The most effective management strategy for tomato spotted wilt orthotospovirus (TSWV) has been the use of resistant tomato (Sw-5b+) cultivars. Unfortunately, resistance-breaking isolates (RB) have emerged in many countries. In 2019, TSWV-like symptoms were observed in protected crops of a tomato (Sw-5b+) cultivar in two separate locations in Serbia. To identify variations associated with Serbian TSWV isolates that are able to infect resistant tomato cultivars, 11 samples with TSWV-like symptoms collected from susceptible (Sw-5b−) cultivars from 2011 to 2015, and three collected from the resistant cultivar in 2019 were tested. The presence of TSWV was confirmed serologically in all tomato samples, and partial nucleotide sequences of the NSm and N genes of 14 selected isolates were obtained. The NSm amino acid sequence comparison revealed the presence of two amino acid substitutions (C118Y or T120N) in three isolates originating from tomato (Sw-5b+) indicating their RB phenotype and independent evolutionary events. Isolates originating from tomato (Sw-5b−) cultivars were determined as resistance-inducing isolates (RI). NSm and N gene phylogenetic analyses showed the clustering of Serbian RB and RI isolates regardless of their phenotype. The N gene tree also showed the clustering of Serbian TSWV isolates in different subpopulations within the European clade indicating two independent introduction events and gene flow between southern and northern regions of the country. Bioassay confirmed that three RB isolates had the ability to break the Sw-5b-mediated resistance in tomato, but were unable to overcome the Tsw resistance gene in pepper. This first detection of the Sw-5b resistance-breaking in Serbia shows the necessity of further monitoring to prevent the establishment of RB TSWV isolates.
