LAUSR

Tomato (Solanum lycopersicum L.), an important vegetable widely grown in the world, is susceptible to a number of emerging viruses. In September 2018, a severe disease outbreak, including mosaic, mottling, and plant stunting was observed on grafted tomato plants in a greenhouse (∼8 acres) in southern California, U.S.A. Initial screening of one symptomatic sample was positive for pepino mosaic virus (PepMV) and tomato mosaic virus (ToMV) using enzyme-linked immunosorbent assay (ELISA). Two types of particles, including flexuous potexvirus-like and rigid tobamovirus-like, were observed under an electron microscope. However, reverse transcription polymerase chain reaction (RT-PCR) tests using primers that are specific to ToMV and tomato mottle mosaic virus (ToMMV) (Sui et al. 2017) were negative. We suspected an involvement of tomato brown rugose fruit virus (ToBRFV) (Luria et al. 2017; Salem et al. 2016). A new set of primers specific to ToBRFV was designed and validated to have no cross-reaction to ToMV and ToMMV. Using this primer set, ToBRFV-F (5503), 5′-GAAGTCCCGATGTCTGTAAGG-3′, and ToBRFV-R (6344), 5′-GTGCCTACGGATGTGTATGA-3′, an expected PCR product (842 bp) was amplified and sequenced from this first sample but not from a healthy control. In a follow-up survey, 20 additional symptomatic samples were tested and shown to be all positive for PepMV using ELISA or qRT-PCR (Ling et al. 2007), and 14 of the same 20 samples were also positive for ToBRFV using RT-PCR. Sanger sequencing of seven amplicons generated a consensus 799-bp sequence after primer trimming, designated as isolate ToBRFV-US (accession no. MK109002). A BLASTn search of the NCBI database revealed 99.6% sequence identity to two other ToBRFV isolates from Jordan (KT383474; Salem et al. 2016) and Israel (KX619418; Luria et al. 2017). In pathogenicity assays, four tomato ‘Moneymaker’ seedlings (susceptible to tobamoviruses) were mechanically inoculated with an inoculum prepared from the first positive sample, and similar symptoms were visible. The target pathogen, ToBRFV, was confirmed on the inoculated plants using RT-PCR and amplicon sequencing. In a separate survey in October 2018, a disease outbreak was also observed on tomato in a greenhouse facility in Baja California, Mexico. An extensive crop health screen using ELISA tests to a panel of 19 common tomato viruses, as well as appropriate RT-PCR tests, were conducted on 14 collected samples. A mixed infection of multiple viruses was identified. In addition to common infection by PepMV, seven of 14 samples were also positive for tomato spotted wilt virus. Weak positives were observed for tobacco mosaic virus or ToMV when tested by ELISA. However, when using genus-specific primers for tobamoviruses (Tob-Uni1 and Tob-Uni2) (Letschert et al. 2002), amplicon sequencing revealed the presence of ToBRFV in both Californian and Mexican samples, with no mixed infection by any other tobamoviruses. Using the ToBRFV-specific primers, amplicon sequences from 14 Mexican samples resulted in a consensus sequence, designated as isolate ToBRFV-MX (accession no. MK109003). The two sequences of ToBRFV-US and ToBRFV-MX isolates were similar (99.6% sequence identity). Identification of the first ToBRFV outbreaks in the United States and Mexico, as well as its ability to break the Tm-2² resistance (Luria et al. 2017), raises a serious concern for the tomato industry. How ToBRFV was introduced to the United States and/or Mexico has not been determined. Eradication efforts are underway in both countries to contain and control these disease outbreaks.