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.