Abstract
In recent years, tomato mottle mosaic virus (ToMMV) has become one of the most important viral pathogens affecting solanaceous crop production in Yunnan, Hainan, and Shandong provinces of China, often causing huge yield reductions. To provide farmers and vegetable industry with reliable and easy-to-use ToMMV detection methods, we immunized BALB/c mice with purified ToMMV and obtained six hybridoma cell lines (i.e., 2D6, 9C12, 26A10, 3A4, 23A4 and 17B11) that secrete anti-ToMMV monoclonal antibodies (MAbs) through the hybridoma technology. Using these MAbs as the detection antibody, we developed three serological assays: antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and tissue print enzyme-linked immunosorbent assay (tissue print-ELISA) for ToMMV detection. Our test results showed that these three newly developed serological methods can be used to specifically detect ToMMV infection in plant samples, but not tobacco mosaic virus, tomato mosaic virus, cucumber green mottle mosaic virus and cucumber mosaic virus. Sensitivity analyses further showed that ACP-ELISA and dot-ELISA can be used to detect ToMMV infection in plant crude extracts diluted at 1:81,920 and 1:40,960 (weight/volume, g/mL), respectively. Surprisingly, the detection limit of the developed dot-ELISA was 26 times higher than that of traditional RT-PCR. Using field-collected plant samples, we have demonstrated that these three new serological methods are accurate and easy-to-use for large-scale detection of ToMMV in fields.
Highlights
Tomato (Solanum lycopersicum) is an important vegetable crop with high economic values
tomato mottle mosaic virus (ToMMV) virion purification ToMMV infection in tomato plants used for virion purification was confirmed by Reverse transcript-polymerase chain reaction (RT-PCR) (Fig. 1a)
Production and characterization of ToMMV‐specific Monoclonal antibody (MAb) On the third day after the fourth immunization, splenocytes were isolated from the immunized BALB/c mice and used for hybridoma preparation
Summary
Tomato (Solanum lycopersicum) is an important vegetable crop with high economic values. Tomato mottle mosaic virus (ToMMV) has been reported to infect. Like tobacco mosaic virus (TMV), the ToMMV genome consists of a singlestranded positive-sense RNA, with a methylguanosine [m7G(5’)pppG] cap and a tRNA-like structure at. ToMMV genomic RNA contains four open reading frames (ORFs), encoding two replication-related proteins of 126 and 183 kDa, a move protein (MP) of 29.8 kDa and a coat protein (CP) of 17.5 kDa (Li et al 2013; Sui et al 2017). ToMMV shares the highest nucleotide sequence similarity (84.3%) with tomato mosaic virus (ToMV) (Li et al 2013; Li et al 2017). The deduced amino acid sequences of the 126 and183 kDa replication-related proteins, MP and CP of ToMMV share 94.3%, 94.7%, 81.4% and 91.4% identities with the corresponding proteins of ToMV, respectively (Nagai et al 2019; Rodriguez-Mendoza et al 2019)
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