Tomato brown rugose fruit virus (ToBRFV) is an emerging and rapidly spreading tobamovirus that is causing substantial damage to tomato crops. Since its first description in tomato plants in Israel in 2014 and initial identification in Jordan in 2015 (Luria et al., 2017, Salem et al., 2016), ToBRFV has been reported to quickly expand to at least 35 countries across four continents, including Europe, Africa, America, and Asia (Zhang et al., 2022). ToBRFV naturally infects tomato and pepper plants, with tomato as the primary natural host. Infection of ToBRFV causes typical symptoms including mottle, mosaic, and deformed leaves, and necrotic spotted or brown rugose fruits in infected tomato plants. Mild symptoms such as mild mosaic leaves or asymptomatic infection can also be observed in some tomato cultivars (Ilyas et al., 2022, Zhang et al., 2022). ToBRFV is readily transmitted via mechanical contact. It can also be transmitted by infected fruits and contaminated seeds, and the contamination rate of seeds extracted from ToBRFV-infected tomato fruits can reach 100%. Although the transmission rate from ToBRFV-contaminated seeds to their seedling is low, ranging from 0.08% to 2.8%, the distribution of such seeds to distant places allows for long-distance dissemination of ToBRFV, which may act as an important source of primary inoculum and be further dispersed via mechanical contact (Davino et al., 2020, Klap et al., 2020a, Salem et al., 2022). In laboratory conditions, ToBRFV can infect more than 40 plant species belonging to four families (Zhang et al., 2022).

Like other tobamoviruses, ToBRFV possesses a positive-sense, single-strand RNA genome of approximately 6.4 kb, encoding four open reading frames (ORFs). ORF1 and ORF2 encode replication-related proteins of 126 kDa and 183 kDa, respectively. ORF3 encodes a movement protein (MP) of 30 kDa. ORF4 encodes a 17.5-kDa coat protein (CP) required for viral assembly and transmission (Salem et al., 2016). Phylogenetic analysis of complete genome sequences of ToBRFV and other representative tobamoviruses indicates the closest relationship of ToBRFV with tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), and tomato mottle mosaic virus (ToMMV) (Salem et al., 2016, Zhang et al., 2022). Previously, the Tm-2 and Tm-22 resistance genes have conferred long-standing resistance to these tobamoviruses. However, ToBRFV could break such resistance, and neither resistant commercialized tomato cultivars nor effective chemicals are available to control the disease caused by ToBRFV (Hak and Spiegelman, 2021, Luria et al., 2017, Yan et al., 2021). Currently, ToBRFV has been considered as one of the most important tomato-infecting viruses and is included in the alert list by the European and Mediterranean Plant Protection Organization (EPPO) and quarantine pests of several countries, which has attracted great concern from tomato growers and vegetable production industries.

Rapid, specific, and sensitive detection methods are required for early robust detection of ToBRFV, which is essentially important in preventing the introduction and expansion of ToBRFV. To date, several assays, including reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and CRISPR/Cas12-based assays, have been developed to detect the RNA of ToBRFV and are useful in accurate detection of ToBRFV in laboratories (Alon et al., 2021, Bernabe-Orts et al., 2021, Luigi et al., 2022, Sarkes et al., 2020). However, most of these assays require specialized equipments or technical skills and are not suitable for onsite detection. Enzyme-linked immunosorbent assay (ELISA) designed against the CP of plant viruses allows for high-throughput detection of plant samples (Rubio et al., 2020). An ELISA-based diagnostic kit and immunostrip for ToBRFV detection have been commercialized by Agdia (USA). However, cross-reaction with other closely related tobamoviruses, including TMV, ToMV, and ToMMV, was observed for both the Agdia ELISA kit and Immunostrip (Eads et al., 2023). Cross-reaction with TMV and ToMV was also observed for the DAS-ELISA kit developed by LOEWE (https://loewe-info.com/10919–2/).

In this study, we raised sensitive and specific monoclonal antibodies against ToBRFV using purified ToBRFV virions as the immunogen. We developed a dot-ELISA-based assay for sensitive, robust, and high-throughput detection of ToBRFV at a low cost. We also developed a colloidal gold immunochromatography strip (CGICS) that is suitable for rapid and sensitive onsite detection of ToBRFV without the need of special equipment or technical skills.