First Field Report of Tomato Brown Rugose Fruit Virus (ToBRFV) Naturally Infecting Rapeseed ( <i>Brassica napus</i> ) in China

Tomato brown rugose fruit virus (ToBRFV), a tobamovirus causing significant damage to tomato and pepper crops, has been primarily associated with Solanaceae species. However, its potential presence in non-solanaceous crops under natural conditions remains poorly characterized (Salem et al. 2023). In May 2024, over 60% of Solanum lycopersicum plants cultivated in an approximately 500 m² open field in Shandong Province, China, exhibited symptoms ranging from mild leaf curling to severe fruit deformation. Five symptomatic plants were randomly sampled, and all tested positive for ToBRFV using reverse transcription-recombinase aided amplification with lateral flow strip (RT-RAA-LFS) assays, following the method of Cao et al. (2023). Five months later, in October, Brassica napus plants grown in the same rotation field exhibited virus-like symptoms, including leaf wrinkling and interveinal chlorosis. Total RNA was extracted from five symptomatic B. napus plants. Four overlapping genomic fragments were amplified using reverse transcription PCR, and the 5′ and 3′ untranslated regions (UTRs) were obtained using a cDNA synthesis kit (Wang et al. 2025). The complete viral genome (6378 bp) was assembled and deposited in the China National GeneBank (accession: CNP0007029). Sequence alignment revealed 99.70%, 99.71%, and 99.78% nucleotide identity with Chinese (GenBank accession no. PV701254.1), Canadian (GenBank accession no. OQ674195.1), and Mexican (GenBank accession no. OM892691.1) ToBRFV isolates, respectively. To assess infectivity, sap extracted from symptomatic B. napus leaves (100 mg tissue per 1ml of 1× PBS) was mechanically inoculated onto B. napus cv. ‘Wanchoutai-Suman’, S. lycopersicum cv. ‘Fenguan’, and Nicotiana benthamiana. Three individuals per plant species were inoculated, and all exhibited typical symptoms, such as chlorosis and leaf deformation, within 7 to 14 days. ToBRFV infection was confirmed by reverse transcription quantitative PCR (RT-qPCR) using primers and probes from EPPO 2021 and western blot analysis of newly emerged symptomatic leaves in all three species. Negative control plants inoculated with 1× PBS showed no symptoms and tested negative by RT-qPCR. This represents the first confirmed field case of ToBRFV naturally infecting a Brassicaceae species. While natural infections have previously been reported in Solanaceae and, less frequently, in Amaranthaceae, Apocynaceae, and Asteraceae (Salem et al. 2022), the detection of ToBRFV in B. napus significantly broadens the known host range of the virus. The prior detection of ToBRFV in tomato plants in the same field suggests that crop rotation without effective field sanitation may facilitate cross-family transmission. In this context, the presence of B. napus as a rotation crop may have allowed the virus to persist and spill over beyond its usual hosts. The exact transmission route remains uncertain, but contaminated tools (Skelton et al. 2023), irrigation water or soil (Mehle et al. 2023), and insect-mediated mechanical transmission (Caruso et al. 2024) are all plausible sources. B. napus is widely planted in China and often rotated with tomato, so its role as an intermediate or symptomless host should be considered. These findings suggest that virus monitoring should also include non-solanaceous crops, especially in mixed-cropping systems where proximity may increase the risk of cross-species transmission.