Acalypha australis as a Potential Reservoir Host of Hop Stunt Viroid in Vineyards

Abstract

Vineyard-associated weeds can serve as reservoirs for grapevine pathogens, but their role in virus and viroid ecology remains unclear. This study aims to examine the presence of grapevine-associated viruses and viroids in symptomatic Acalypha australis plants growing under grapevines in Yeongdong, South Korea. RT-PCR screening conducted on neighboring grapevines revealed mixed infections with multiple viruses and viroids. In A. australis, hop stunt viroid (HSVd), grapevine rupestris stem pitting-associated virus, and grapevine virus E were detected via RT-PCR, with additional support from nanopore sequencing. Sequence and phylogenetic analyses showed that the HSVd isolates from A. australis and grapevine collected from the same vineyard were genetically identical. Mechanical inoculation confirmed HSVd infection in healthy A. australis plants. Additional screening identified HSVd infection in A. australis from Yeongcheon and Haman, and in neighboring grapevines. These findings suggest that A. australis may serve as a potential reservoir host of HSVd in vineyard agroecosystems.

Grapevine (Vitis spp.) is one of the economically valuable fruit crops worldwide and is susceptible to many viruses and viroids. More than 100 virus species have been reported to infect grapevine, and such infections may result in reduced vine vigor, vineyard longevity, yield, and fruit quality. Dissemination is largely associated with the presence of infected propagules. In addition, certain viruses that affect grapevines can be transmitted through insects or nematodes (Fuchs, 2020, 2025; Martelli, 2017). Among grapevine-associated subviral pathogens, hop stunt viroid (HSVd) is an important pathogen due to its broad host range, global distribution, and ability to persist in perennial hosts. Previous studies suggest that grapevine can serve as a long-term reservoir for HSVd and that mechanical means of transmission may contribute to viroid dissemination through pruning and other cultural practices (Hadidi et al., 2022; Kawaguchi-Ito et al., 2009; Marquez-Molins et al., 2021; Sano et al., 2001).

Weeds and wild plants in agroecosystems can serve as alternative hosts or ecological bridges for plant viruses or viroids, but their role in grapevine pathogen ecology remains insufficiently understood. Non-Vitis hosts have been observed for several grapevine pathogens, including grapevine fanleaf virus in Bermuda grass and grapevine Pinot gris virus in various non-Vitis plants (Demian et al., 2022; Izadpanah et al., 2003). Because grapevines are frequently affected by mixed infections, combining RT-PCR with high-throughput sequencing can help characterize complex infection profiles. This study aims to investigate symptomatic Acalypha australis plants growing under grapevines and evaluate their potential role as reservoir hosts of grapevine-associated viruses and viroids, with particular emphasis on HSVd.

HSVd, together with two grapevine-associated viruses, was identified in field-collected A. australis using RT-PCR, nanopore sequencing, Sanger sequencing, and phylogenetic analysis. We further evaluated the biological significance of these pathogens through mechanical inoculation of healthy A. australis plants (Fig. 1). Our findings provide evidence that A. australis can harbor HSVd under vineyard conditions and highlight the potential importance of vineyard-associated weeds in grapevine pathogen ecology.

Fig. 1.

Schematic overview of the workflow used in this study. Symptomatic Acalypha australis plants growing under grapevines were screened for grapevine-associated viruses and viroids using RT-PCR, nanopore sequencing, Sanger sequencing, pairwise nucleotide identity analysis, and phylogenetic analysis. Mechanical inoculation was performed to assess infectivity in healthy A. australis plants, while further regional screening was conducted using A. australis samples from Yeongcheon and Haman, South Korea.

The symptomatic A. australis plants growing under grapevines were collected in June 2025 at a vineyard located in Yeongdong-gun, Chungcheongbuk-do, South Korea (Fig. 2A). Leaves from five symptomatic A. australis plants were pooled for RNA extraction. To compare pathogen occurrence between A. australis and neighboring grapevines, leaf samples were also collected from eight grapevines growing close to the symptomatic A. australis plants. Total RNA was extracted using the Beniprep Super Plant RNA Extraction Kit (InvirusTech, Gwangju, Korea), following the manufacturer’s instructions.

Fig. 2.

Detection of grapevine-associated viruses and viroids in grapevine and field-collected A. australis from the Yeongdong vineyard. (A) Field images of symptomatic A. australis growing under grapevines. (B) RT-PCR screening summary of eight grapevine samples for 11 grapevine-associated viruses and viroids. Numbers indicate positive samples among the eight tested grapevines. (C) RT-PCR screening results of field-collected A. australis showing the presence of HSVd, GRSPaV, and GVE. (D) Reference-based nanopore read mapping analysis supporting the detection of HSVd, GRSPaV, and GVE in A. australis. M, 100 bp ladder; HSVd, hop stunt viroid; GRSPaV, grapevine rupestris stem pitting-associated virus; GVE, grapevine virus E.

