Molecular Detection of Tomato Brown Rugose Fruit Virus on Imported Seeds in Greenhouse Production System in Fiji, Global Genetic Diversity, and Patterns of Evolution

The pathogen known as tomato brown rugose fruit virus (ToBRFV; genus Tobamovirus , family Virgaviridae ) is a recently emerged RNA pathogen with rapid international spread that threatens tomato ( Solanum lycopersicum ) production worldwide. In this study, we combined field surveillance, high-throughput sequencing, and comparative genomics to investigate patterns of genomic variation and evolution in ToBRFV at a global scale and to characterize a ToBRFV isolate recently detected in Fiji. The Fijian isolate, obtained from symptomatic tomato plants grown in a secured greenhouse from imported seed in 2023, shared high nucleotide identity (> 98%) and close phylogenetic relatedness with isolates from Canada, China, Palestine, and the Netherlands. Analysis of 77 full-length ToBRFV genome sequences revealed 53 haplotypes (haplotype diversity Hd = 0.987), 187 segregating sites, and low overall nucleotide diversity (π = 0.00239), consistent with a genetically compact but rapidly disseminating virus population. Among the four main coding regions, the RNA-dependent RNA polymerase (RdRp) proteins (126 and 183 kDa) displayed higher genetic variability than the proteins responsible for movement (MP) and encapsidation (CP), likely reflecting the larger size and functional complexity of the RdRp region. Selection analyses indicated that ToBRFV evolution is dominated by strong purifying selection acting on RdRp, with limited evidence for positively selected sites, and only a low-impact recombination signal detected across the dataset. Protein disorder predictions further showed that RdRp, MP, and CP harbor distinct intrinsically disordered regions, which may contribute to host interactions and viral adaptability. Importantly, ToBRFV was not detected in open-field tomato cultivation in Fiji, and destruction of infected greenhouse plants at the end of the production cycle has so far prevented further detection at the study site, supporting seed transmission as the most likely pathway. Together, these results improve our understanding of ToBRFV population structure and evolutionary constraints and provide a molecular foundation for early detection and risk management strategies, particularly for seed-borne spread.