Inhibitory Effects of Garlic Extract on Hop Stunt Viroid in Micropropagated Grapevine Plantlets

Article information

Plant Pathol J. 2025;41(1):51-63
Publication date (electronic) : 2025 February 1
doi : https://doi.org/10.5423/PPJ.OA.10.2024.0169
Department of Applied Biology, Chonnam National University, Gwangju 61185, Korea
*Corresponding author. Phone) +82-62-530-2075, FAX) +82-62-530-2069, E-mail) jraed2@jnu.ac.kr
Handling Editor: Eui-Joon Kil
Received 2024 October 28; Revised 2024 December 2; Accepted 2024 December 12.

Abstract

Hop stunt viroid (HSVd) is a major pathogen that affects grapevine health and causes substantial economic losses in grape cultivation. Many studies have been conducted to control grapevine diseases, but effective control methods after plant infections remain lacking. This study aimed to assess the antiviral potential of garlic extract, a natural substance that inhibits HSVds in grapevine plants after micropropagation. Garlic extract was diluted 1,000-fold and applied to grapevine plants, and its effect on HSVd accumulation was evaluated using reverse transcription polymerase chain reaction and digital PCR. The results showed that HSVd accumulation was significantly reduced, with an inhibition rate of 74.45%; meanwhile, higher garlic extract concentrations resulted in contamination and plant damage. Nanopore sequencing confirmed that the integrity of the HSVd genome was compromised after treatment. Furthermore, garlic extract inhibited the HSVd and promoted plant growth by enhancing shoot and root development. Additionally, inhibition of the HSVd was sustained in regenerated grapevine plants. Moreover, the garlic extract showed inhibitory effects against HSVds in natural host cucumber plants. These results suggest that garlic extract could be a cost-effective and sustainable alternative for viroid control in grapevine cultivation, providing long-term protection and broader antiviral activity across plant species.

In total, 80.1 million tons of grapevines were produced in 2022 worldwide (OIV, 2022). In Korea, grapevines are one of the six major fruits, with a total cultivation area of 14,655 ha, yielding 188,771 tons (Statistics Korea, 2022). Most grapes produced are consumed as fresh fruit or used in wine, but they can also be processed into raisins, juice, and seed oils (Campos et al., 2021). Alternatively, some grape varieties are grown for ornamental purposes and used in food, cosmetics, and pharmaceuticals (Abiri et al., 2020).

Grapevine varieties are affected by biotic stresses, such as bacteria, viruses, and insects, as well as abiotic stresses, such as extreme temperatures and drought. These stresses reduce the quality and lifespan of grapevines, leading to significant economic losses in the grape and wine industries (Butiuc-Keul and Coste, 2023). Viruses and virus-like organisms can alter the chemical and sensory quality of grapes and wine and cause developmental and morphological abnormalities in grape organs, leading to increased and severe economic losses (Sudarshana et al., 2015). A total of 86 viruses have been isolated from grapes, which have been noted to cause economic losses by impairing fruit and plant growth (Fuchs, 2020).

The hop stunt viroid (HSVd), which belongs to the genus Hostuviroid and the family Pospiviroidae, is prevalent in most grapevines worldwide (Di Serio et al., 2014; Kappagantu et al., 2017). In a 2011 study of grape samples in Korea, the HSVd had the highest infection rate among four viruses and two viroids tested, with 40% of the samples found to be infected (Kim et al., 2011). The HSVd is known to infect several plant species, including cucumber (Cucumis sativus L.), grapevine (Vitis), citrus (Citrus), plum (Prunus domestica), pomegranate (Punica granatum), peach (Prunus persica), apricot (Prunus armeniaca), jujube (Ziziphus jujuba), and almond (Prunus dulcis) (Jo et al., 2017; Zhang et al., 2009). The symptoms of HSVd infection include various growth-retarding effects, such as stunting, yellowing, and loss of vigor (Zhang et al., 2020). Notably, when grapevines infected with the HSVd were also co-infected with the grapevine fleck virus (GFkV) and grapevine leafroll-associated virus 3 (GLRaV-3), symptoms including leaf mosaic, severe deformities, and poor fruit quality, alongside a 30% reduction in yield were observed (Rural Development Administration, 2018).

Grapevines are propagated through methods such as grafting rootstock and scion, in vitro propagation for mass production, and using dormant hardwood cutting from winter canes (Thomas and Schiefelbein, 2004; Torregrosa et al., 2001; Waite et al., 2015). While these methods facilitate easy reproduction, they also increase the risk of rapid virus and viroid spread. Indeed, the HSVd is primarily transmitted through grafting using infected scions but can also be spread mechanically through workers, tools, and equipment (Hadidi et al., 2022; Rural Development Administration, 2018). Subsequently, it is essential to use healthy propagation materials in viticulture since controlling these infections is challenging because they can easily spread from infected to healthy vines, causing significant damage alongside other viruses (Bota et al., 2014; Hadidi et al., 2022).

Various methods, such as thermotherapy, meristem tip culture, micrografting, chemotherapy, cryotherapy, and electrotherapy, alongside a combination of techniques, are employed to produce virus and viroid-free grapevine materials (Bi et al., 2018; Diab et al., 2011; Guţǎ et al., 2019; Hu et al., 2020; Lazo-Javalera et al., 2016; Panattoni and Triolo, 2010; Panattoni et al., 2007, 2011; Skiada et al., 2013; Turcsan et al., 2020). Thermotherapy, which involves exposing plants to high temperatures for a specific duration, inhibits viral replication and degrades viral RNA (Křižan et al., 2009; Panattoni and Triolo, 2010). Meristem tip culture can eliminate viral pathogens present in the parent plant while maintaining genetic stability, and micrografting of in vitro meristems or shoot tips enables rapid regeneration of plants from meristems (Diab et al., 2011; Lazo-Javalera et al., 2016; Turcsan et al., 2020). Chemotherapy employs antiviral compounds such as ribavirin and tiazofurin to eliminate the viruses or inhibit their replication (Panattoni et al., 2011; Skiada et al., 2013). However, thermotherapy can induce heat stress, and these methods often have low efficacy for viruses that are difficult to remove, may be toxic to plants, and can be time-consuming. In addition, meristem tip culture is potentially ineffective against certain viruses, particularly viruses that perform systemic or latent infections and those with a low regeneration rate (Gambino et al., 2006; Hu et al., 2020; Panattoni et al., 2007; Skiada et al., 2013).

