First report of calla lily chlorotic spot virus causing mottling on Angelica sinensis (Oliv.) Diels in China

Angelica sinensis (Oliv.) Diels is a traditional Chinese medicinal plant cultivated in Gansu, Qinghai, and Yunnan in China (Wei et al., 2016). In June 2021, A. sinensis samples exhibiting leaf mottling and chlorotic spots on leaves were collected from Heqing, Yunnan, China. Within the 1.2-hectare area surveyed, the symptom incidence rate was approximately 30%. TEM ultra-thin leaf section revealed spherical virions approximately 80-120 nm in diameter, with a morphology characteristic of orthotospoviruses (Zhang et al. 2016). To further identify the pathogen, high throughput sequencing (HTS) was conducted (Illumina Novaseq 6000) following previously described methods (Hu et al., 2023). The HTS analysis generated 102.63 million raw reads, which resulted in 102.05 million clean reads, with a mean length of 150 nucleotides (nt) after removing low-quality reads (Q30>93.14%, N50=873bp). De novo assembly (SPAdes v3.15.4) produced 8,185 virus-related contigs (>500bp), of which 2,086 contigs were mapped to Orthotospovirus sequences. Based on these contigs, the complete genome sequence of calla lily chlorotic spot virus (CCSV) was successfully assembled and named CCSV-AS, comprising three segments: S segment (3,197 bp; GenBank accession no. PV178716) encoding the N (278aa) and NSs (461aa) proteins; M segment (4,735 bp; GenBank accession no. PV178715) encoding the NSm (310aa) and Gn/Gc (1124aa) proteins; L segment (8,192 bp; GenBank accession no. PV178713) encoding the RdRp (2650aa) protein. Next, total RNA was extracted from one of the symptomatic sample leaves using the EASY Spin Plus Complex Plant RNA Kit (Aidlab Biotechnologies, Beijing, China). Complementary DNA (cDNA) was synthesized using the HiFi Script gDNA Removal RT Master Mix (CoWinBio scences, Beijing, China). Futher, The specific primers, CCSV-NF (5'-ATGTCTAACGTCAGAGGTTTAACAC-3') and CCSV-NR (5'-TTAAAAAGTTAAATCACCACTCTTC-3'), were used to amplify the N gene of CCSV-AS from symptomatic samples. The PCR conditions included an initial denaturation at 94℃ for 5min, 35 cycles of denaturation at 94℃ for 10s, annealing at 54℃ for 30s, extension at 72℃ for 1 min, and a final extension at 72℃ for 5 min. Expected amplicons (834 bp) were obtained and sequenced by Sanger method (Fig. S3). BLAST analysis revealed that the N gene sequence of the CCSV-AS shared the highest nucleotide sequence identity (96.28%) with the CCSV LJ-1 isolate from Nicotiana tabacum (GenBank accession No. KT004452). Phylogenetic analyses were conducted using the N protein sequences of CCSV-AS and other orthotospoviruses with MEGA7.0 software. According to the phylogenetic tree, CCSV isolates were divided into two distinct clades. CCSV-AS clustered with several Yunnan and Taiwan CCSV isolates as the major branch. In contrast, the two isolates from Zhejiang clustered separately in another branch. Collectively, these results confirmed CCSV infection A. sinensis in China. A. sinensis is mainly propagated by seeds. CCSV is thrips-transmitted through a persistent-propagative manner (Chen et al., 2005), and was first identified in calla lily in Taiwan, China in 2003 (Chen et al., 2003). It later reported to infect tobacco and spider lily in Yunnan, China in 2012 (Liu et al., 2012) and lettuce in Zhejiang in 2018 (Wu et al., 2018). To date, CCSV has been documented in 28 plant species globally (Wu et al., 2018). However, to our knowledge, this is the first report of CCSV infecting A. sinensis. These findings will contribute to the development of management strategies for CCSV in A. sinensis cultivation.