First Report of Tobacco Mild Green Mosaic Virus and Tomato Brown Rugose Fruit Virus Infecting Capsicum annuum in Jordan

Pepper (Capsicum annuum L.) is one of the major vegetable crops in Jordan. During the winter growing seasons of 2015 and 2016, virus symptoms including stunting of young plants, puckering, and yellow mottling of leaves in sweet pepper plants grown under plastic houses in Jordan Valley were observed. The most obvious symptoms were the misshapen fruits that affected the market value of the crop, which resembled recent descriptions of tomato brown rugose fruit virus (ToBRFV) on tomato fruits (Salem et al. 2016). Symptoms on mechanically inoculated indicator hosts including Chenopodium quinoa, Datura stramonium, D. metel, Nicotiana glutinosa, and N. tabacum, and serological testing using double-antibody sandwich ELISA (antiserum A128 of the PLAVIT collection at IPSP-CNR, Italy) suggested the presence of a tobamovirus. Total RNA was extracted from fruits and leaves of 39 symptomatic pepper plants, using an SV-Total RNA Extraction kit (Promega, U.S.A.). Samples were tested by reverse transcription polymerase chain reaction (RT-PCR) for the most common tobamoviruses infecting pepper, including tomato mosaic virus, tobacco mosaic virus, tobacco mild green mosaic virus (TMGMV), and pepper mild mottle virus (Takeuchi et al. 2005). In addition, generic primers for detection of tobamoviruses were also used (Dovas et al. 2004). In RT-PCR, amplicons of the expected product size (400 and 728 bp) using the generic tobamovirus and TMGMV-specific primers, respectively, in all symptomatic plant samples were obtained, but such amplicons were not obtained from healthy plant extracts or water negative controls. RT-PCR products of the RdRp (400 bp) and CP (728 bp) partial regions were purified and ligated into pGEM T-Easy Vector (Promega), and two clones for each PCR product were sequenced and deposited in NCBI GenBank (accession nos. MK816313 to 16). BLASTN analysis showed that these nucleotide sequences had 96 to 99% identity to TMGMV genome sequences (JX534224 and MH730962) in NCBI GenBank. Furthermore, total RNA was extracted with TRIzol reagent (Invitrogen, U.S.A.) according to the manufacturer’s specifications. After ribosomal RNA depletion, the cDNA library was constructed using a TruSeq RNA Sample Prep kit (Illumina, U.S.A.) and sequenced by Illumina HiSeq X-ten platform (Biomarker, China). Raw sequencing data were analyzed using CLC Genomics Workbench 9.5 (Qiagen, Denmark). After raw reads were processed, a total of 80,818,734 paired-end reads of 150 bp were obtained, generating 266,412 contigs (>200 nt) with de novo assembly by CLC Genomics Workbench 9.5. BLASTN analysis of the assembled contigs revealed the presence of two virus-derived contigs: TMGMV (6,414 nt, MK648158) and ToBRFV (6,388 nt, MK648157), which represented a nearly full-length genome. To confirm the ToBRFV identity, all samples were tested by RT-PCR using two pairs of primers: ToBRFV F1 (5′-GTATTTTTGTTTTACAACATATACCAAC-3′) and ToBRFV R1 (5′-AGTGCGAATGTGATTTAAAACTGTGAA-3′), and ToBRFV F7 (5′-GGAAGAAGTCCCGATGTCTGTAAGGCTT-3′) and ToBRFV R7 (5′-GATGCAGGTGCAGAGGACCATTGTAAAC-3′), designed on ToBRFV genome (KT383474; Salem et al. 2016). Twenty-two out of 39 tested samples originated specific amplicons (1,300 and 697 bp for RdRp and CP, respectively). RT-PCR products of two samples were purified and sent for direct sequencing, and results were deposited in NCBI GenBank (MK834288, MK834289, MK834294, and MK834295), revealing nucleotide identity of 99 to 100% to ToBRFV for both RdRp and CP (KT383474 and KX619418). To our knowledge, this is the first report of TMGMV and ToBRFV infecting pepper in Jordan. The occurrence of these tobamoviruses that are transmitted through seeds, in Jordan Valley, the main area for pepper production, may represent a potential threat to other susceptible vegetables and requires careful monitoring to avoid future outbreaks and significant yield losses.