Grapevine samples were screened via RT-PCR for 11 grapevine-associated viruses and viroids commonly detected in Korean vineyards. The 11 viruses and viroids include HSVd, grapevine yellow speckle viroid 1 (GYSVd-1), grapevine yellow speckle viroid 2 (GYSVd-2), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine fleck virus (GFkV), grapevine virus B (GVB), grapevine virus E (GVE), Australian grapevine viroid (AGVd), grapevine Pinot gris virus (GPGV), grapevine geminivirus A (GGVA), and grapevine fabavirus (GFabV). Primer sequences and expected amplicon sizes are listed in Supplementary Table 1 (Al Rwahnih et al., 2016; Astruc et al., 1996; Gambino et al., 2014; Jo et al., 2017a, 2017b; Kishan et al., 2023; Lee et al., 2023; Manduláné Farkas et al., 2014). RT-PCR was performed using SuPrimeScript RT-PCR Premix (GeNet Bio, Daejeon, Korea) with reverse transcription at 50°C for 30 min, initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, primer-specific annealing for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 5 min. Among the 11 targets, all except AGVd were detected in grapevine samples (Fig. 2B). HSVd, GRSPaV, GFkV, and GVE were detected in all eight grapevine samples, whereas GYSVd-1, GYSVd-2, GVB, GPGV, GGVA, and GFabV were found in some samples, indicating complex mixed infections in this vineyard.

The symptomatic A. australis samples were analyzed using primer sets identical to those described in Supplementary Table 1. RT-PCR detected HSVd, GRSPaV, and GVE, yielding amplicons of the expected sizes: 303 bp for HSVd, 226 bp for GRSPaV, and 749 bp for GVE (Fig. 2C). However, no amplification was observed in the other tested targets, suggesting that field-collected A. australis may harbor HSVd together with two grapevine-associated viruses under vineyard conditions.

RT-PCR amplicons of HSVd, GRSPaV, and GVE from grapevine and field-collected A. australis were purified and subjected to Sanger sequencing. For HSVd, full-length sequences were obtained by cloning. Four independent grapevine-derived HSVd clones were sequenced and designated HSVd-Vv (LC938827.1), HSVd-Vv-2 (LC944877.1), HSVd-Vv-3 (LC944878.1), and HSVd-Vv-4 (LC944879.1). For A. australis, one representative HSVd clone from the pooled RT-PCR amplicon was sequenced and designated HSVd-Aa-F (LC938828.1). For GRSPaV and GVE, partial RdRP sequences were obtained from grapevine and field-collected A. australis. The representative sequences were designated GRSPaV-Aa-F (LC940384.1), GRSPaV-Vv (LC940385.1), GVE-Vv (LC940382.1), and GVE-Aa-F (LC940383.1). Three additional grapevine-derived clones were also obtained for each virus and designated GRSPaV-Vv-2 to GRSPaV-Vv-4 (LC944880.1–LC944882.1) and GVE-Vv-2 to GVE-Vv-4 (LC944883.1–LC944885.1). Nanopore sequencing was conducted using the Oxford Nanopore MinION system as described by Kim et al. (2025), with slight modifications. Raw signals were basecalled using Guppy v6.5.7, while FASTQ reads were trimmed and quality-filtered in Geneious Prime v2026.1.2. Reads with a mean quality score <Q7 were eliminated. The quality-filtered reads were analyzed using EPI2ME WIMP, BLASTn, and mapping to reference sequences in Geneious Prime for HSVd, GRSPaV, and GVE (Kearse et al., 2012). The mapped reads were examined for read number, nucleotide identity, and reference coverage to support the RT-PCR-based identification of grapevine viruses and viroids in A. australis.

Sequencing resulted in 35,856 quality-filtered reads, which were uploaded to the SRA database with an accession number of SRR39421772 (Supplementary Table 2). Reads associated with HSVd, GRSPaV, and GVE were classified based on taxonomic identification, BLASTn search, and reference-based mapping in the A. australis library, supporting the RT-PCR-based detection of these pathogens (Fig. 2D, Supplementary Table 3). Since the mapped reads showed relatively low nucleotide identities to the reference sequences, the nanopore data were considered supportive evidence, together with target-specific RT-PCR and Sanger sequencing, rather than as stand-alone confirmation.

