First report of Tomato brown rugose fruit virus infecting tomato in Florida, USA

Abstract

Florida is a leading producer of fresh-market tomatoes (Solanum lycopersicum) in the US, a $262 million industry for the state. In October 2019, eight tomato plants showing chlorotic, cupped leaves and stunted foliage (Fig. 1) were collected from a c. 1.6 ha community garden in Alachua County, Florida. The symptoms observed were characteristic of Tomato yellow leaf curl virus (TYLCV). Samples were tested by PCR using TYLCV-specific primers by Navot et al. (1992). Three samples tested positive for TYLCV. Lateral flow immunoassay tests (“immunostrip assays”; Agdia, USA) for Pepino mosaic virus and Cucumber mosaic virus were negative. However, an immunostrip for Tobacco mosaic virus (TMV) gave a faint positive result for one of three TYLCV-positive sample, suggesting a mixed infection. Tobamovirus-like rigid rod-shaped particles of ∼300 nm in length, were also observed in leaf dips from this plant using an LVEM5 transmission electron microscope (DeLong America, USA). Tests for common tobamoviruses, i.e. TMV, Tomato mosaic virus, Tomato mottle mosaic virus, (Sui et al., 2017), Tobacco mild green mosaic virus (Cohen et al., 2006), and Tomato brown rugose fruit virus (ToBRFV) (Ling et al., 2019) were conducted using specific primers in RT-PCR with total RNA extracted using RNeasy® Plant Mini kits (Qiagen, Germany). Additionally, conventional PCR primers, DPI-782(5859nt-5879nt) = 5′-TAGCGAAGTGTGGAAACCTG-3′ and DPI-783(6154nt-6183nt) = 5′-GGTGCAGAGGACCATTGTAA-3′ (amplicon size of 342 bp), and primers and probes for a real-time RT-PCR Taqman assay DPI-784(6029nt-6049nt) = 5′-CCGAAACGTTAGACGCTACTC-3′, DPI-785(6106nt -6127nt) = ACCTGTTCCTTTGACCAA TTCT-3′, and DPI-787(6060nt-6083nt) TaqMan probe = 5′-ATG CAACGGTGGCTATAAGGA-3’) from the coat protein ∼5800-6130 nt region were developed for ToBRFV. Six of eight samples, including three TYLCV-positive samples, were positive for ToBRFV. The sequenced PCR products (GenBank Accession No. MW322766) were 98 to 99 % identical to ToBRFV isolates from Jordan and Mexico (KT383474, MK319944). The identification of ToBRFV was further confirmed by USDA-APHIS-PPQ-S&T laboratory (Laurel, Maryland, USA) by RT-PCR and sequencing. The Ct values of the real-time assay were in the range of 27 to 31. The weak positive result from the immunostrip, relatively high Ct values and co-infection with TYLCV suggest that ToBRFV was present in a low titre in these samples. ToBRFV was also detected in tomatoes imported from Mexico and for sale in grocery stores in Naples and Gainesville by University of Florida and Florida Department of Agriculture and Consumer Services (FDACS) around the same time (Batuman et al., 2020), Immediately following the survey, all solanaceous plants in the organic garden were destroyed. FDACS is taking all precautionary measures to test tomatoes produced in Florida. Further studies are needed to better understand the risk factors from this virus in Florida. The authors would like to thank Department of Plant Industry (DPI), Florida Department of Agriculture and Consumer Services (FDACS) for all assistance with the project.