Resistance Broken: Integrated Genomic and Phenotypic Insights into Tomato Spotted Wilt Virus Strains Overcoming Sw-5b and Tsw Resistance

Background Tomato spotted wilt orthotospovirus (TSWV) is a globally significant plant pathogen infecting more than 1,000 species and causing billion-dollar crop losses annually. The emergence of resistance-breaking (RB) strains has undermined single-gene resistance conferred by Sw-5b in tomato and Tsw in pepper, threatening durable resistance strategies. However, genomic and phenotypic characterization of novel RB strains remains limited. Results We characterized six novel TSWV strains—four tomato RB strains (Tom-BL1, Tom-BL2, Tom-CA, Tom-MX) and two pepper strains (RB Pep-BL and non-RB Pep-Non-RB)—using phenotypic assays, full-genome sequencing, and population genetic analyses. Tomato RB strains triggered hypersensitive response (HR) in resistant pepper but not in resistant tomato, whereas Pep-BL uniquely overcame both Sw-5b and Tsw resistance without HR. Pep-BL caused earlier and more severe symptoms in resistant pepper (12 DPI) than tomato (22 DPI) and accumulated to higher titers in resistant tomato than other RB strains. Comparative genomics revealed 93–98% (L), 90–98.5% (M), and 90–98% (S) nucleotide identity with reported isolates. Phylogenetic analyses showed clustering by geography, with Tom-CA grouping distinctly with a Mexican RB isolate, while Pep-BL clustered with Pep-Non-RB. No reassortment was detected, though recombination was observed in Tom-CA, Tom-BL1, and Pep-BL. Population genetics revealed low nucleotide diversity and evidence of purifying selection, consistent with thrips-mediated bottlenecks. Codon-level analyses identified novel substitutions in NSm (C118F, K125R) and NSs (Y462H) that may contribute to RB phenotypes. Conclusions This study provides the first comprehensive genomic, phylogenetic, and phenotypic characterization of multiple novel RB TSWV strains, including the discovery of Pep-BL, the first reported strain capable of breaking both Sw-5b and Tsw resistance. By linking unique mutations with dual resistance-breaking capacity, our findings advance understanding of TSWV evolution and lay the groundwork for functional validation studies and the development of sustainable resistance strategies.