First Report of Chilli Veinal Mottle Virus Infecting <i>Datura innoxia</i> in Yunnan, China

Chilli veinal mottle virus (ChiVMV) is a member of the genus Potyvirus in the family Potyviridae and has been reported to infect various Solanaceae crops in Asia (Zhao et al., 2014). Datura innoxia (Solanaceae), an annual herb, is native to Central/South America (Mohammed et al., 2017). In July 2023, virus-like symptoms such as mosaic, mottling, and leaf deformation were observed on several D. innoxia plants near a tobacco field, infected by viral pathogens in Jianshui County, Yunnan Province, China, with an incidence of 30%. Leaves from four plants (3 symptomatic, 1 asymptomatic) were collected for biological and molecular characterization. Sap from symptomatic leaves was stained with a 1% (w/v) phosphotungstic acid solution and potyvirus-like particles were observed (approximately 700-800×13 nm) by transmission electron microscope (TEM). Total RNA was extracted from symptomatic leaf samples using TRIzol Reagent (Invitrogen, U.S.A.) and subjected to RNA-Seq on the Illumina NovaSeq 6000 platform. After trimming and quality control of the raw data using Trinity software, 19,854,780 high-quality clean reads were assembled into 89,781 unigenes. Thirty contigs were obtained from comparing unigenes and mapped to a specific sequence of the ChiVMV using BLASTx, with an E-value cutoff of 10-5 while no other viruses were detected. The near full-length sequence of ChiVMV-Di (GenBank accession no. PQ466185) was verified through RT-PCR amplification and sanger sequencing, BLASTn analysis of the complete genomic sequence showed that ChiVMV-Di shared the highest nucleotide identity (98.75%) with the isolate YN-tobacco (JX088636) from China. Phylogenetic analysis further confirmed that ChiVMV-Di clustered most closely with Chinese isolates. RT-PCR was conducted on symptomatic/asymptomatic D. innoxia leaves using ChiVMV-specific primers (ChiVMV-F: 5'-ACATTGACCCATATCTGAAGGCACTC-3'; ChiVMV-R: 5'-GGGAACCACACTGAGGAATATGTATGA-3') targeting the complete CP gene. A ~1043 bp amplicon (containing partial Nib and 3'UTR) was obtained from all symptomatic samples and verified by sequencing. BLASTn analysis of the complete CP gene sequence revealed 93.5-99.4% nt identity with other ChiVMV isolates. To assess the infectivity of ChiVMV, healthy D. innoxia (n=12) and Nicotiana benthamiana (n=6) seedlings were mechanically rub-inoculated with sap extracted from the symptomatic leaves of infected D. innoxia. Mock-inoculated controls were treated with healthy D. innoxia leaf sap. All virus inoculated plants developed mosaic symptoms and leaf deformation on systemic leaves 8 days post-inoculation. No virus-like symptoms were observed in the control plants. All symptomatic samples tested positive for ChiVMV by RT-PCR with the specific primers described above, while the control tested negative. This confirmed that the symptomatic D. innoxia is infected with ChiVMV. This study provides the first molecular evidence of natural ChiVMV infection in D. innoxia in China, expanding its known host range beyond the previous single report from India (Kaur et al., 2018). Given the prior detection of ChiVMV in solanaceous crops like tobacco, tomato, and pepper within the same county, the presence of infected D. innoxia near tobacco fields poses a significant risk to tobacco cultivation. Therefore, management strategies, including weed control and vector monitoring, are recommended to prevent the spread of ChiVMV.