First report of cowpea mild mottle virus naturally infecting Salvia miltiorrhiza in China

Abstract

Salvia miltiorrhiza Bge, also called Danshen, is a perennial Chinese herbal medicine belonging to the family Lamiaceae and has been used for the treatment of cardiovascular and cerebrovascular diseases (Jung et al. 2020). Henan province is one of the main and genuine production areas in China, approximately 20,000 ha of S. miltiorrhiza are cultivated annually. It is propagated through root tubes, and long-term asexual propagation leads to severe viral diseases. The accumulation of viruses caused severe losses in yield and quality of S. miltiorrhiza. Cucumber mosaic virus (CMV), tobacco mosaic virus (TMV) and soybean geminivirus B (SGVB) had been reported to infect S. miltiorrhiza (Yao et al. 2022; Qin et al. 2025). In April 2025, viral diseases of S. miltiorrhiza from six cultivation areas in Henan province were surveyed by using the five-point sampling method (Yang et al. 2021) and the results indicated a high disease incidence (average 71%). Seven S. miltiorrhiza samples exhibiting mosaic, mottle and chlorosis were collected from Yuzhou city of Henan province, China. To investigate the potential viral infection, a library was constructed from one pooled sample containing a small portion of each leaf from seven samples and high-throughput sequencing (HTS) was performed on the Illumina Nova Seq6000 sequencing system (Berry Genomics Corporation). The HTS analysis generated 77,591,670 clean reads and assembled 181 virus-related contigs (＞200 bp). BLASTn analysis indicated that nine contigs with length from 210 to 5781 nt mapped to cowpea mild mottle virus (CPMMV) sharing 97.87%-100% nucleotide sequence identity with the isolates in NCBI database. To confirm the infection of CPMMV, RNAs were extracted from seven symptomatic leaf samples and RT-PCR assays were carried out by using the primer pairs CPMMV-CPF/CPMMV-CPR designed according to the sequences of isolates in GenBank. The expected coat protein gene products (840 bp) were obtained from seven S. miltiorrhiza leaf samples and confirmed by sanger sequencing. To further characterize the CPMMV S. miltiorrhiza isolate, the nearly complete genomic sequence of CPMMV-Sm5 isolate was obtained by RT-PCR amplification of six overlapping fragments using the specific primer pairs 20F/2010R, 1700F/3780R, 3520F/5570R, 5200F/7200R, 6650F/7910R, 6740F/8190R based on the full-length genomic sequences of isolates in GenBank. The CPMMV-Sm5 isolate is 8,200 nt in length and deposited in GenBank (accession number PZ135698). BLASTn analysis revealed the genomic sequence of CPMMV-Sm5 has the highest nucleotide identity (98.99%) with the CPMMV-HN-SQ isolate (MW354940). Phylogenetic analysis demonstrated that CPMMV-Sm5 isolate was clustered in the same branch with the isolates belonging to Fabaceae from China and closest to CPMMV-HN-SQ isolates (MW354940). CPMMV was first reported from cowpeas in Ghana in 1973 (Brunt et al. 1973). To date, it has been described to infect Fabaceae, Solanaceae, Amaranthaceae, Cucurbitaceae, Chenopodiaceae, and Lamiaceae (Zanardo et al. 2017). In China, this virus had been detected from cowpea, soybean, sesame, millettia speciosa, macroptilium atropurpureum, sesbania cannabina and physalis angulata (Xu et al. 2024; Li et al. 2025a, 2025b). To our knowledge, this study is the first report of CPMMV infecting S. miltiorrhiza and expanding its known host range. Further research will be conducted to assess the impact of CPMMV on the S. miltiorrhiza and developing integrated strategies to prevent its transmission.