diagnose TYLCSV exclusively, two primer pairs were designed to anneal specifically to the replication-associated protein gene (Rep) of TYLCSV from Morocco. To detect TYLCV exclusively, a primer pair previously described to amplify the intergenic region (IR) of TYLCV was used. The PCR primers were tested for their effectiveness using DNA clones of the TYLCSV from Morocco and of the TYLCV from the Dominican Republic. PCR using these primers offers a rapid means to detect the TYLCV complex and to distinguish between the two TYLCV species present in Morocco.