First Report of Tobacco Vein Banding Mosaic Virus Naturally Infecting Black Nightshade (Solanum nigrum) in China

Abstract

Black nightshade (Solanum nigrum L. var. humile [Bernh.]) is a common solanaceous plant that is an arable weed and also used in herbal medicine in China (Wang et al. 2017b; Zhao et al. 2018). The weed occurs commonly in a wide variety of habitats, including orchards, fields, villages, and roadsides in southwest provinces in China. In May 2019, about 3% of black nightshade plants growing in a tomato field at Kunming city in Yunnan province in China were found showing typical symptoms of viral disease (chlorosis, mosaic, leaf twisting, and plant stunting). To identify the suspected virus, one plant with severe symptoms was chosen for small RNA sequencing. Total RNA was extracted from leaves of symptomatic tissues by TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA), and a small RNA library was constructed and then sequenced by high-throughput sequencing (HTS) on an Illumina HiSeq 2500 platform. The analysis generated approximately 11.37 million Illumina reads (15 to 40 nt in length), of which more than 0.082 million reads were mapped to the tobacco vein banding mosaic virus (TVBMV) genome (reference sequence accession no. NC_009994.1). No other viruses and viroids were detected. Further observation by transmission electron microscopy showed that the negatively stained crude sap of two symptomatic plants contained flexuous, filamentous virus particles about 600 to 900 nm in length, typical of a potyvirus. To further confirm the results, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA), first-strand cDNA was synthesized from 1 µg of total RNA using an oligo dT primer and a random primer mix, and then fragments of the TVBMV-CP gene were amplified with specific primers (F, 5′-AATGACGAACAGACAGTTGATGCTG-3′; R, 5′-CACGCCACTCACACCAAGTAGG-3′; designed from the CP gene of an HTS-generated clone) by 2×T5 Super PCR Mix (Tsingke BioTech, China). After cloning into a pGEM-T vector (Promega Biotech, U.S.A.), six clones of each fragment were randomly chosen for Sanger sequencing (Tsingke BioTech). The clone sequences were identical, and this partial TVBMV sequence was then submitted to GenBank (accession no. MN331841). This new sequence was most similar to Chinese isolate XDBB-01 (95.1% nucleotide identity to the CP mRNA, accession MG880266.1, and 98.2% amino acid identity to the corresponding coat protein AYG97953.1). Ten additional black nightshade plants with similar symptoms and another three asymptomatic plants were randomly selected and tested for the presence of TVBMV using the same primers. All 10 symptomatic plants tested positive for TVBMV, whereas the asymptomatic plants tested negative by reverse transcription PCR (RT-PCR) (data not shown). Transmissibility of TVBMV from black nightshade to tomato (cultivar KP13) by aphids (Myzus persicae), which were present widely in the tomato fields in the region, was tested according to the method reported by Yu et al. (2007). At 21 days postinoculation, 10 out of 20 inoculated plants showed leaf twisting symptoms. RT-PCR confirmed the presence of TVBMV in these plants but not in plants without symptoms. Results from three biological repeats were consistent, demonstrating that black nightshade can serve as an effective inoculum source for TVBMV. TVBMV has been reported to infect tobacco, potato, Datura stramonium, wild eggplant, tomato, and sesame in China (Wang et al. 2017a). To our knowledge, this is the first report of TVBMV infecting S. nigrum in natural field conditions. Our findings indicate that S. nigrum in the field could be a reservoir of TVBMV to infect tomato and cause viral disease in tomato fields.