Effect of ozone on inactivation of purified pepper mild mottle virus and contaminated pepper seed

Authors: Stommel, John; Dumm, Judith; Hammond, John

Submitted to: PhytoFrontiers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/16/2020
Publication Date: 1/27/2021
Citation: Stommel, J.R., Dumm, J.M., Hammond, J. 2021. Effect of ozone on inactivation of purified pepper mild mottle virus and contaminated pepper seed. Phytofrontiers. 1: 85-93. https://doi.org/10.1094/PHYTOFR-09-20-0020-R.
DOI: https://doi.org/10.1094/PHYTOFR-09-20-0020-R

Interpretive Summary: Viral diseases of crop plants that are transmissible by contaminated seed result in significant crop loss without efficacious treatments to rid seed of virus. One such disease is pepper mild mottle virus (PMMoV) which is localized to the seed coat of sweet and hot pepper. Conventional treatments reduce but do not eliminate the incidence of seed-born PMMoV. We evaluated use of ozone as a treatment to disinfest pepper seed naturally contaminated with PMMoV. Exposure of purified virus and contaminated seed demonstrated that ozone can reduce concentrations of viable PMMoV and that virus destruction increased with length of ozone exposure time. Levels of viral inactivation were insufficient to recommend ozone as a standard treatment to substantially reduce PMMoV incidence in contaminated lpepper seed lots. These results will benefit seed producers and plant researchers.

Technical Abstract: Pepper mild mottle virus (PMMoV) of the genus Tobamovirus is a major viral pathogen of pepper (Capsicum spp.). PMMoV is readily mechanically transmitted and is also seed transmissible. Trisodium phosphate is commonly used as a seed treatment to reduce the level of active virus in PMMoV contaminated seed. Ozone has shown efficacy in disinfecting seed of fungal pathogens and disrupting bacterial and viral pathogens on a variety of substrates. The purpose of this study was to evaluate the efficacy of ozone and chemical treatment on inactivation of PMMoV on contaminated pepper seed harvested from PMMoV infected plants, and to assess treatment impact on seed germination. Infectivity of treated seed preparations was evaluated via bioassay with mechanical inoculation of Nicotiana benthamiana. Symptoms of PMMoV systemic infection were not observed in bioassays of trisodium phosphate treated seed preparations. However, sufficient viable PMMoV remained on ozone treated seed to cause infection in bioassays. ELISA confirmed PMMoV infection. Neither treatment had a significant effect on pepper seed germination. Ozone treatment of purified PMMoV was assessed to further determine the extent, if any, of PMMoV inactivation by ozone in aqueous and dried preparations. At low PMMoV concentrations (0.01 mg/mL), 14 Hr ozone exposure eliminated PMMoV infectivity as determined by N. benthamiana bioassays with ELISA confirmations. At higher concentrations of purified PMMoV (0.1 mg/mL), ozone treatment was insufficient to prevent infection in bioassays. Degradation of PMMoV was quantified via bioassay using the local lesion host N. glutinosa, and real time PCR quantification of PMMoV in ozone treated purified PMMoV preparations. Lesion counts as well as PMMoV concentration were reduced in response to ozone exposure and the magnitude of PMMOV degradation increased as ozone exposure time increased. Even though PMMoV concentration was reduced by ozone exposure and infection was eliminated at low PMMoV concentration, bioassays using naturally infected seed and purified PMMoV preparations at relatively higher concentrations demonstrated that ozone is not efficacious as a standard treatment to substantially reduce the level of infective PMMoV in contaminated pepper seed.