New Resources for the Specific and Sensitive Detection of the Emerging Tomato Brown Rugose Fruit Virus

Abstract

Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test.

Development and Validation of a One-Step Reverse Transcription Real-Time PCR Assay for Simultaneous Detection and Identification of Tomato Mottle Mosaic Virus and Tomato Brown Rugose Fruit Virus

“…The ToBRFV and ToMMV duplex assay (Duplex B) included in the validation process according to [38] was evaluated in terms of analytical sensitivity and specificity, selectivity, repeatability, and reproducibility. The LODs obtained for both leaf (10 −6 /10 −5 ) and seed (10 −5 ) matrices and for both target viruses were in line with those of other real-time RT-PCR tests for the detection of tobamovirus species [20,40,41]. In addition, the performance criteria when testing dilution series (leaf or seeds samples) showed that there was no relevant reduction in LOD and/or primer efficiency (E) for the duplex assay compared to the single assays.…”

Development and Validation of a One-Step Reverse Transcription Real-Time PCR Assay for Simultaneous Detection and Identification of Tomato Mottle Mosaic Virus and Tomato Brown Rugose Fruit Virus

“…In fact, the other pre-tested sets developed in this study and targeting CP region, reacted unspecifically with CGMMV, a tobamovirus that infects Cucurbitaceae plant species, and a very limited number of Solanaceae species [43]. (10 -6 /10 -5 ) and seed (10 -5 ) matrices and for both target viruses were in line with those of other real-time RT-PCR tests for the detection of tobamovirus species [22,44,45]. In addition, the performance criteria when testing dilution series (leaf or seeds samples) showed that there was no relevant reduction in LOD and/or primer efficiency (E) for the duplex assay compared to the single assays.…”

PM 7/146 (2) Tomato brown rugose fruit virus

“…Outcomes of the TPS are available through the EPPO database on diagnostic expertise (https://dc.eppo.int/validation_data/validationlist). In this revised version of the protocol, two additional tests have been included: Panno et al (2019b) which was evaluated in VALITEST, and Bernabé‐Orts et al (2021) which targets a different part of the ToBRFV genome compared to the tests evaluated in the framework of VALITEST.…”