Garlic is an annual bulb plant of the Alliaceae family, which is native to Central and South Asia and typically grown in dry and hot climates (Rouf et al., 2020). Widely recognized as a functional food and seasoning herb, garlic is known globally for its powerful preventive and therapeutic properties (Adetumbi and Lau, 1983; Tsai et al., 1985). Further, garlic is commonly used to treat colds, fevers, coughs, and asthma and has demonstrated antiviral activity against animal viruses such as adenovirus, coronavirus, dengue, and influenza A (Chen et al., 2011; Hall et al., 2017; Khanal et al., 2018; Rasool et al., 2017; Taghavi et al., 2023). Antiviral effects of garlic have also been reported against viruses, including potato virus Y (PVY) and grapevine leafroll-associated virus-2 (GLRaV-2) (Wang et al., 2020). Organosulfur compounds in garlic are the biologically active components responsible for its pungent odor. Garlic contains more than 30 types of sulfur-containing compounds, with alliin (S-allyl-L-cysteine sulfoxide) noted as the most abundant (Chung, 2006). The proposed mechanisms for antiviral activity against various viruses include inhibiting the viral life cycle, enhancing the immune response of the host, and reducing cellular oxidative stress (Adetumbi and Lau, 1983; El-Saber Batiha et al., 2020; Kyo et al., 2001; Tsai et al., 1985; Yi and Su, 2013).

This study explores the potential use of garlic extract as a cost-effective and environmentally friendly strategy to inhibit HSVd, reducing the viroid accumulation in infected grapevine plantlets and promoting plant growth. The results of this study could provide a practical approach to viroid control, benefiting grapevine cultivation and potentially other crops affected by viroid infections.

Materials and Methods

Sample preparation and RNA extraction

Micropropagated grapevine (Vitis vinifera cv. ‘188.08’) plantlets provided by the National Institute of Horticultural Science, Korea, were cultured in vitro on a medium supplemented with activated charcoal. Vitis vinifera cv. ‘188.08’ is commonly used in virus inhibition experiments involving grapevine tissue cultures due to its high susceptibility to various grapevine viruses, making it an ideal model for studying virus-host interaction and evaluating antiviral treatments (Wang et al., 2003). The medium comprised 2.2 g/l Linsmaier & Skoog medium, 2 g/l activated charcoal, 10 g/l sucrose, and 9 g/l plant agar and was adjusted to a pH of 5.8 before being autoclaved at 121°C for 15 min. The cultured grapevines were grown in a growth chamber under a 16/8 h light/dark photoperiod, with a light intensity of 2,000 1ux. Total RNA was extracted from the leaves of the micropropagated plantlets grown in vitro using the Super Plant RNA Extraction kit (In VirusTech Co., Gwangju, Korea) following the manufacturer’s protocol. The quality and quantity of the extracted RNA samples were assessed using a spectrophotometer (BioDrop, Biochrom Ltd., Cambridge, UK).

Nanopore library preparation and sequencing

A library was constructed using the quantified RNA with the Direct cDNA Sequencing kit (SQK-LSK114), following a previously described protocol (Lee et al., 2022). In brief, host ribosomal RNA (rRNA) was removed using the QIAseq FastSelect-rRNA Plant kit (Qiagen, Hilden, Germany), and cDNA was synthesized using the Maxima H Minus Double-Stranded cDNA Synthesized kit (Thermo Fisher Scientific, Waltham, MA, USA). End-repair and dA-tailing were performed on the double-stranded cDNA using the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs, Ipswich, MA, USA). Adapter ligation was constructed using the Ligation Sequencing kit V14 (SQK-LSK114, Oxford Nanopore Technologies, Oxford, UK). The nanopore library was purified using the AMPure XP Reagent (Beckman Coulter, Brea, CA, USA) and was loaded into a Flongle flow cell (R10.4.1) on the MinION device (Oxford Nanopore Technologies). The flow cell was analyzed for 24 h using the MinKNOW software (version 21.11.7), and the reads were compared to NCBI Viral Genome data using a BLASTn search. Q10 MinION reads were aligned, and consensus sequences were generated using medium sensitivity settings in the ‘Map to Reference’ function of Geneious Prime.

HSVd detection using reverse transcription polymerase chain reaction

The presence of plant viruses was confirmed through reverse transcription polymerase chain reaction (RT-PCR) using the SuPrimeScript RT-PCR premix (Genet Bio, Daejeon, Korea) according to the manufacturer’s instructions. The HSVd genome sequence was obtained from nanopore sequencing, and primers were designed accordingly using Prime 5.0 software. The primers were synthesized by Bionics (Daejeon, Korea), and the primer sequences used are listed in Supplementary Table 1. The RT-PCR reaction mixture was prepared to a final volume of 20 μl:10 μl of SuPrimerScript RT Premix (Genet Bio), 2 μl of RNA template, 2 μl of each of the 10 μM forward and reverse primers, and 6 μl of DEPC-treated water. The amplification protocol included a reverse transcription step at 50°C for 30 min, an initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 56°C for 60 s, and 72°C for 30 s, with a final extension at 72°C for 10 min. To compare the HSVd concentrations at 0 days post-treatment (dpt) and after 28 days post garlic extract treatment, the grapevine RNA underwent 25 cycles of RT-PCR. The subsequent RT-PCR products were stained using RedSafe nucleic acid staining solution (iNtRON Biotechnology Inc., Seongnam, Korea), electrophoresed on 1.5% agarose gels, and visualized under UV light.

