Detalle de: First Evidence of Tomato Yellow Leaf Curl Virus-Israel IS76 Recombinant Isolates Associated with Severe Yellow Leaf Curl Epidemics in Resistant Tomatoes in Spain

Tomato yellow leaf curl is one of the most devastating diseases affecting tomato crops worldwide. In 2017, tomato plants showing severe disease symptoms were reported in Almería province, one of the main tomato-producing areas of Spain. The tomato varieties affected are resistant to tomato yellow leaf curl virus (TYLCV), which is the main cause of the disease in Spain, because they harbor the Ty-1/Ty-3 gene. A survey was conducted in six greenhouses, and a total of 33 samples were collected from symptomatic Ty-1/Ty-3 tomato plants. Total DNA was extracted from the samples and used in polymerase chain reactions (PCRs) with the universal begomovirus primer pair TY1/TY2, which amplifies a 580-bp fragment of the begomovirus coat protein gene (Accotto et al. 2000). Digestion of the resulting PCR products with the restriction enzyme AvaII distinguishes TYLCV from tomato yellow leaf curl Sardinia virus (TYLCSV; another begomovirus species causing tomato yellow leaf curl disease in Spain) by restriction fragment length polymorphism (Accotto et al. 2000). This analysis showed the presence of TYLCV in all samples. Samples were pooled (one pool from each plot), and circular DNA was amplified by the rolling circle amplification (RCA) technique from plant DNA extracts using phi29 DNA polymerase (TempliPhi, GE Healthcare, Chicago, IL). RCA products were digested with SacI, and whole genomes were cloned in pGEM-5Zf (+) vectors (Promega, Madison, WI). Nucleotide sequences for the whole genome were determined for five independent clones (one clone per pool) using Sanger sequencing (accession nos. MH931766 to MH931770). A BLAST analysis showed that two of the clones (accession nos. MH931769 and MH931770) are isolates of the Israel strain of TYLCV (TYLCV-IL), with a percentage of nucleotide identity of 99.6% between them and 99.7% with the Spanish isolate TYLCV-IL_Mu_5.1_AG:15 (accession no. MH680956), which has recently been identified in association with severe TYLCV epidemics in resistant tomatoes in Murcia (Torre et al. 2018). The other three clones (accession nos. MH931766 to MH93768) shared a 99.9% nucleotide sequence identity between each other and had 99.7% nucleotide sequence identity with the TYLCV-IL IS76 G2-6 recombinant isolate (accession no. LN846599) recently described by Belabess et al. (2015). TYLCV-IL IS76-like isolates were found in Morocco associated with severe epidemics in Ty-1/Ty-3 tomato crops and, characteristically, had a TYLCSV-specific fragment of approximately 76 nucleotides in the intergenic region (Belabess et al. 2015). To detect recombination events, we carried out an analysis of the sequences from Almeria using the RDP4 program package (Martin et al. 2015), a manual sequence alignment, as well as phylogenetic analyses, all confirming the presence of the TYLCSV-specific region. Apart from Morocco, recombinant isolates similar to TYLCV-IL IS76 were only identified in Sicily, and the Sicilian isolate had a larger recombination event (Panno et al. 2018). This is the first evidence of recombinant isolates of the TYLCV-IL IS76-type in Spain and outside Morocco. Given the apparently rapid spread of TYLCV-IL IS76 isolates in Morocco, efforts are required to monitor their potential global spread.