Oxford Nanopore Sequencing as a useful tool for investigating the population dynamics of invasive begomoviruses in Sicily

Tomato yellow leaf curl disease (TYLCD) is a major viral disease severely affecting tomato crops in the Mediterranean region, leading to reduced crop yield and significant economic losses. The disease is caused by monopartite begomoviruses belonging to the Geminiviridae family, primarily tomato yellow leaf curl Sardinia virus (TYLCSV) and tomato yellow leaf curl virus (TYLCV), which often co-infect tomato plants, promoting the emergence of recombinant viral genomes. To investigate the diversity and evolutionary dynamics of these viruses, symptomatic plants collected from agricultural sites in Sicily between 2020 and 2022, along with archived plant samples from 1994 to 1999, were analyzed. For each collection site, leaves from symptomatic plants were pooled to form representative samples. Total nucleic acids were extracted and subjected to rolling circle amplification to enrich circular viral genomes. The amplified products were sequenced using Oxford Nanopore Technologies (ONT) long-read sequencing to obtain full-length viral genomes. Bioinformatic analyses revealed that archived samples exclusively contained TYLCSV-related sequences, confirming its historical predominance in Sicilian agroecosystems. Recent samples, by contrast, no longer contained TYLCV or TYLCSV parental genomes but were dominated by TYLCV-derived recombinants such as TYLCV-IS141- and TYLCV-IS76-like variants, indicating a temporal shift in the structure of the viral population. Furthermore, a distinct group of newly emerged recombinants, provisionally referred to as TYLCV-IMS54, was identified in the most recent samples. Their genome comprises a TYLCV backbone, a 54-nucleotide segment from TYLCSV located downstream of the stem-loop region, and an 341-nucleotide region derived from TYLCV-Mild. These results demonstrate the importance of continuous viral population monitoring through ONT-based sequencing to detect emerging variants that may influence disease management strategies in tomato crops and highlight the central role of recombination in shaping begomovirus populations. IMPACT STATEMENTTomato yellow leaf curl disease (TYLCD) is one of the most damaging viral diseases affecting tomato crops in the Mediterranean basin, yet the long-term dynamics of its causal agents and the role of recombination remain challenging due to the genome plasticity of these viruses. This study provides an updated and comprehensive picture of the begomovirus population structure in Sicily, a key agricultural region for tomato production, by analyzing both contemporary and historical plant samples. Through the application of ONT long-read sequencing combined with RCA and bioinformatic analyses, this research identified persistent recombinant genotypes including a distinct group of newly emerged recombinants, named TYLCV-IMS54. These findings expand current knowledge on the genetic variability and evolutionary processes shaping begomovirus populations in Sicilian agroecosystems. The detection of recombinant genomes highlights the enduring role of recombination in begomovirus diversification. By integrating sequencing data with population and phylogenetic analysis, this work offers valuable insights into the epidemiology and management of TYLCD in regions heavily impacted or newly colonized by these viral pathogens. The study also underscores the importance of continuous molecular surveillance using ONT-based platforms to enable early detection of emerging recombinant variants, with significant implications for plant virology, crop protection and agricultural biosecurity strategies. DATA SUMMARYRaw reads are deposited in the Sequence Read Archive (SRA) of NCBI (https://www.ncbi.nlm.nih.gov/sra) with BioProjects ID PRJNA1226414 and PRJNA1273745; the recombinant TYLCV-IMS54 sequence is available in GenBank with Accession Number PQ873011. Parental TYLCV-strains used in this study could be retrieved in GenBank with the following Acc. Nos: DQ144621 for TYLCV, NC_003828 for TYLCSV, KJ913682 for TYLCV-Mild. Recombinant strains could be retrieved in GenBank with the following Acc. Nos.: LN846609 for TYLCV-IS76 and AF271234 for TYLCMaV. The authors confirm that all supporting data, code and protocols have been provided within the article or through supplementary data files.