First Report of Hydrangea chlorotic mottle virus Infecting Hydrangea macrophylla in China

Hydrangea chlorotic mottle virus (HdCMV), a member of the genus Carlavirus (family Betaflexiviridae), was first reported in Hydrangea macrophylla (H. macrophylla) in the United States in 2005 (Machado Caballero et al. 2009) and has also been detected in New Zealand and South Korea. In this study, we report the first detection of HdCMV in H. macrophylla in China using the Pacific Biosciences single-molecule real-time (PacBio) high-throughput technology. In 2020, total RNA was extracted from a pooled sample of root, stem, leaf, lateral bud, terminal bud, pedicel, and flower organ tissues of H. macrophylla 'Bailmer' plants collected in Beijing, using TRIzol reagent (TIANGEN Biotech, Beijing, China). Sequencing conducted on the PacBio Sequel platform (SRA accession: PRJNA849710; Qi et al., 2022) generated 26,078,511 subreads with an average length of 1,934 bp. The raw reads were processed using SMRT Link v8.0.0 to generate high-quality circular consensus sequencing (CCS) reads. After removing primers, barcodes, poly(A) tails, and concatemers, clustering of full-length non-chimeric (FLNC) reads produced 72,848 high-quality isoforms (51-13,524 bp). Functional annotation of these isoforms was performed by BLASTX alignment against the NCBI non-redundant (nr) protein database, and the sequences homologous to genomes of Hydrangea ringspot virus and HdCMV were identified. Among these, this set of 53 isoforms showed 91.7%-99.4% nucleotide sequence identity to the reference isolate NZ (GenBank accession no. EU754720.2; 8,433 bp), with sequence lengths spanning 7,887-9,925 bp and covering 99.9% of the isolate NZ genome (mean depth: 52.9×). The complete genomic sequence of HdCMV with 8,432 bp was amplified using primers designed according to the isoforms, verified by Sanger sequencing, and deposited as isolate Hyd-CN3 (PX718916). Hyd-CN3 shared the highest nucleotide sequence identity (97.2%) with HdCMV isolate NZ. Phylogenetic analysis based on the CP gene revealed that the isolate had a close relationship with the HdCMV isolates from other countries. According to the coat protein (CP) sequence, a pair of specific primers (HdCMV-F/R) with producing a 406-bp amplicon during the RT-PCR, was designed for detecting Hyd-CN3 infection in H. macrophylla. In 2025, a survey conducted across four major hydrangea-producing regions revealed that HdCMV was detected in 156 of 185 sampled plants (84.3%) using RT-PCR, with infection rates of 78.1% (100/128) in Yunnan Province, 100% (24/24) in Guizhou Province, 95.8% (23/24) in Jiangxi Province and 100% (9/9) in Sichuan Province. The H. macrophylla leaves infected with HdCMV exhibited a symptom spectrum ranging from asymptomatic infection‌ to leaf distortion, blistering, and chlorotic mottling. To our knowledge, this is the first confirmed report of HdCMV in H. macrophylla in China, thus expanding its geographical range. Given the high infection rate of HdCMV in the production area, effective measures must be taken to eliminate or suppress its spread to avoid significant commercial losses.