Garlic extract and treatment

The crude garlic extract was prepared by dissolving 5 g of dry garlic powder in 1 liter of acetic acid solution (acidity 4.5%), diluted 1,000-fold using DEPC-treated water, and applied to treat the grape plantlets following micropropagation (Wang et al., 2020). The diluted garlic extract was filter-sterilized using a 0.22 μm filter, and 1 ml was administered per plantlet by spraying. All treatments were carried out on a clean bench under sterile conditions. Grapevine plantlets were sprayed at 7-day intervals, and samples were collected at 28 dpt. RT-PCR was performed using specific primers to compare HSVd accumulations before and after treatment.

Digital PCR analysis of the inhibitory effect of garlic extract on the HSVd in micropropagated grapevine plantlets

Micropropagated grapevine plantlets treated with garlic extract were sampled, and RT-PCR confirmed the inhibition effect on HSVd. Absolute quantification of HSVd was performed using nanoplate-based digital PCR (dPCR) with RNA extracted from grapevine plantlets. Primers and a probe were designed using Primer 5.0 software, based on the HSVd sequence obtained from Nanopore sequencing and synthesized by Bionics. The forward and reverse primers and the probe are listed in Table 1. The total RNA quality was evaluated using a BioDrop spectrometer (Biochrom, Ltd.), and dPCR was performed using a QIAcuity One 2-plex digital PCR system (Qiagen). The reaction mixture was prepared according to the manufacturer’s instructions: 10 μl of 4× concentrated QIAcuity Probe Master mix (Qiagen), 900 nM forward and reverse primers and 250 nM probe, and RNA template, to a final volume of 40 μl. The prepared reaction mixture was transferred to QIA cuity26k 24-well Nanoplates (Qiagen) and aliquoted using the Qiagen standard priming profile. The dPCR protocol included enzyme activation at 95°C for 2 min, followed by denaturation at 95°C for 15 s and annealing/extension at 60°C for 30 s for 45 cycles. Images were acquired using an exposure time of 500 ms (FAM channel), and segmentation data were analyzed using QIAcuity Suite Software V2.0.20.

Evaluation of the inhibitory effect of garlic extract on HSVd infection in cucumber plants

Cucumbers were grown in pots in a growth chamber under controlled conditions: a constant temperature of 24°C and a 14/10 h (light/dark) photoperiod. One-week-old seedlings with two fully expanded cotyledons were used to examine the inhibitory effect of garlic extract on the HSVd. The same concentration of garlic extract used for treating the micropropagated grapevines was mixed with the HSVd inoculum obtained from the infected cucumber plant leaves in a 1:1 volume ratio and incubated for 1 min. At 28 day post-inoculation (dpi), the growth of untreated plants, plants treated only with HSVd inoculum, and plants treated with HSVd inoculum incubated with garlic extract were compared. HSVd presence was confirmed using RT-PCR.

Results

HSVd identification by nanopore sequencing in grapevine plantlets grown in vitro

Oxford nanopore sequencing was performed on a library of micropropagated grapevine plantlets grown in vitro (Fig. 1). A total of 336,727 raw reads were obtained, with an average read length of 330.9 bp, a maximum read length of 7,438 bp, and a minimum read length of 66 bp (Supplementary Table 2). The WIMP workflow identified the HSVd from the sequenced reads. Genome assembly was performed using the HSVd reference genome (NC001351) and presented 100% coverage and 73.7% identity (Fig. 1B). Nanopore sequencing identified only HSVd in the in vitro grapevine plantlets, with no evidence of other major grapevine viruses based on genome assembly. Next, RT-PCR was performed using the HSVd gene-specific primer set to confirm the presence of the HSVd. The expected amplicon size was confirmed by RT-PCR (Fig. 1C). The amplicon was then sequenced, validated by Sanger sequencing, and deposited in GenBank (LC844705).

Fig. 1

Detection of hop stunt viroid (HSVd) infection in grapevine plantlets in vitro using nanopore sequencing. (A) Grapevine plantlets grown in vitro. (B) HSVd reads mapped to the HSVd reference genome. (C) Reverse transcription polymerase chain reaction validation using HSVd-specific diagnostic primers.

Optimization of the garlic extract treatment concentration

To determine the optimal concentration of the garlic extract required to inhibit the HSVd in micropropagated grapevine plantlets, garlic extract was dissolved in acetic acid solution and diluted 10-fold, 100-fold, and 1,000-fold using DEPC-treated water. Each garlic extract concentration was sprayed on the grapevine plantlets at 7-day intervals. RT-PCR was performed 28 dpt using specific primer sets (Supplementary Table 1). The accumulation of HSVd was not reduced at 28 dpt on the grapevine plantlets administered the 10-fold and 100-fold garlic extract dilutions. However, the accumulation of HSVd decreased dramatically after treatment with the 1,000-fold garlic extract dilution compared to the pretreatment load (Fig. 2A). Additionally, plant damage was observed on the plantlets administered the 10-fold and 100-fold treatments owing to the medium becoming too easily contaminated (Fig. 2B). Therefore, the 1,000-fold garlic extract concentration was selected for further experiments on HSVd inhibition.

Fig. 2

Optimization of garlic extract concentration for hop stunt viroid (HSVd) inhibition. (A) Reverse transcription polymerase chain reaction analysis confirmed the inhibitory effect of garlic extract diluted 10-, 100-, and 1,000-fold. At 0 days post-treatment (dpt): plants before garlic extract treatment; 28 dpt: plants at 28 dpt with garlic extract. (B) Grapevine plantlets were treated with 10-fold, 100-fold, and 1,000-fold dilutions of garlic extract.

