Abstract
BackgroundTomato brown rugose fruit virus (ToBRFV) is a highly infectious tobamovirus that causes severe disease in tomato (Solanum lycopersicum L.) crops. In Italy, the first ToBRFV outbreak occurred in 2018 in several provinces of the Sicily region. ToBRFV outbreak represents a serious threat for tomato crops in Italy and the Mediterranean Basin.MethodsMolecular and biological characterisation of the Sicilian ToBRFV ToB-SIC01/19 isolate was performed, and a sensitive and specific Real-time RT-PCR TaqMan minor groove binder probe method was developed to detect ToBRFV in infected plants and seeds. Moreover, four different sample preparation procedures (immunocapture, total RNA extraction, direct crude extract and leaf-disk crude extract) were evaluated.ResultsThe Sicilian isolate ToB-SIC01/19 (6,391 nt) showed a strong sequence identity with the isolates TBRFV-P12-3H and TBRFV-P12-3G from Germany, Tom1-Jo from Jordan and TBRFV-IL from Israel. The ToB-SIC01/19 isolate was successfully transmitted by mechanical inoculations in S. lycopersicum L. and Capsicum annuum L., but no transmission occurred in S. melongena L. The developed real-time RT-PCR, based on the use of a primer set designed on conserved sequences in the open reading frames3, enabled a reliable quantitative detection. This method allowed clear discrimination of ToBRFV from other viruses belonging to the genus Tobamovirus, minimising false-negative results. Using immunocapture and total RNA extraction procedures, the real-time RT-PCR and end-point RT-PCR gave the same comparable results. Using direct crude extracts and leaf-disk crude extracts, the end-point RT-PCR was unable to provide a reliable result. This developed highly specific and sensitive real-time RT-PCR assay will be a particularly valuable tool for early ToBRFV diagnosis, optimising procedures in terms of costs and time.
Highlights
Tomato (Solanum lycopersicum L.) is one of the most important horticultural crops worldwide
Greenhouse tomato production in Italy is affected by important losses that are caused by several different viruses: Pepino mosaic virus (Davino et al, 2017b, 2008; Tiberini et al, 2011), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (Panno et al, 2012), Tomato leaf curl New Delhi virus (Panno et al, 2019), Tomato yellow leaf curl virus—Tomato yellow leaf curl Sardinia virus and their recombinants (Davino et al, 2009, 2012; Panno, Caruso & Davino, 2018) and Tomato brown rugose fruit virus (ToBRFV) detected at the end of 2018 (Panno, Caruso & Davino, 2019)
The only two available tools to contain ToBRFV worldwide are early diagnosis and the implementation of preventive measures in crop management, which can be a valuable aid in reducing the introduction and subsequent ToBRFV spread in other countries
Summary
Tomato (Solanum lycopersicum L.) is one of the most important horticultural crops worldwide. The developed real-time RT-PCR, based on the use of a primer set designed on conserved sequences in the open reading frames, enabled a reliable quantitative detection. This method allowed clear discrimination of ToBRFV from other viruses belonging to the genus Tobamovirus, minimising false-negative results. Using direct crude extracts and leaf-disk crude extracts, the end-point RT-PCR was unable to provide a reliable result. This developed highly specific and sensitive real-time RT-PCR assay will be a
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