First Report of Tomato Mosaic Virus on <i>Hydrangea macrophylla</i> in China

Tomato mosaic virus (ToMV), a member of the genus Tobamovirus within the family Virgaviridae, was first reported in the United States in 1899 (Naik et al. 2025). ToMV exhibits a broad host range, infecting plants from multiple families, including Solanaceae, Rosaceae, and Malvaceae (Naik et al. 2025). In 2024, numerous Hydrangea macrophylla plants exhibiting virus-like symptoms, including leaf chlorosis, mosaic patterns, distortion, and size reduction, were observed in several production areas in Yunnan and Sichuan Provinces, China. Sampling was conducted to assess disease incidence, and assessments indicated 1-10% incidence rates. To identify the causative agent, symptomatic leaves were pooled from individual plants at two production sites in Yunnan Province and two in Sichuan Province, and subjected to high-throughput sequencing. For this, total RNA was extracted using the RNA Prep Pure Plant Plus Kit (TIANGEN BIOTECH, Beijing, China). A ribosomal RNA-depleted library was constructed using TruSeq DNA Sample Prep Kit (Illumina, San Diego, CA, USA). Sequencing was performed on the Illumina NovaSeq platform with 150-bp paired-end reads, yielding 93,452,496 clean reads (SRA accession: PRJNA1237933). Subsequently, the reads mapped to the Hydrangea genome database (Nashima et al. 2021) using minimap2 (v2.28) were removed. The remaining 1,450,584 reads were de novo assembled into contigs (ranging from 300 to 6,832 nt) using MEGAHIT (v1.2.9). The contigs were compared against the NCBI RefSeq-viral database (v1.1, 2023.11) using DIAMOND-BlastP (v2.1.11). Contigs corresponding to hydrangea chlorotic mottle virus (HdCMV) and hydrangea ringspot virus (HdRSV) were identified, both of which have been previously reported in Hydrangea (Song et al. 2016). In addition, a 6,382-nt contig (Hyd-CD1, GenBank PQ997932) showed 99.2% nucleotide sequence identity to the ToMV Queensland isolate (NC_002692). To determine the ToMV genome sequence, we performed RT-PCR amplification followed by Sanger sequencing, which yielded a 6,382-nt consensus sequence (isolate Hyd-T1, PV590581) from a symptomatic Hydrangea sample (Sichuan36). The sequence showed 99.7% identity to the loquat isolate Pi20 (PP915779) in Sichuan and phylogenetically clustered with it. To validate ToMV infection in H. macrophylla across different regions, twelve symptomatic samples (nine from Sichuan and three from Yunnan) were ToMV-positive by ELISA (MEIMIAN) and positive for the ToMV coat protein-gene by RT-PCR (167 bp, Sanger sequencing-verified). Multiplex RT-PCR with primers for HdRSV, HdCMV and ToMV revealed all samples were co-infected with all tested viruses. Extended screening of 100 field samples confirmed this co-infection pattern, with no single ToMV infections detected. Pearson correlation analysis revealed a significant positive correlation (r = 0.77, p < 0.01, n = 100) between symptom severity (leaf chlorosis, curling, and narrowing, standardized 0-3 scale) and ToMV RNA levels quantified by triplicate RT-PCR using AlphaImager 2200 (v.3.2.). This study presents the first report of natural infection of Hydrangea by ToMV in China. The virus not only compromises the ornamental value of Hydrangea but may also spread mechanically to other susceptible hosts through nationwide trade of potted plants, posing a potential threat to agricultural production security. Therefore, effective management strategies are needed to mitigate its spread.