Effect of garlic extract on the growth of grapevine plantlets in vitro

After treating micropropagated grapevine plantlets with garlic extract, the lengths of the shoots and roots were measured. The plantlets treated with garlic extract exhibited similar growth rates to the control plantlets treated with DEPC-treated water. However, there was a notable difference in the average shoot height and root length between the two groups (Fig. 3A). The average shoot length of the plantlets administered garlic extract was 5 cm longer, and the average root length was 4 cm longer compared to the control sample (Fig. 3B, Supplementary Table 3). These results indicate a significant increase in the growth of shoots and roots in the treated plants compared to control plants, which suggests that garlic extract promotes shoot and root elongation in grapevine plantlets grown in vitro.

Fig. 3

Comparison of the growth of grapevine plantlets treated with garlic extract and DEPC. (A) Growth of grapevine plantlets. Distilled water: plantlets treated with DEPC; garlic extract: plantlets treated with garlic extract. (B) Comparison of shoot and root lengths. *P < 0.05, **P < 0.01.

Inhibition effects of garlic extract on HSVd in grapevine plantlets grown in vitro

To investigate whether garlic extract inhibits HSVd, grapevine plantlets were treated with 1,000-fold-diluted garlic extract under in vitro conditions (Fig. 4A). The inhibition effect was confirmed by treating 99 plantlets with a 1,000-fold dilution garlic extract, which demonstrated an inhibitory effect on HSVd. RT-PCR was performed on plantlets treated with 1,000-fold garlic extract for 28 days to determine the presence of HSVd. Gel electrophoresis confirmed a decrease in HSVd accumulation after treatment with garlic extract (Fig. 4B). In total, 74 of the 99 plantlets showed reduced HSVd accumulation compared to the day 0 levels, presenting an inhibition rate of 74.45% (Supplementary Table 4). No toxicity was observed relating to plant growth and proliferation in the plantlets treated for 28 days, and no contamination was detected (Fig. 4A). Since no HSVd symptoms were evident in the plants, visual symptoms could not be compared with the control plants.

Fig. 4

Inhibitory effect of 1,000-fold diluted garlic extract on hop stunt viroid (HSVd). (A) Grapevine plantlets were treated with the diluted garlic extract. (B) Reverse transcription polymerase chain reaction results following treatment with the garlic extract. dpt, days post-treatment.

Quantification of HSVd by dPCR

Following the treatment of micropropagated grapevine plantlets with garlic extract for 28 days, the inhibitory effect on HSVd was evaluated through absolute quantification of HSVd levels using dPCR. HSVd was quantified in 33 plantlets, and RT-PCR confirmed a reduction in HSVd accumulation. The dPCR results revealed that HSVd accumulation was decreased by between 41.4% and 100% in all 33 garlic extract-treated grapevine plantlets compared to 0 dpt (Fig. 5). Of the 30 plantlets, three exhibited a 70% reduction in HSVd accumulation, while 11 plantlets showed a decrease between 71% and 90%. Furthermore, HSVd accumulation was reduced by more than 90% in 19 plantlets (42.2%) (Supplementary Table 5).

Fig. 5

Verification of the inhibitory effect of garlic extract using digital PCR (dPCR). (A) Grapevine plantlets treated with garlic extract. (B) Quantification of hop stunt viroid (HSVd) in garlic extract-treated grapevine plantlets using dPCR. dpt, days post-treatment.

Comparison of HSVd reads obtained after garlic extract treatment using nanopore sequencing

Nanopore sequencing was performed on libraries prepared from grapevine plantlets infected with HSVd and treated with garlic extract for 28 days. The libraries were generated from RNA samples (28 dpt) that had previously demonstrated a 100% reduction in HSVd accumulation and RNA sample (0 dpt), as confirmed by RT-PCR. In total, an equal amount of cDNA (24.0 ng) was sequenced for each plantlet by nanopore sequencing.

The nanopore sequencing reads from both 0 dpt and 28 dpt were mapped to the reference HSVd genome (NC001351) (Fig. 6). At 0 dpt, the sequencing results yielded 3,024 reads, with 100% query coverage and a nucleotide identity of 73.2%. In contrast, the results at 28 dpt showed a dramatic reduction in viroid-associated reads, with only 233 reads mapped to the HSVd reference genome, and a significant drop in nucleotide identity to 37.4 and 100% query coverage (Fig. 6A and B). This decrease in the number of reads and the nucleotide similarity suggests that garlic extract substantially alters the genomic integrity of HSVd. The sequencing results strongly indicate that garlic extract reduces the overall load of HSVd and affects the genomic structure of the viroid at the nucleotide level.

Fig. 6

Detection of hop stunt viroid (HSVd) before and after garlic extract treatment using nanopore sequencing. (A) Mapping of HSVd reads to the reference genome. (B) The number of HSVd reads was mapped before and after garlic treatment. dpt, days post-treatment.

Validation of the inhibitory effect of garlic extract on HSVd in cucumber plants

To determine whether garlic extract directly inhibits HSVd rather than inducing a defense response in plants, cucumber plants, the natural host of HSVd, were mechanically inoculated with a 1:1 (v:v) mixture of HSVd-infected grapevine sap and garlic extract, as well as with HSVd alone. Disease symptoms were subsequently examined (Fig. 7). When HSVd was inoculated into cucumber plants, no visible symptoms were observed on the systemic leaves; however, the plants exhibited stunted growth at 28 dpi (Supplementary Fig. 1). At 28 dpi, cucumber plants treated with the HSVd-garlic extract mixture showed growth rates similar to healthy plants, with no leaf symptoms. In contrast, cucumber plants inoculated with HSVd alone exhibited significantly reduced growth rates (Fig. 7A). These experiments were performed twice independently with three replicates and consistently yielded the same results. Therefore, these results suggest that garlic extract has an antiviral effect on the HSVd. To confirm this, HSVd accumulation was examined using RT-PCR. The RT-PCR results showed that the HSVd was detected only in samples inoculated with HSVd alone, while no HSVd was found in the cucumber plants treated with garlic extract and HSVd mixture (Fig. 7B).