The pairwise nucleotide identity and phylogenetic analyses were performed using the Sanger-derived sequences. Alignments were generated in MEGA 12, and maximum-likelihood trees were constructed using the Tamura-Nei model with 1,000 bootstrap replicates. HSVd-Vv and HSVd-Aa-F shared 100% nucleotide identity across the full-length HSVd sequence, and HSVd-Aa-F clustered with HSVd-Vv and representative HSVd isolates from grapevine and other hosts (Fig. 3A). These findings indicate that grapevine and A. australis from the same vineyard harbored genetically identical HSVd isolates. All four grapevine-derived HSVd clones, including HSVd-Vv and HSVd-Vv-2 to HSVd-Vv-4, shared 100% pairwise nucleotide identity, indicating complete sequence conservation within the analyzed grapevine-derived HSVd population.

Fig. 3.

Pairwise nucleotide identity and phylogenetic analyses of HSVd, GRSPaV, and GVE sequences obtained from grapevine and field-collected Acalypha australis. (A) Full-length HSVd sequences. (B) Partial RdRp sequences of GRSPaV. (C) Partial RdRp sequences of GVE. Phylogenetic trees and corresponding identity matrices are shown for each pathogen. Grapevine-derived sequences obtained in this study are indicated by black circles (●), whereas A. australis-derived sequences are indicated by black triangles (▲). Vv (including Vv-2 through Vv-4) and Aa denote sequences derived from grapevine and A. australis, respectively.

For GRSPaV and GVE, pairwise nucleotide identity and phylogenetic analyses based on partial RdRp sequences showed that the A. australis-derived sequences were very similar to the corresponding grapevine-derived sequences from the same vineyard (Fig. 3B, C). The grapevine-derived GRSPaV clones showed 95.33–98.90% pairwise nucleotide identity, whereas the grapevine-derived GVE clones showed 95.22–100% identity, indicating the presence of limited sequence variation among grapevine-derived viral clones. Since only partial sequences were analyzed, further genomic and biological studies are needed to explain their relevance in A. australis.

To determine whether the pathogens detected in field-collected A. australis could infect healthy A. australis plants, two seedlings obtained from seeds provided by the National Institute of Biological Resources (NIBR), Republic of Korea, were mechanically inoculated with the leaf sap extract of symptomatic A. australis using 0.01 M sodium phosphate buffer (pH 7.4) with carborundum. Mock-inoculated plants received only buffer treatment. At 21 days post-inoculation (dpi), HSVd-positive A. australis plants showed reduced growth, smaller leaves, compact growth, and mild leaf curling or deformation compared with mock-inoculated plants (Fig. 4A). Newly developed systemic leaves were analyzed via RT-PCR using HSVd-, GRSPaV-, and GVE-specific primers. HSVd was detected in both inoculated plants, whereas GRSPaV and GVE were not detected under the conditions tested (Fig. 4B). These findings suggest that HSVd can infect A. australis and may be associated with mild growth suppression and foliar deformation.

Fig. 4.

Mechanical inoculation and sequence analysis of HSVd in A. australis. (A) Phenotypes of mock-inoculated controls and HSVd-positive A. australis plants at 3 weeks post-inoculation. (B) RT-PCR detection of HSVd, GRSPaV, and GVE in mechanically inoculated A. australis plants. (C) Alignment of HSVd-Aa-F and HSVd-Aa-I with NC_001351.1. HSVd-Aa-F and HSVd-Aa-I shared 100% identity. (D) Secondary structure prediction of NC_001351.1 and HSVd-Aa-F/I. The minimum free energies were -123.20 and -114.00 kcal/mol, respectively. Green, red, and black arrows indicate substitutions, insertions, and deletions, respectively. M, DNA ladder; H, healthy mock-inoculated plant; lanes 1 and 2, inoculated plants; NTC, no-template control; PC, positive control.

The HSVd sequence recovered from experimentally inoculated A. australis was designated HSVd-Aa-I (GenBank accession no. LC938829.1). HSVd-Aa-F and HSVd-Aa-I showed 100% nucleotide identity across the GenBank-deposited 303-nt sequence (Fig. 4C). For secondary-structure analysis, HSVd-Aa-F/I and the NCBI HSVd reference sequence NC_001351.1 were reoriented to the same starting position, and gap characters introduced during alignment were excluded. Mfold analysis showed that both sequences formed typical rod-like conformations, with minimum free energies of −123.20 and −114.00 kcal/mol for NC_001351.1 and HSVd-Aa-F/I, respectively (Fig. 4D). Nucleotide substitutions, insertions, and deletions in HSVd-Aa-F/I relative to NC_001351.1 are shown in Fig. 4D. These findings support the stability of the HSVd sequence after experimental passage in A. australis.

