High throughput sequencing reveals mixed infections of impatiens necrotic spot virus and tomato brown rugose fruit virus in bell pepper crops in Mexico

Pepper crops are affected by several viruses worldwide. In September 2022, symptoms of mosaic, deformation and ringspot were observed on bell pepper leaves and fruits in a commercial greenhouse located in Queretaro, Mexico. Symptoms were indicative of infection by an orthotospovirus and/or a tobamovirus, but some plants exhibited increased severity compared to what is typically observed for these viruses in pepper plants. Six out of a total of 10 symptomatic plants tested positive for tomato brown rugose fruit virus (ToBRFV) using ImmunoStrips during initial testing of field plant samples. Due to the severity of the damage, the six ToBRFV-positive samples were independently subjected to high throughput sequencing (HTS) analysis. Sequences of ToBRFV and impatiens necrotic spot virus (INSV) were identified from two of the HTS-analyzed plants, whereas the other four plants were infected with ToBRFV alone. HTS results were validated by reverse transcription-PCR employing specific primers amplifying genomic fragments of those viruses. Finally, using ELISA, INSV presence was confirmed in the two coinfected bell peppers in addition to the four original plants not analyzed by HTS. This is the first report of a natural mixed infection of INSV and ToBRFV in bell pepper in Mexico and potentially around the world, raising the possibility of a synergistic effect.