First Report of the Resistance Breaking (RB) Strains of Tomato Spotted Wilt Virus (TSWV) Infecting Tomatoes with the Sw -5b Resistance Gene in Alabama

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In 2023, one tomato plant showing symptoms typical of tomato spotted wilt virus (TSWV; Orthotospovirus tomatomaculae), including necrotic spots on older leaves, concentric rings, stunting, and progressive wilting, was submitted from Marengo Co., Alabama (AL), to the Vector Entomology Laboratory for analysis. In 2024, three additional samples with the same symptoms were received from the Chandler Mountain area of northeast AL (St. Clair Co.). Leaf tissues from symptomatic plants collected in 2023-24 were processed for Sanger sequencing of TSWV NSm as described (Shehata et al. 2025a). NSm sequences from 2023 (four colonies sequenced from a single sample; PX634739-742) lacked resistance-breaking (RB) mutations. In contrast, 2024 NSm sequences (five colonies sequenced from three samples; PX634743-747) had V49A, C118Y, and V141I substitutions. The C118Y is associated with Sw-5b RB in tomato in Spain (Lopez et al. 2011). In 2025, growers in the same production region reported a severe outbreak of TSWV. To determine whether TSWV-RB with the C118Y mutation was circulating, and to test the Sw-5b RB phenotype under greenhouse conditions, we collected 10 samples with different TSWV-resistance backgrounds from three farms (Farms I-III). Seedlings of Sw-5b resistant cultivar ‘Mountain Merit’ and TSWV susceptible cultivar ‘Beefsteak’ were grown under greenhouse conditions, and 25 seedlings from each variety were mechanically inoculated with TSWV-infected leaf tissues from Farm III (Shehata et al. 2025b) at 17d after emergence. At 13d post-inoculation, plants from both varieties showed typical symptoms of TSWV. Infection was confirmed using Enzyme-Linked Immunosorbent Assay (ELISA) (AgDia, Elkhart, IN), while healthy controls were negative. Moreover, NSm was sequenced from both 2025 field samples and mechanically inoculated plants, resulting in 20 sequences (PX634748-767): 12 from field samples (eight sequenced from individual samples across three farms and four from a pooled Farm-III inoculum) and eight from mechanically inoculated plants (four colonies per variety). Consistent with 2024, C118Y and the other two mutations were present in 2025. Another mutation (I130V) appeared in 55% of 2025 sequences (n=11/20), indicating ongoing evolution of RB strains in AL. Moreover, AL’s 2024-25 sequences shared 4/6 mutations (I163V, V290I, N293S, K296Q) with Texas RB strains lacking C118Y (Chinnaiah et al. 2023), indicating both local evolution and regionally shared lineages circulating in AL. A phylogenetic tree was constructed (Shehata et al. 2025c) to assess the relatedness of AL NSm sequences to RB and peanut-derived isolates from AL and Georgia (GA). AL’s 2023 tomato isolates, which lack the C118Y and other mutations, clustered within Sub-clade 6.3 together with peanut isolates, suggesting RB variants were likely absent from tomato in AL that year. In contrast, AL’s 2024-25 tomato isolates formed six host-associated sub-clades within Sub-clade 6.7, together with single isolates from North Carolina and GA (OP832375 and KU179600), consistent with largely local diversification of RB-associated lineages. These genomic and phylogenetic data provide the first evidence of TSWV-RB strains overcoming Sw-5b-mediated resistance in tomato in AL, making AL the third southeast state in the United States to document a severe outbreak of TSWV-RB (Macedo et al., 2024). This underscores an urgent need for more durable TSWV management and resistance strategies.

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