To determine whether HSVd-positive A. australis plants were limited to the Yeongdong vineyard, additional A. australis samples were collected from grapevine farms in Yeongcheon-si, Gyeongsangbuk-do, and Haman-gun, Gyeongsangnam-do, South Korea (Fig. 5A). At each location, 17 A. australis samples, representing all accessible plants at the time of sampling, were screened by RT-PCR using primer sets specific to the 10 grapevine-associated viruses and viroids detected in the Yeongdong grapevine samples. Among the tested targets, only HSVd was detected in the additional A. australis samples, with detection rates of 6/17 in Yeongcheon and 4/17 in Haman (Fig. 5B). Amplicons from all HSVd-positive samples were subjected to direct Sanger sequencing, and a representative HSVd sequence from each region was deposited in GenBank (Yeongcheon isolate, accession no. LC940386.1; Haman isolate, accession no. LC941255.1). These findings indicate that HSVd-positive A. australis plants occur in multiple grape-growing regions in South Korea, although further surveys are needed to determine their incidence and geographical distribution.

Fig. 5.

Regional screening of HSVd in A. australis and grapevine samples from Yeongcheon and Haman. (A) Representative field images of A. australis growing near grapevines. (B) RT-PCR analysis of HSVd infection in individual A. australis samples. HSVd was identified in 6 of 17 Yeongcheon samples and 4 of 17 Haman samples. (C) RT-PCR analysis of HSVd in nearby grapevine samples. All 14 grapevine samples were positive for HSVd, and grapevine 18S rRNA was amplified as an internal control. M, DNA ladder; H, healthy A. australis; NTC, no-template control.

To determine whether HSVd was also present in grapevines near the A. australis sampling sites, representative grapevine leaf samples were collected. Because it was not feasible to examine all grapevines on each farm, four samples from Yeongcheon and 10 from Haman were analyzed by RT-PCR using HSVd-specific primers. All 14 grapevine samples were positive for HSVd (Fig. 5C). Three representative amplicons were confirmed as HSVd-derived sequences via Sanger sequencing (data not shown). Grapevine 18S rRNA was amplified from all samples as an internal control for RNA integrity and RT-PCR performance, and primer information is provided in Supplementary Table 1 (Gambino et al., 2008).

The present study provides molecular and biological evidence that A. australis can harbor HSVd under vineyard conditions and is susceptible to HSVd infection through mechanical inoculation. HSVd was detected in field-collected A. australis, and the HSVd sequence found in A. australis was identical to that from grapevine collected from the same vineyard. Moreover, HSVd was recovered from experimentally inoculated A. australis plants with complete sequence conservation. Additional HSVd-positive A. australis samples were identified in two other grape-growing regions. Together, these findings support the potential role of A. australis as a reservoir host of HSVd in vineyard agroecosystems. This interpretation is consistent with the broad host range of HSVd and its ability to persist in perennial plants and spread through infected plant material or mechanical injury (Hadidi et al., 2022; Kawaguchi-Ito et al., 2009; Marquez-Molins et al., 2021; Sano et al., 2001).

Although natural HSVd infection was confirmed in A. australis plants growing near infected grapevines, and experimental susceptibility was demonstrated by mechanical inoculation, the actual transmission route from grapevine to A. australis under field conditions remains unknown. Therefore, the role of A. australis as a potential reservoir host of HSVd should be interpreted cautiously until direct grapevine-to-A. australis transmission is experimentally confirmed. Further studies, including mechanical inoculation using HSVd-infected grapevine sap, tool-mediated transmission assays, and controlled field surveys, will be needed to establish this epidemiological link.

Although additional grapevine-derived clones were analyzed in this study, the sequence comparison for A. australis was based on representative sequences obtained from pooled symptomatic plants. Therefore, possible within-host or within-population sequence variation of HSVd, GRSPaV, and GVE in A. australis could not be fully resolved. Future studies using multiple clones or amplicon deep sequencing from individual grapevine and A. australis plants will be needed to clarify the population-level relationships of these viruses and viroids between the two hosts.

In contrast, GRSPaV and GVE were detected in field-collected A. australis but were not detected after mechanical inoculation under the conditions tested. GRSPaV is mainly associated with infected grapevine propagation material, whereas vitiviruses such as GVE may also be associated with transmission by mealybugs or soft-scale insects (Meng and Rowhani, 2017; Nakaune et al., 2008; Hommay et al., 2022a, 2022b). Therefore, the detection of GRSPaV and GVE in A. australis may reflect close ecological contact with infected grapevines, possible exposure to vineyard-associated hemipteran insects, or a transient/low-level association rather than stable systemic infection. Overall, these findings indicate that vineyard-associated weeds may contribute to grapevine virus and viroid ecology and should be considered in HSVd monitoring and management strategies.

Electronic Supplementary Material

Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).