Fig. 7

Inhibition effect of garlic extract against hop stunt viroid (HSVd) in pot-grown cucumber plants. (A) Comparison of cucumber plant growth and symptoms at 25 days post-inoculation. Healthy: untreated plants; garlic extract: plants inoculated with HSVd treated with garlic extract; Positive: plants inoculated with HSVd only. (B) Detection of HSVd in cucumber plants using reverse transcription polymerase chain reaction.

The efficiency of HSVd inhibition in regenerated grapevine plantlets in vitro

Nine grapevine plantlets, in which HSVd inhibition had been confirmed by RT-PCR and dPCR analyses, were randomly selected. The shoot tips of these plantlets were excised and cultured under normal conditions (Fig. 8C). Subsequently, dPCR also confirmed the continuous inhibition of HSVd in the first generation of regenerated plants, and inhibition was again further verified after four rounds of regenerated plants using RT-PCR. The absence of HSVd amplification in the gel electrophoresis analysis was considered evidence of the sustained inhibitory effect of garlic extract. Notably, no HSVd amplification was detected in the agarose gel analysis across all grapevine plantlets regenerated up to the fourth generation (Fig. 8A). Next, dPCR analysis was employed to quantify the HSVd inhibition further. In the control plantlets treated with DEPC, HSVd was detected at a concentration of approximately 400 copies/μl. Comparatively, in plantlets treated with garlic extract, HSVd levels either decreased or remained at 0 copies/μl as the generations progressed (Fig. 8B, Supplementary Table 6). These results demonstrate that the inhibitory effect of garlic extract on HSVd was maintained under normal conditions without additional treatment.

Fig. 8

Sustained hop stunt viroid (HSVd) inhibition was confirmed in grapevine plantlets treated with garlic extract across four generations of regenerated plants. (A) Detection of HSVd by reverse transcription polymerase chain reaction. At 0 days post-treatment (dpt): plants before garlic extract treatment; 2nd generation: second regeneration of grapevine plantlets with confirmed HSVd inhibition; 4th generation: fourth regeneration of grapevine plantlets with confirmed HSVd inhibition. (B) Quantification of HSVd by digital PCR. (C) Fourth-generation regenerated plants showing sustained HSVd inhibition following garlic extract treatment.

Discussion

The production of viroid-free grapevine plantlets is critical for maintaining healthy vineyards and ensuring high-quality fruit production. Viroids, including HSVd, are notorious pathogens in grapevines, leading to reduced plant vigor and yields and poor fruit quality (Gambino et al., 2014). Therefore, establishing viroid-free plantlets is essential for mitigating the spread of these pathogens and ensuring long-term vineyard sustainability. Indeed, several methods have been employed previously to eliminate viruses and viroids from infected plant materials, including thermotherapy, cryotherapy, and meristem cultures (Barba et al., 2017). However, these techniques are often labor-intensive and time-consuming, have low efficacy of elimination rate, and may result in suboptimal plant regeneration. Additionally, viroid re-infection is a constant threat due to environmental transmission routes, such as mechanical tools and vectors, thereby further complicating disease management.

Given these challenges, there is a pressing need for alternative, effective, and practical approaches applicable to routine use. Natural antiviral compounds, such as those found in garlic, offer a promising solution for viroid inhibition. Garlic extract, known for its broad-spectrum antimicrobial properties, has shown potential as an antiviral agent in various plant and human pathogens (Curtis et al., 2004; Ishikawa et al., 2006; Shojai et al., 2016). However, the application of garlic extract in viroid management, particularly for HSVd in grapevine plantlets, remains largely unexplored. This study aimed to address this gap by evaluating the inhibitory effects of garlic extract on HSVd accumulation in micropropagated grapevine plantlets to identify a natural, cost-effective alternative to conventional viroid elimination techniques.

The current study investigated the inhibitory effect of garlic extract on HSVd accumulation in micropropagated grapevine plantlets, highlighting its potential as a natural antiviral agent. The results demonstrated that a 1,000-fold dilution of garlic extract significantly reduced HSVd accumulation, while higher garlic extract concentrations led to contamination and plant damage. The greater efficacy of HSVd reduction at a 1,000-fold dilution compared to higher concentrations of garlic extract could be attributed to several factors. At this dilution, the active compounds may achieve an optimal balance between bioavailability and reduced phytotoxicity, allowing effective inhibition of HSVd without harming the plant tissues. Additionally, lower concentration might minimize contamination risk observed at higher concentrations, contributing consistent antiviroid effects. Furthermore, the high concentration of sulfur compounds in the 10-fold or 100-fold garlic extract may lead to nutrient imbalance within the plant. This could inhibit plant growth or weaken the HSVd inhibition mechanism by disrupting essential metabolic pathways (Inamori et al., 1992). These findings suggest that dilution plays a critical role in modulating the efficacy of garlic extract, and further research is needed to elucidate the mechanisms underlying this concentration-dependent manner. This indicates that careful optimization of the garlic extract concentration is crucial for achieving antiviral efficacy and plant health.

The observed reduction in HSVd accumulation, confirmed through RT-PCR and dPCR, suggests that garlic extract directly inhibits HSVd replication. A 74.45% inhibition rate in treated plantlets further indicated that the treatment was largely effective, with some variability across individual samples. This variability might be attributed to physiological differences among the plantlets or the uneven distribution of the extract during application. Furthermore, nanopore sequencing provided strong evidence of disruption in the integrity of the HSVd genome following garlic extract treatment. This disruption is likely due to the antiviral compounds in garlic, such as alliin, which have been reported to interfere with nucleic acid replication in other viral pathogens (Omar and Al-Wabel, 2010).

Interestingly, the garlic extract inhibited the HSVd and promoted plant growth, as evidenced by a significant increase in shoot and root lengths compared to control plants. This dual effect of garlic extract, both antiviral and grow-promoting, suggests its potential as a multi-functional treatment in grapevine cultivation, particularly under in vitro conditions, where maintaining plant health and controlling pathogens are essential.

