Leguminous crops belonging to the family Leguminosae are rich in proteins and minerals and have a high nutritional value (Belhassen et al., 2019). An interesting characteristic of these crops is their ability to form symbiotic relationships with root nodule bacteria, reducing the need for chemical nitrogen fertilizers, a feature not shared by other plant varieties. This makes them a crucial source of nourishment for both humans and livestock. Broad beans (Vicia faba) and peas (Pisum sativum L.) belong to this family. According to data from the FAOSTAT database, broad beans are produced in more than 60 countries, with China and Ethiopia being the main producers, a global production of 596 million tons in 2021 (FAOSTAT, 2021a, FAOSTAT, 2021b). Similarly, peas are produced in more than 90 countries, with Russia and Canada as the main producing nations, resulting in a global production of 1.24 billion tons in 2021.

Many viruses that infect these leguminous crops and cause extensive damage to have been reported, one of which is broad bean true mosaic virus (Comovirus fabae; BBTMV) (CABI, 2022b, CABI, 2022c, Harveson et al., 2021; CABI, 2022a; Schwartz et al., 2005; Sutic et al., 1999). BBTMV belongs to the order Picornavirales, the family Secoviridae, the subfamily Comovirinae, and the genus Comovirus, with isometric particles approximately 25 nm in diameter (Gibbs et al., 1968; International Committee on Taxonomy of Viruses, 2017). BBTMV comprises two genome segments of linear positive sense ssRNA, with RNA1 (approximately 6 kb) and RNA2 (approximately 3.5 kb) completely sequenced (International Committee on Taxonomy of Viruses (ICTV), 2017; Petrzik, 2010). As of April 2023, the NCBI GenBank database contains records of three BBTMV isolates, but genetic information on this virus is limited.

BBTMV infects broad beans and peas, resulting in mosaic symptoms on the leaves and reduced crop yields (Sutic et al., 1999). BBTMV infection has been reported to reduce broad bean yield by 11.25% (Kumari and Makkouk, 2007). However, some cultivars of these plants are infected with BBTMV but show no symptoms during their early growth stages (Mali et al., 2003). Furthermore, BBTMV symptoms can be easily mistaken for those caused by the broad bean stain virus (Comovirus viciae; BBSV), which belongs to the same genus, because of the similarity in symptoms, virus particle morphology, and the possibility of co-infection with BBSV (Gibbs et al., 1968). BBTMV was first reported in broad beans in Germany in 1953 and has since spread throughout China, Syria, Lebanon, Europe, and Africa, including the main production areas for these legumes (CABI, 2022a; Kumari and Makkouk, 2007; Quantz, 1953).

BBTMV is transmitted by insect vectors, such as Apion vorax and other weevils, mechanical transmission, and seed transmission in broad beans (Eman et al., 2012, Sastry, 2013). Seed transmission rates of the virus have been reported to range from 0% to 28% but vary between plant cultivars (Mali et al., 2003, Sastry, 2013). Seed transmission has also been experimentally reported in vetch (V. sativa) and hairy vetch (V. villosa) (Mali et al., 2003). In the presence of highly transmission-efficient vectors, such as A. vorax, even a few infected seeds can rapidly spread the infection to healthy plants, resulting in severe crop losses (Sastry, 2013). Preventing the introduction of infected seeds is vital to mitigate the damage caused by BBTMV in agricultural products. Therefore, Japan designates BBTMV as a quarantine pest and requires prior inspections of broad bean seeds imported from regions where the virus is present.

Various serological and molecular methods have been used to detect and identify BBTMV. Serological detection methods, such as enzyme-linked immunosorbent assay (ELISA) and tissue blot immunoassay (TBIA), have been developed (Eman et al., 2012). In contrast, genetic detection methods include the RT-PCR method using primers that can detect the genus Comovirus, including BBTMV, and the subfamily Comovirinae (containing the genera Comovirus and Fabavirus) (Perez-Egusquiza et al., 2014, Ye et al., 2015). When using such a universal primer set, sequence analysis of the RT-PCR products is essential for identification of the virus species. Until now, ELISA has been the main method for diagnosing BBTMV (Eman et al., 2012, Kawanna and Fegla, 2015, Kumari and Makkouk, 2007). However, ELISA tests are often longer and less sensitive than RT-PCR (Kaur et al., 2020, Patel et al., 2023). Therefore, in this study, we developed an RT-PCR-based detection method using newly designed BBTMV-specific primers to detect BBTMV with higher sensitivity. In addition, we have tested the practicality of this new detection method against BBTMV in broad bean seeds.