First Report of Soybean Geminivirus B Infecting Salvia miltiorrhiza in China.

Abstract

Salvia miltiorrhiza Bge (Family Lamiaceae) is a traditional Chinese herbal medicine; its roots and rhizomes are used as medicines (Zhang et al. 2023). The plant has been vegetatively propagated using root tubers, and long-term asexual reproduction can lead to the accumulation of plant viruses, which may cause severe diseases. Tobacco mosaic virus and cucumber mosaic virus have been reported to infect S. miltiorrhiza (Yao et al. 2022), and their prevalence has become increasingly serious in China. From June to September 2023, 125 samples of S. miltiorrhiza symptomatic leaves were randomly collected in Henan province, China. A small portion of each leaf of above 125 collected samples was mixed into a total sample (~30 g) and sent to Berry Genomics Corporation (Beijing, China) for high-throughput sequencing (HTS) examination. Ribosomal RNA was removed by RNAprep Pure Plant Plus kit (TIANGEN Biotech, Beijing, China), and the NEBNext Ultra RNA Library Prep kit for Illumina (NEB, Ipswich, MA, USA) was used to build a cDNA library. The sequencing procedure was performed on the Illumina Nova Seq6000 sequencing system (Berry Genomics Corporation). Wuhan Biowefind Co., Ltd. (Wuhan, China) handled the subsequent processing and analysis of sequencing data. A total of 64,043,098 paired-end reads (150 nt) were obtained, and 63,559,676 clean reads were assembled by de novo (IDBA-UA, version 1.1.3) to generate 131,718 contigs (＞200 bp). There were 58,300 reads that covered 85.8% mapping to the soybean geminivirus B genome (SGVB, GenBank MH428830.1). Via BLAST analysis, 12 contigs with length from 228-882 nt were associated with CMV of a 90.4-99.7% similarity; 464 contigs with length from 202-8864 nt were associated with Betaflexiviridae of a 52.82-73.72% similarity; 2 contigs with length from 1849-6181 nt were associated with Alphaflexiviridae of a 33.4~75.3% similarity; 30 contigs with length from 205-7164 nt were associated with Fimoviridae of a 59.17~70.09% similarity. Seventeen contigs with length from 217-1046 nt were associated with SGVB of a 87.5~100% similarity. To identify the presence of the virus, the DNA of 125 samples were extracted using the EZ-10 Spin Column Plant Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China). The DNA samples were subjected to PCR using the SGVB-specific primer set DSSGVBF/DSSGVBR (a 457-nt fragment), designed based on the HTS results. Results revealed 80 positive samples out of 125 (detection rate, 64%); no bands were detected in ddH2O used as a negative control. A pair of back-to-back primers (DSSGVBF1/DSSGVB1) was designed based on the sequences of the 457-nt fragments to amplify the complete sequence of SGVB. Complete sequences of seven isolates (33#, 130#, 131#, 148#, 149#, 152#, and 155#) were obtained from the positive samples with the DSSGVBF1/DSSGVB1 primer set. It was obtained by sanger sequencing (Sangon Biotech, Zhengzhou, China). The genome sequences of these isolates were 2625 nt long and were deposited in GenBank (accession no. PQ141695, PQ163462-163467). BLASTn analysis revealed that the homology of these seven isolates with the soybean geminivirus B isolate Kong (MH428830.1) was 93.94-97.37%, and the consistency between the seven isolates was 94.0-99.9%. In addition, eight nucleotides were inserted between nt sites 518-525, resulting in the CP position range from 705-1184 reported in SGVB isolate Kong changed to 424-1194 in this study, and the length changing from 477 nt to 768 nt. However, the effect on CP function is unclear. As a novel virus assigned to the Mastrevirus genus, family Geminiviridae, SGVB has only been reported in soybean crops in South Korea (Choi et al. 2022). This study revealed that S. miltiorrhiza is a new host of SGVB. To the best of our knowledge, this is the first report of SGVB-infected S. miltiorrhiza and SGVB in China. In future, we will determine how this virus was likely moved from potential soybean to S. miltiorrhiza via its insect vectors and study its pathological implications on S. miltiorrhiza in China.