This work provides evidence of the significant contribution of recombination to the genetic diversification of emerging begomovirus populations. In southern Spain, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV) are distinct geminivirus species that coexist in the field and contribute to the tomato yellow leaf curl disease epidemic. A natural recombinant between TYLCSV and TYLCV has been detected and an infectious clone of a recombinant isolate (ES421/99) was obtained and characterized. Analysis of its genome showed that the recombination sites are located in the intergenic region in which a conserved stem-loop structure occurs and at the 3'-end of the replication enhancer protein open reading frame. ES421/99 exhibited a novel pathogenic phenotype that might provide it with a selective advantage over the parental genotypes. This agrees with results from field studies which revealed that the recombinant strain is becoming prevalent in the region in which it was detected.

We examined the native plant host Solanum nigrum as reservoir of genetic diversity of begomoviruses that cause the tomato yellow leaf curl disease (TYLCD) emerging in southern Spain. Presence of isolates of all the species and strains found associated with TYLCD in this area was demonstrated. Mixed infections were common, which is a prerequisite for recombination to occur. In fact, presence of a novel recombinant begomovirus was demonstrated. Analysis of an infectious clone showed that it resulted from a genetic exchange between isolates of the ES strain of Tomato yellow leaf curl Sardinia virus and of the type strain of Tomato yellow leaf curl virus. The novel biological properties suggested that it is a step forward in the ecological adaptation to the invaded area. This recombinant represents an isolate of a new begomovirus species for which the name Tomato yellow leaf curl Axarquia virus is proposed. Spread into commercial tomatoes is shown.

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.

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.

Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Málaga virus are monopartite begomoviruses (genus Begomovirus, family Geminiviridae) that infect common bean (Phaseolus vulgaris), causing bean leaf crumple disease (BLCD). This disease was found to be widespread in southern Spain and causes stunted growth, flower abortion, and leaf and pod deformation in common bean plants. Commercial yield losses of up to 100% occur. In the present study, we have identified and characterized a resistance trait to BLCD-associated viruses in the common bean breeding line GG12. This resistance resulted in a complete absence of BLCD symptoms under field conditions or after experimental inoculation. Our analysis showed that virus replication was not inhibited. However, a severe restriction to systemic virus accumulation occurred in resistant plants, suggesting that cell-to-cell or long-distance movement were impaired. In addition, recovery from virus infection was observed in resistant plants. The reaction of P. vulgaris lines GG12 (resistant) and GG14 (susceptible), and of F(1), F(2), and backcross populations derived from them, to TYLCV inoculation suggested that a single dominant gene conferred the BLCD resistance described here.

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Epidemics of tomato yellow leaf curl disease (TYLCD) caused by tomato yellow leaf curl-like begomoviruses (genus Begomovirus, family Geminiviridae) severely damage open field and protected tomato crops worldwide. Intensive application of insecticides against the whitefly vector Bemisia tabaci is generally used as control strategy to reduce TYLCD impact. This practice, however, is frequently ineffective and has a negative impact on the environment and human health. TYLCD-resistant varieties are commercially available, but cultivation of susceptible traditional tasting ones is also requested if possible. For susceptible tomatoes, here we show that using whitefly optical barriers by means of UV-blocking plastics in protected crops can contribute to reducing TYLCD damage and increasing commercial fruit yield. Moreover, induction of systemic acquired resistance by application of the elicitor of plant defense acibenzolar-S-methyl was effective to reduce yield losses when viral pressure was moderate. Interestingly, combining both practices in protected tomato crops can result in a significant TYLCD control. Therefore, these control practices are proposed to be used commercially as management alternatives to include in integrated management of TYLCD.

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