The validation of the inhibitory effect of garlic extract on the HSVd in cucumber plants, a natural host of the HSVd, further supports the broad-spectrum antiviral properties of garlic extract. Interestingly, the complete inhibition of HSVds in garlic-treated cucumber plants suggests that garlic extract has a strong viroid-inhibiting activity across different plant species, raising the possibility of its application beyond grapevines.

A key aspect of understanding the antiviral properties of garlic extract lies in exploring its underlying mechanism of action against HSVd. Garlic extract contains a variety of bioactive compounds (sulfur-containing compounds) such as alliin, allicin, ajoene, S-allyl cysteine, S-allyl mercaptocysteine, diallyl sulfide, and diallyl disulfide, many of which work synergistically to provide wide-ranging antimicrobial and antiviral effects, making it a potent natural remedy for various pathogens (Rouf et al., 2020). These sulfur-containing compounds are known to interfere with the cellular redox balance, disrupting various biochemical pathways essential for pathogen replication (Weber et al., 1992). In the context of HSVd, it is plausible that these compounds directly inhibit the replication or assembly of HSVd. Unlike typical viruses, viroids lack a protective coat protein and rely entirely on the machinery of the host plant for replication. This makes viroids particularly susceptible if the replication processes in the host cell are disrupted. The sulfur-containing compound’s activity to generate reactive oxygen species may contribute to oxidative stress within the cells of the host plant, thereby damaging the RNA genome of the HSVd and impairing its ability to replicate (Iciek et al., 2009). This oxidative environment could also later affect the structural integrity of the RNA genome, as the nanopore sequencing results demonstrated a marked reduction in the post-treatment genome integrity and nucleotide identity of the HSVd. Allicin, one of the major sulfur compounds in garlic, inhibits replication by binding to and modifying cysteine residues in proteins, which are crucial for pathogen activity (Borlinghaus et al., 2014). Additionally, reactive oxygen species generated by sulfur compounds can directly damage the genomes of pathogens by modifying guanine, which is highly susceptible to oxidative stress (Dvořáková et al., 2015). Allicin and ajoene are also known to bind directly to pathogen genomes, chemically altering nucleic acid bases and destroying the secondary structure of RNA, thereby preventing the pathogen from functioning properly (Ariga and Seki, 2006).

While many studies on the antiviral properties of garlic extract in plants focus on its ability to stimulate defense mechanisms, such as the induction of pathogenesis-related (PR) genes, such as PR-1, our results suggest a different primary mode of action of HSVd inhibition in micropropagated grapevine plantlets. In previous studies, garlic extract has been shown to elicit systemic acquired resistance in plants by upregulating defense-related genes and enhancing antioxidant enzyme activities, thereby inhibiting the replication of grapevine viruses (Zhang et al., 2020). Our key observation comes from experiments where a mixture of garlic extract and HSVd-infected grapevine sap was mechanically inoculated into cucumber plants, a natural host of HSVd.

Notably, even after a single treatment with garlic extract, the inhibitory effect on HSVd persisted across four generations of regenerated grapevine plantlets without additional treatments. The consistent absence of HSVd in these plantlets confirmed the long-term efficacy of garlic extract. The sustained inhibition of HSVd across multiple generations suggests that garlic extract may act directly on the HSVd rather than merely triggering a defense response in the host plant. This indicates that the antiviral effect of garlic extract is not transient but can provide prolonged protection against HSVd, potentially reducing the need for frequent treatments.

In conclusion, although garlic extract may induce a moderate defense response in grapevine plantlets, the primary antiviral effect against HSVd seems to result from direct interaction with the viroid RNA, likely mediated by bioactive sulfur compounds. Further studies involving detailed molecular analysis, such as single sulfur-compound-viroid binding assays or oxidative damage assays, will be necessary to elucidate this mechanism fully. Nevertheless, our findings highlight that garlic extract can be used as a potent and practical inhibitor of HSVd, offering a promising alternative to traditional viroid control methods in grapevines and potentially other crops. Moreover, using natural products such as garlic extract for plant disease management has implications for sustainable agriculture and offers a low-cost and eco-friendly solution to the ongoing challenge of viroid infections.

Notes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Electronic Supplementary Material

