Genomic Diversity of Tomato Brown Rugose Fruit Virus in Canadian Greenhouse Production Systems

Tomato brown rugose fruit virus (ToBRFV) is a recently emerged viral pathogen in the Tobamovirus genus first observed in 2014 in the Middle East that has since spread worldwide, causing significant losses in greenhouse tomato production. ToBRFV is easily mechanically transmitted and can escape the durable Tm-2² resistance gene, facilitating its global spread. Seed companies have identified novel sources of resistance and introduced these resistance traits into commercial cultivars. The identity, number, and mechanisms of these putative novel resistance genes are largely unknown but could be exerting selective pressures on ToBRFV. Here, we report 15 new ToBRFV genomic sequences from Canadian greenhouse production systems in susceptible and novel resistant or tolerant cultivars collected since 2023. We combined these sequences with five other Canadian ToBRFV genomes previously deposited in Genbank and a further five consensus sequences derived from metagenomic-based wastewater monitoring sequence data and conducted phylogenetic analysis. Most Canadian sequences grouped together when compared with 332 publicly available international sequences, but several isolates appeared distantly related, suggesting multiple introductions to Canadian production systems. High sequence identity between samples suggest movement of ToBRFV between independent greenhouses, highlighting areas where biosecurity can be improved. Several novel non-synonymous polymorphisms identified in the p126 and movement protein (MP) open reading frames (ORFs) were unique to Canadian sequences and associated with infection of novel resistant tomato cultivars. Many polymorphisms in the p126 ORF are located in a region of the protein associated with Tm-1 resistance-breaking isolates of tomato mosaic virus and ToBRFV, but have not been previously reported. Four novel polymorphisms in MP were also identified and do not appear to be associated with sites previously identified as interacting with Tm-2² and could be related to other unknown resistance genes. Together, these results confirm the difficulties in preventing the transmission of ToBRFV, identify putative adaptations to novel and existing resistance genes, and emphasize the urgent need for the cloning and characterization of these new sources of resistance to ToBRFV.