Validation of a reverse transcription PCR detection method for tobamovirus maculatessellati, in tomato (Solanum lycopersicum L.) and pepper (Capsicum annuum L.)

Authors: Padmanabhan, Chellappan; Gilliard, Andrea; Ling, Kai Shu; Rivera Rivera, Yazmin

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2025
Publication Date: 1/29/2025
Citation: Padmanabhan, C., Gilliard, A.C., Ling, K., Rivera Rivera, Y.N. 2025. Validation of a reverse transcription PCR detection method for tobamovirus maculatessellati, in tomato (Solanum lycopersicum L.) and pepper (Capsicum annuum L.). Frontiers in Plant Science. https://doi.org/10.3389/fpls.2025.1535175.
DOI: https://doi.org/10.3389/fpls.2025.1535175

Interpretive Summary: Tomato is a globally important crop consumed by billions of people. Among a large number of virus species infecting tomato, tobamoviruses are one of the most devastating viruses infecting tomato and pepper plants. In 2013, a new species of tobamovirus named tomato mottle mosaic virus (ToMMV), was identified by the USDA-ARS laboratory in Charleston. Since the first identification, this seed-borne pathogen has been identified in many countries around the world and poses a serious threat to tomato and pepper productions in the U.S. Without a validated standard test, ToMMV could affect international trade of seed and produce of tomato and pepper. In collaboration with scientists at the USDA-APHIS, we performed extensive validation tests of a previously described reverse transcription polymerase chain reaction (RT-PCR) assay that was developed in the USDA-ARS laboratory in Charleston. This assay showed 100% specificity to a diverse collection of ToMMV isolates, without cross reaction with non-target viruses and viroids commonly infecting tomato and pepper plants. The RT-PCR can detect ToMMV consistently on contaminated seed or leaf tissues, thus to prevent the spread and introduction of this emerging and economically important tobamovirus. This validated RT-PCR test will help establish a standard method for government regulatory agencies, seed health testing laboratories and plant disease diagnostic clinics on ToMMV detection.

Technical Abstract: The solanaceous-infecting tobamoviruses are closely related and hence it can be challenging to detect them using serological or molecular methods, particularly when present in a mixed infection. Tomato mottle mosaic virus (ToMMV) is a newly identified tobamovirus that poses serious risk to tomato (Solanum lycopersicum L.) and pepper (Capsicum annuum L.) production worldwide. Species-specific identification is crucial to prevent the entry and establishment of plant pathogens and protect the billion-dollar tomato industry. In this study, we report the validation of a previously described reverse transcription polymerase chain reaction (RT-PCR) assay that amplifies a 289 bp fragment of the coat protein coding region of ToMMV genome. This assay has 100% specificity for ToMMV. Inclusivity tests were performed against a diverse collection of six ToMMV isolates in North America. Exclusivity tests showed no cross reaction with eleven non-target viruses and seven viroids commonly found on tomato and pepper host plants. The detection limit of the one-step RT-PCR was determined to be at 10^-5 (or 0.25pg/ml) dilution in plant samples, with its amplicon sequence confirmed by Sanger sequencing. The RT-PCR can detect ToMMV consistently on contaminated seed or leaf tissues. This validated assay could serve as a standard method for detecting ToMMV in seed health testing and for plant disease diagnosis, thus to prevent inadvertent introduction and spread of this emerging and economically important tobamovirus in tomato and pepper fields.