# Oxford nanopore sequencing as a useful tool for investigating the population dynamics of invasive begomoviruses in Sicily

## TL;DR

This study uses Oxford Nanopore sequencing to track changes in begomovirus populations in Sicilian tomato crops over time, revealing new recombinant variants.

## Contribution

The study demonstrates the utility of ONT sequencing for monitoring begomovirus evolution and detecting emerging recombinant variants in agricultural settings.

## Key findings

- Archived Sicilian samples were dominated by TYLCSV, while recent samples showed a shift to TYLCV-derived recombinants.
- A new recombinant variant, TYLCV-IMS54, was identified with a unique genomic structure combining elements from multiple viruses.
- ONT sequencing proved effective for tracking viral population dynamics and recombination events in begomoviruses.

## Abstract

Tomato yellow leaf curl disease 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 analysed. 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 mainly contained TYLCSV-related sequences, confirming its historical predominance in Sicilian agroecosystems. Recent samples, by contrast, 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-nt segment from TYLCSV located downstream of the stem-loop region and a 341-nt 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.