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

References

Abiri K., Rezaei M., Tahanian H., Heidari P., Khadivi A.. 2020;Morphological and pomological variability of a grape (Vitis vinifera L.) germplasm collection. Sci. Hortic. 266:109285.
Adetumbi M. A., Lau B. H. S.. 1983;Alliumsativum (garlic): a natural antibiotic. Med. Hypotheses 12:227–237.
Ariga T., Seki T.. 2006;Antithrombotic and anticancer effects of garlic-derived sulfur compounds: a review. Biofactors 26:93–103.
Barba M., Hosakawa M., Wang Q.-C., Taglienti A., Zhang Z.. 2017. Viroid elimination by thermotherapy, cold therapy, tissue culture, in vitro micrografting, or cryotherapy. Viroids and satellites In : Hadidi A., Flores R., Randles J. W., Palukaitis P., eds. p. 425–435. Elsevier. Oxford, UK:
Bi W.-L., Hao X.-Y., Cui Z.-H., Pathirana R., Volk G. M., Wang Q.-C.. 2018;Shoot tip cryotherapy for efficient eradication of grapevine leafroll-associated virus-3 from diseased grapevine in vitro plants. Ann. Appl. Biol. 173:261–270.
Borlinghaus J., Albrecht F., Gruhlke M. C. H., Nwachukwu I. D., Slusarenko A. J.. 2014;Allicin: chemistry and biological properties. Molecules 19:12591–12618.
Bota J., Cretazzo E., Montero R., Rosselló J., Cifre J.. 2014;Grapevine fleck virus (GFkV) elimination in a selected clone of Vitis vinifera L. cv. Manto Negro and its effects on photosynthesis. OENO One 48:11–19.
Butiuc-Keul A., Coste A.. 2023;Biotechnologies and strategies for grapevine improvement. Horticulturae 9:62.
Campos G., Chialva C., Miras S., Lijavetzky D.. 2021;New technologies and strategies for grapevine breeding through genetic transformation. Front. Plant Sci. 12:767522.
Chen C.-H., Chou T.-W., Cheng L.-H., Ho C.-W.. 2011; In vitro anti-adenoviral activity of five Allium plants. J. Taiwan Inst. Chem. Eng. 42:228–232.
Chung L. Y.. 2006;The antioxidant properties of garlic compounds: allyl cysteine, alliin, allicin, and allyl disulfide. J. Med. Food 9:205–213.
Curtis H., Noll U., Störmann J., Slusarenko A. J.. 2004;Broad-spectrum activity of the volatile phytoanticipin allicin in extracts of garlic (Allium sativum L.) against plant pathogenic bacteria, fungi and oomycetes. Physiol. Mol. Plant Pathol. 65:79–89.
Di Serio F., Flores R., Verhoeven J. T. J., Li S.-F., Pallás V., Randles J. W., Sano T., Vidalakis G., Owens R. A.. 2014;Current status of viroid taxonomy. Arch. Virol. 159:3467–3478.
Diab A. A., Khalil S. M., Ismail R. M.. 2011;Regeneration and micropropagation of grapevine (Vitis vinifera L.) through shoot tips and axillary buds. Int. J. Adv. Biotechnol. Res. 2:484–491.
Dvořáková M., Weingartová I., Nevoral J., Němeček D., Krejčová T.. 2015;Garlic sulfur compounds suppress cancerogenesis and oxidative stress: a review. Sci. Agric. Bohem. 46:65–72.
El-Saber Batiha G., Magdy Beshbishy A., Wasef L. G., Elewa Y. H. A., Al-Sagan A. A., Abd El-Hack M. E., Taha A. E., Abd-Elhakim Y. M., Prasad Devkota H.. 2020;Chemical constituents and pharmacological activities of garlic (Allium sativum L.): a review. Nutrients 12:872.
Fuchs M.. 2020;Grapevine viruses: a multitude of diverse species with simple but overall poorly adopted management solutions in the vineyard. J. Plant Pathol. 102:643–653.
Gambino G., Bondaz J., Gribaudo I.. 2006;Detection and elimination of viruses in callus, somatic embryos and regenerated plantlets of grapevine. Eur. J. Plant Pathol. 114:397–404.
Gambino G., Navarro B., Torchetti E. M., La Notte P., Schneider A., Mannini F., Di Serio F.. 2014;Survey on viroids infecting grapevine in italy: identification and characterization of Australian grapevine viroid and grapevine yellow speckle viroid 2. Eur. J. Plant Pathol. 140:199–205.
Guţǎ I.-C., Buciumeanu E.-C., Tǎtaru L. D., Oprescu B., Topalǎ C. M.. 2019;New approach of electrotherapy for grapevine virus elimination. Acta Hortic. 1242:697–701.
Hadidi A., Sun L., Randles J. W.. 2022;Modes of viroid transmission. Cells 11:719.
Hall A., Troupin A., Londono-Renteria B., Colpitts T. M.. 2017;Garlic organosulfur compounds reduce inflammation and oxidative stress during dengue virus infection. Viruses 9:159.
Hu G., Dong Y., Zhang Z., Fan X., Ren F.. 2020;Efficiency of chemotherapy combined with thermotherapy for eliminating grapevine leafroll-associated virus 3 (GLRaV-3). Sci. Hortic. 271:109462.
Iciek M., Kwiecień I., Włodek L.. 2009;Biological properties of garlic and garlic-derived organosulfur compounds. Environ. Mol. Mutagen. 50:247–265.
Inamori Y., Muro C., Tanaka R., Adachi A., Miyamoto K., Tsujibo H.. 1992;Phytogrowth-inhibitory activity of sulfur-containing compounds. I. Inhibitory activities of thiazolidine derivatives on plant growth. Chem. Pharm. Bull. 40:2854–2856.
Ishikawa H., Saeki T., Otani T., Suzuki T., Shimozuma K., Nishino H., Fukuda S., Morimoto K.. 2006;Aged garlic extract prevents a decline of NK cell number and activity in patients with advanced cancer. J. Nutr. 136(3 Suppl):816S–820S.
Jo Y., Chu H., Kim H., Cho J. K., Lian S., Choi H., Kim S.-M., Kim S.-L., Lee B. C., Cho W. K.. 2017;Comprehensive analysis of genomic variation of hop stunt viroid. Eur. J. Plant Pathol. 148:119–127.
Kappagantu M., Nelson M. E., Bullock J. M., Kenny S. T., Eastwell K. C.. 2017;Hop stunt viroid: effects on vegetative growth and yield of hop cultivars, and its distribution in central Washington State. Plant Dis. 101:607–612.
Khanal S., Ghimire P., Dhamoon A. S.. 2018;The repertoire of adenovirus in human disease: the innocuous to the deadly. Biomedicines 6:30.
