First Report of Apple Stem Grooving Virus Infecting <i>Acalypha australis</i> in China

Acalypha australis, commonly known as Asian copperleaf, is a Euphorbiaceae species widely distributed across East and Southeast Asia. It is recognized both as an important traditional medicinal herb and a common field weed in China. From May to July in 2024 and 2025, approximately 30% of A. australis plants in a mixed-crop field in Ningbo City, Zhejiang Province, displayed mosaic and curling on newly expanded leaves, with infected plants exhibiting dwarfism. Foliar Symptoms remained milder for the rest of the season. To investigate the potential viral agent, symptomatic leaves were collected from six plants (~30mg each) and pooled. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, USA) and subjected to high-throughput sequencing (HTS). The cDNA library was constructed using a TruSeq RNA Sample Preparation Kit (Illumina) and 150 nt paired-end reads were generated on an Illumina NovaSeq 6000 platform. Data analysis was performed using CLC Genomic Workbench 11 (QIAGEN) with default parameters. A total of 40,415,434 paired-end reads were obtained, and 145,453 contigs (57 - 26,843 nt) were generated de novo and compared with sequences in the NCBI database using BLASTn and BLASTx. A total of 41,946 reads were mapped to the apple stem grooving virus (ASGV; Capillovirus mali) genome with an average coverage of 955×. A contig of 6507 nt was aligned to ASGV (GenBank accession no. MK481988) by BLASTn with the highest nucleotide sequence identity of 89.8%, suggesting that the samples were infected with ASGV. No other plant viral sequence was identified by HTS analysis. Subsequent RT-PCR confirmed ASGV in all six aforementioned plants. One plant was selected to obtain the complete genome sequence of the virus by Sanger sequencing of RT-PCR products amplified with ASGV-specific primers (Suppl. Table 1) and the 5’-termini obtained via the commercial SUPERSWITCH RACE cDNA Synthesis Kit (Tiosbio, Beijing, China). The complete genomic sequence of the isolate (designated as ASGV-Nb) from A. australis was 6488 nt long, excluding the poly(A) tail, has the expected two predicted ORFs and was deposited to the GenBank under the accession number PX433544. Phylogenetic analysis of polyprotein of ASGV-Nb and other ASGV isolates showed that it was most closely related to two South Korean isolates from Cnidium officinale (BBK03475 and BBK03478), with which it shared 90.8% amino acid identity. To investigate the host range of this ASGV isolate, an infectious cDNA was constructed and agroinfiltrated into leaves of Nicotiana. benthamiana (n = 5), N. glutinosa (n = 5), pepper (n = 5) and apple (n = 3) plants. Mosaic and curling symptoms appeared on leaves of N. benthamiana and pepper at 21 and 7 days post-inoculation (dpi), respectively. No obvious symptoms were observed on N. glutinosa or apple even three months after inoculation. Interestingly, ASGV was detected by RT-PCR in all inoculated plants. The only viruses previously reported from A. australis are the begomoviruses tomato yellow leaf curl virus (TYLCV; Begomovirus coheni) and papaya leaf curl China virus (PaLCCNV; B. caricachinaense) (Ji et al. 2013; Tang et al. 2018). However, neither TYLCV nor PaLCCNV was detected in any of our samples by PCR. ASGV has a wide host range but mainly infects fruit trees (Shokri et al. 2023, Xia et al. 2025), and to our knowledge, this is the first report of the virus on A. australis, which expands the host range of ASGV.