RESUMEN
ABSTRACT The evolution of the plant single-stranded DNA virus Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae) has been monitored for 8 years after its appearance in southern Spain. Variation within three genomic regions of 166 TYLCSV isolates collected from three locations was assessed by single-strand conformation polymorphism (SSCP) analysis. According to SSCP, the intergenic region (IR) was the most variable. Low genetic diversity was found within the population and geographical or temporal differences were not evident. Nucleotide sequences of specific genomic regions of haplotypes identified by SSCP indicated close relationships among them. Therefore, the Spanish TYLCSV population appears to represent a single, undifferentiated population. The analysis of IR sequences for a subsample of 76 randomly chosen isolates confirmed the limited genetic diversity revealed by the SSCP analysis. A tendency to a lineal increase in diversity over time was observed in Málaga and Almería subpopulations; however, no accumulation of mutations in single isolates was evident. Negative selection to variation seems to operate to conserve certain regions of the genome. Thus, the low genetic diversity found in the studied TYLCSV population might be the result of a founder effect with subsequent selection against less fit variants arising by mutation.
RESUMEN
Tomato yellow leaf curl virus (TYLCV, formerly TYLCV-Is) and Tomato yellow leaf curl Sardinia virus (TYLCSV, formerly TYLCV-Sar) are geminivirus species of the genus Begomovirus that cause the disease known as tomato yellow leaf curl. In Spain, TYLCV and TYLCSV have coexisted in field and greenhouse tomato (Lycopersicon esculentum) crops since 1996 (2). TYLCV is also the causal agent of the leaf crumple disease of common bean (Phaseolus vulgaris) (1), a species that TYLCSV is unable to infect (2). Analysis of field samples from common bean plants affected by leaf crumple disease collected in Almería (southeastern Spain) during 1999 showed that, unexpectedly, several samples hybridized with TYLCV- and TYLCSV-specific probes prepared to the intergenic region (IR) as previously described (1). Polymerase chain reactions (PCR) performed with total nucleic acids extracted from one of these samples (ES421/99) using primer pairs specific to the IR of TYLCV (MA-30/MA-31) or TYLCSV (MA-14/MA-15) (1) gave no amplification product. However, the combination of MA-30 (5' end of TYLCV IR) and MA-15 (3' end of TYLCSV IR) produced a PCR DNA product of the expected size (351 bp). Direct DNA sequencing of this product (GenBank Accession No. AF401478) indicated the presence of a chimeric IR in ES421/99. Comparison of the obtained sequence with those available for isolates reported from Spain showed that the 5' side (149 nt) from the stem-loop structure conserved in the IR of all geminiviruses was 99% identical to the corresponding region of TYLCV (GenBank Accession No. AF071228) and only 62% identical to TYLCSV (GenBank Accession No. Z25751). In contrast, the 3' side (124 nt) from the stem-loop was 98% identical to the corresponding region of TYLCSV and only 57% identical to TYLCV. The 33-nt region involved in the stem-loop was 100% identical to TYLCV and showed one nucleotide change in the loop with respect to TYLCSV. Therefore, this DNA sequence data showed evidence of the occurrence in ES421/99 of a natural recombination between TYLCV and TYLCSV. The biological and epidemiological consequences of the presence of this new interspecific recombinant have yet to be determined. References: (1) J. Navas-Castillo et al. Plant Dis. 83:29, 1999. (2) S. Sánchez-Campos et al. Phytopathology 89:1038, 1999.
RESUMEN
Severe outbreaks of tomato yellow leaf curl disease occurred during summer and autumn 1999 in tomato (Lycopersicon esculentum Mill.) crops in the Vecindario Region of Gran Canaria (Canary Islands, Spain) and Agadir (southwestern Atlantic coast of Morocco). Symptoms of the disease included upward curling of leaflet margins, reduction of leaflet area, and yellowing of young leaves, as well as stunting and flower abortion. High populations of whiteflies, Bemisia tabaci Gen., were present on tomatoes in Agadir, and analysis of adult individuals by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) identified them as the biotype Q. Samples were collected from symptomatic tomato plants: 5 plants from Gran Canaria and 22 from three areas in Agadir, (7 from Agadir/1, 12 from Agadir/2, and 3 from Agadir/3) in the Koudya Region. Samples were analyzed for Tomato yellow leaf curl virus (TYLCV) Sar or Is (genus Begomovirus, family Geminiviridae) infection by squash blot hybridization under high stringency conditions with digoxigenin-labeled DNA probes specific to TYLCV-Sar or -Is, as described previously (1,3). The TYLCV-Sar probe hybridized to the five samples from Gran Canaria, and the TYLCV-Is probe hybridized to the 22 samples from Agadir. The TYLCV-Sar probe also hybridized to the three samples from Agadir/3. Primer pairs MA-14/MA-15 and MA-30/MA-31, designed for specific amplification of the intergenic region (IR) of TYLCV-Sar or -Is reported from Spain, respectively (1), were used in PCR to amplify one sample each from Gran Canaria, Agadir/1, and Agadir/3. A fragment of the expected size was obtained from the samples from Gran Canaria and Agadir/3 using MA14/MA15 (342 bp) and from the two samples from Agadir using MA30/MA31 (357 bp). PCR products were directly sequenced (GenBank Accession nos. AF215819 to AF215822). The nucleotide sequences of the IR fragments amplified from the Gran Canaria and Agadir/3 sample using MA-14/MA-15 indicated their closest relationship (99.0 and 96.7% identity, respectively) was to the corresponding region of a TYLCV-Sar isolate reported from Spain (GenBank Accession no. L27708). The nucleotide sequences of the IR fragments amplified from the Agadir/1 and Agadir/3 samples using MA-30/MA-31 indicated their closest relationship (98.1% identity) was to the corresponding region of the TYLCV-Is isolate reported from Spain (GenBank Accession no. AF071228). Based on the hybridization and sequence data, we conclude that the symptomatic plants from Gran Canaria were infected by TYLCV-Sar, those from Agadir/1 and Agadir/2 were infected by TYLCV-Is, and those from Agadir/3 had mixed infections with TYLCV-Is and TYLCV-Sar. The presence of TYLCV-Is in Morocco has been described recently (2). However, this is the first report of TYLCV-Sar in the Canary Islands and Morocco and extends its geographic range beyond the Iberian Peninsula and Italy. References: (1) J. Navas-Castillo et al. Plant Dis. 83:29, 1999. (2) M. Peterschmitt et al. Plant Dis. 83:1074, 1999. (3) S. Sánchez-Campos et al. Phytopathology 89:1038, 1999.