Kim J. S., Lee S. H., Choi H. S., Kim M. K., Kwak H. R., Nam M., Kim J. S., Choi G. S., Cho J. D., Cho I. S., Chung B. N.. 2011;Occurrence of virus diseases on major crops in 2010. Res. Plant Dis. 17:334–341. (in Korean).
Křižan B., Ondrušiková E., Holleinová V., Moravcová K., Bláhová L.. 2009;Elimination of grapevine fanleaf virus in grapevine by in vivo and in vitro thermotherapy. Hortic. Sci. 36:105–108.
Kyo E., Uda N., Kasuga S., Itakura Y.. 2001;Immunomodulatory effects of aged garlic extract. J. Nutr. 131:1075S–1079S.
Lazo-Javalera M. F., Troncoso-Rojas R., Tiznado-Hernández M. E., Martínez-Tellez M. A., Vargas-Arispuro I., Islas-Osuna M. A., Rivera-Domínguez M.. 2016;Surface disinfection procedure and in vitro regeneration of grapevine (Vitis vinifera L.) axillary buds. SpringerPlus 5:453.
Lee H.-J., Cho I.-S., Jeong R.-D.. 2022;Nanopore metagenomics sequencing for rapid diagnosis and characterization of lily viruses. Plant Pathol. J. 38:503–512.
OIV. 2022. Grape production statistics. International Organisation of Vine and Wine URL https://www.oiv.int/what-we-do/data-discovery-report?oiv [28 October 2024].
Omar S. H., Al-Wabel N. A.. 2010;Organosulfur compounds and possible mechanism of garlic in cancer. Saudi Pharm. J. 18:51–58.
Panattoni A., D’Anna F., Cristani C., Triolo E.. 2007;Grapevine vitivirus A eradication in Vitis vinifera explants by antiviral drugs and thermotherapy. J. Virol. Methods 146:129–135.
Panattoni A., Luvisi A., Triolo E.. 2011;Selective chemotherapy on grapevine leafroll-associated virus-1 and -3. Phytoparasitica 39:503–508.
Panattoni A., Triolo E.. 2010;Susceptibility of grapevine viruses to thermotherapy on in vitro collection of Kober 5BB. Sci. Hortic. 125:63–67.
Rasool A., Khan M-U-R, Ali M. A., Anjum A. A., Ahmed I., Aslam A., Mustafa G., Masood S., Ali M. A., Nawaz M.. 2017;Anti-avian influenza virus H9N2 activity of aqueous extracts of Zingiber officinalis (ginger) and Allium sativum (garlic) in chick embryos. Pak. J. Pharm. Sci. 30:1341–1344.
Rouf R., Uddin S. J., Sarker D. K., Islam M. T., Ali E. S., Shilpi J. A., Nahar L., Tiralongo E., Sarker S. D.. 2020;Antiviral potential of garlic (Allium sativum) and its organosulfur compounds: a systematic update of pre-clinical and clinical data. Trends Food Sci. Technol. 104:219–234.
Rural Development Administration. 2018. Grape-agricultural technology guide 12 Rural Development Administration. Jeonju, Korea: p. 249.
Shojai T. M., Langeroudi A. G., Karimi V., Barin A., Sadri N.. 2016;The effect of Allium sativum (garlic) extract on infectious bronchitis virus in specific pathogen free embryonic egg. Avicenna J. Phytomed. 64:458–467.
Skiada F. G., Maliogka V. I., Katis N. I., Eleftheriou E. P.. 2013;Elimination of grapevine rupestris stem pitting-associated virus (GRSPaV) from two Vitis vinifera cultivars by in vitro chemotherapy. Eur. J. Plant Pathol. 135:407–414.
Statistics Korea. 2022. National statistics on agricultural production Korean Statistical Information Service; URL https://kosis.kr/eng/ [28 October 2024].
Sudarshana M. R., Perry K. L., Fuchs M. F.. 2015;Grapevine red blotch-associated virus, an emerging threat to the grapevine industry. Phytopathology 105:1026–1032.
Taghavi M. R., Tamanaei T. T., Oghazian M. B., Tavana E., Mollazadeh S., Niloofar P., Oghazian S., Hoseinzadeh A., Hesari A., Mohseni M. A., Rezaei S., Haresabadi M.. 2023;Effectiveness of fortified garlic extract oral capsules as adjuvant therapy in hospitalized patients with coronavirus disease 2019: a triple-blind randomized controlled clinical trial. Curr. Ther. Res. Clin. Exp. 98:100699.
Thomas P., Schiefelbein J. W.. 2004;Roles of leaf in regulation of root and shoot growth from single node softwood cuttings of grape (Vitis vinifera). Ann. Appl. Biol. 144:27–37.
Torregrosa L., Bouquet A., Goussard P. G.. 2001. In vitro culture and propagation of grapevine. Molecular biology and biotechnology of the grapevine In : Roubelakis-Angelakis K. A., ed. p. 281–326. Springer. Dordrecht, Netherlands:
Tsai Y., Cole L. L., Davis L. E., Lockwood S. J., Simmons V., Wild G. C.. 1985;Antiviral properties of garlic: in vitro effects on influenza B, herpes simplex and coxsackie viruses. Planta Med. 51:460–461.
Turcsan M., Demian E., Varga T., Jaksa-Czotter N., Szegedi E., Olah R., Varallyay E.. 2020;HTS-based monitoring of the efficiency of somatic embryogenesis and meristem cultures used for virus elimination in grapevine. Plants 9:1782.
Waite H., Whitelaw-Weckert M., Torley P.. 2015;Grapevine propagation: principles and methods for the production of high-quality grapevine planting material. N. Z. J. Crop Hortic. Sci. 43:144–161.
Wang Q., Mawassi M., Li P., Gafny R., Sela I., Tanne E.. 2003;Elimination of grapevine virus A (GVA) by cryopreservation of in vitro-grown shoot tips of Vitis vinifera L. Plant Sci. 165:321–327.
Wang X. Y., Zhang C. W., Huang W. T., Yue J., Dou J. J., Wang L. Y., Wang Q., Cheng Y. Q.. 2020;Crude garlic extract significantly inhibits replication of grapevine viruses. Plant Pathol. 69:149–158.
Weber N. D., Andersen D. O., North J. A., Murray B. K., Lawson L. D., Hughes B. G.. 1992; In vitro virucidal effects of Allium sativum (garlic) extract and compounds. Planta Med. 58:417–423.
Yi L., Su Q.. 2013;Molecular mechanisms for the anti-cancer effects of diallyl disulfide. Food Chem. Toxicol. 57:362–370.
Zhang B., Liu G., Liu C., Wu Z., Jiang D., Li S.. 2009;Characterisation of hop stunt viroid (HSVd) isolates from jujube trees (Ziziphus jujuba). Eur. J. Plant Pathol. 125:665–669.
Zhang Z., