Scopolia, a genus with two species in the family Solanaceae, is native to Europe and Asia . Scopolia japonica Maxim (Japanese belladonna) is distributed in Honshu to Kyushu in Japan and Korean Peninsula in moist forests along mountainous valleys. It forms an inflorescence at the leaf axil in April to May  and becomes dormant during the hot season; therefore, aerial parts grow only in late winter and early spring. The roots and rhizomes of Japanese belladonna, known as “scopolia rhizome”, provide an herbal medicine containing atropine, scopolamine, scopoletin and scopoline, and other compounds that have analgesic and spasmolytic activities [5, 10].
In 2017, leaves with a slight roughness and mottling were collected from a Japanese belladonna plant in Takeda Garden for Medicinal Plant Conservation, Kyoto in western Japan (Fig. 1A). leaves samples from a single S. japonica plant was homogenized with 0.1 M phosphate buffer (pH 7.2) containing 0.1% v/v 2-mercaptopropanol and used to mechanically inoculate indicator plants Nicotiana benthamiana and Chenopodium quinoa. After single-lesion isolations were repeated three times using inoculated leaves of C. quinoa, isolated viruses were propagated in N. benthamiana plants.
To sequence the viral genome, total RNA was extracted from the leaves of N. benthamiana with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Ribosomal RNA was removed using a RiboMinus Plant Kit for RNA-Seq (Thermo Fisher Science, Carlsbad, CA, USA), and the library was prepared using an Ion Total RNA-Seq Kit v2 (Thermo Fisher Science) and the manufacturer’s protocol and sequenced using the Ion GeneStudio S5 System. De novo assembly of sequence reads was performed after removing sequences shorter than 50 nucleotides or longer than 180 nucleotides using CLC Genomics Workbench version 20.0.1. (Qiagen, Valencia, CA, USA). The 5’-terminal sequence of Scopolia mild mottle tobamovirus (SMMoV) was obtained using Takara 5’-Full RACE Core set (cat. 6122) and the manufacturer’s instructions. The 3’-terminal sequence of SMMoV was obtained by adding a poly(A) tail to the RNA using poly(A) polymerase (New England Biolabs, cat. M0276S) and SMARTer RACE 5’/3’ kit (Takara Bio, Kusatsu, Japan).
Phylogenetic analysis of SMMoV and other tobamoviruses was conducted in MEGA X . Multiple sequence alignments and removal of ambiguous alignments including gaps were done using the Clustal W algorithm . Phylogenetic trees were constructed by the neighbor-joining (NJ) method (1,000 bootstrap replications; substitution model, Dayhoff model) . Open reading frames and molecular weights of the predicted proteins were determined using the NCBI ORF Finder  and the Protparam tool of the Expert Protein Analysis System (ExPASy) . N. benthamiana plants that were inoculated with sap from the infected S. japonica plant developed mosaic and C. quinoa developed necrotic spots by 1 week after inoculation. By sequencing analysis using the CLC Genomics workbench, we obtained 850 contigs that were assembled from 1,020,138,918 reads. Annotation of these contigs in BLASTn searches of the NCBI Nucleotide Database showed that the longest one in the obtained contigs corresponded to yellow tailflower mild mottle virus (Genebank accession KJ683937, E-value = 0, Query cover 49%, Percent identity 74%), which belongs to genus Tobamovirus. Other contigs were matched to some viruses, but their query cover ratio was under 40%. Using the ORF finder, we found four ORFs that were predicted to be ORFs of Tobamovirus in BLASTp searches. The ORF1 contains methyltransferase and helicase1, the ORF2 encodes RNA-dependent RNA polymerase, the ORF3 encodes a movement protein, and the ORF4 encodes coat protein, which matches the genome organization of Tobamovirus. The genome sequence of the isolated virus (MAFF accession 260298; DDBJ LC643028) is 6350 nt long. In the genome, four ORFs were identified, as in other tobamoviruses (Fig. 2A). The first gene, p126 (ORF1), extends from nucleotides 70 to 3,429 and encodes a 126-kDa protein. Readthrough translation from the p126 stop codon at nt 3427-3429 to an opal stop codon at nt 4927-4929 was predicted to encode a p183 protein (ORF1-RT). ORF2 (nt 4945-5742) is predicted to encode a 30-kDa protein. ORF3 (nt 5746-6219) encodes a 17.5-kDa protein. GENETYX v13 (GENETYX, Tokyo, Japan) analysis showed that the nucleotide sequence of the full genome of our isolated virus shares 50-71% identity with those of other tobamoviruses and that the range in amino acid sequence identity shared between our virus and other tobamoviruses was 43-70% for replicase protein genes, 30-79% for movement protein genes, 33-73% for coat protein genes (Supplementary table 1). The species demarcation criterion for the genus Tobamovirus is less than 90% whole genome nt sequence identity shared with the other species; thus, our result suggests that scopolia mild mottle tobamovirus (SMMoV) is a new species in genus Tobamovirus. Phylogenetic analysis showed that SMMoV was most closely related to yellow tailflower mild mottle virus , which belongs to subgroup I (host-plant family: Solanaceae) (Fig. 2B and C). To understand its host range, we mechanically inoculated several species of plants with SMMoV and incubated the plants at 25°C until viral symptoms appeared. From 1 to 2 weeks after inoculation, we sampled inoculated plants to detect the CP region of SMMoV using RT-PCR (Takara PrimeScript One Step RT-PCR Kit Ver.2 Dye Plus), SMMoV-specific primers SMMoV-F4938 (GTT ACC TAT GGC TTT GTC ACT) / SMMoV-R5676 (ACC ACT ATT CTT CTT ACC ATT TAC) and the following thermocycling conditions: reverse transcription at 50°C for 30 min and 94°C for 2 min; 30 cycles of PCR at 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min. Among the inoculated plants, SMMoV was detected from Nicotiana rustica, N. glutinosa, C. quinoa, C. amaranticolor, Tetragonia tetragonioides, Solanum lycopersicum (cvs. Momotaro and Rutgers) and Physalis floridana (Fig. 1B and C). But Capsicum annuum (cv. Kagayaki), Cucumis melo (cv. Andesu) and Gomphrena globosa were not infected. Japanese belladonna developed slight yellowing and roughness on upper leaves (Fig. 1D) about 2 weeks after SMMoV inoculation. These results suggested that the host range of SMMoV was mainly confined to Solanaceae plants.
Interaction of secondary metabolites of plants and plant viruses has been studied, and several alkaloids have been suggested to have anti-phytoviruses activity [1, 16]. Since there are few reports of viral disease of genus Scopolia plants [11, 12], our result may provide helpful information for screening for novel phytochemicals to control plant viral diseases.
Since Japanese belladonna is vegetative propagated using the rhizome, any viruses in plant will remain in the infected host for a long period. Several reports have shown deformation of harvested rhizomes and decreased yields after long periods of cultivation [4, 6]. In addition, some tobamoviruses are known to be transmitted through soil. Therefore, the infection cycle and the effect of SMMoV on rhizome quality and yield need further analysis.