First report of and as natural hosts of tomato brown rugose fruit virus.

Tomato brown rugose fruit virus (ToBRFV) is a tobamovirus recently identified on tomatoes in Jordan (Salem et al. 2016). New infections were rapidly reported all over the world becoming a serious threat to tomato production. About 40 species belonging to four families (Amaranthaceae, Apocynaceae, Asteraceae, and Solanaceae) have been reported as experimental hosts (Salem et al. 2023). Tomato and pepper have been reported as natural hosts of ToBRFV but recently Salem and coworkers (2022) detected the presence of the virus in 12 wild species. To identify potential natural hosts of the virus, 10 plants of bindweed (Convolvulus arvensis L.) and 7 of fourleaf allseed (Polycarpon tetraphyllum L.) were collected in the summer 2023 in a tomato greenhouse located in Pachino, Siracuse province (Sicily, Italy), with high-rate infection of ToBRFV. These two species were chosen because predominant among the spontaneous weeds inside the greenhouse. No symptoms ascribable to ToBRFV were observed on bindweed and fourleaf allseed during the surveys. All leaf samples were analyzed for ToBRFV infections by DAS-ELISA with a commercial antiserum (LOEWE Biochemica, Germany), including tomato positive and negative controls. Eight C. arvensis and seven P. tetraphyllum samples out of the total tested positive to ToBRFV. To confirm virus presence, total RNA was extracted from all samples using the RNeasy Plant Mini Kit (QIAGEN) and used as template for RT-PCR with ToBRFV-specific primers (Alkowni et al. 2019). RT-PCR products of the expected size (560bp) confirmed DAS-ELISA results. Amplicons from two isolates of each plant species (Conv-01, Conv-02, Poly-01, and Poly-02) were gel-purified and sequenced in both directions. Sequences were edited and deposited in GenBank (Acc. Num: Conv-01, OP150933; Conv-02, OP193999; Poly-01, OP150934; Poly-02, OP194000). According to sequence analysis, the four isolates shared 100% identity among them and 98.82% identity with the ToBRFV reference sequence (GenBank Accession No. KT383474). To our knowledge, this is the first report of ToBRFV natural infections in C. arvensis and P. tetraphyllum. Since these weeds are common in our tomato production areas, they could act as ToBRFV reservoirs.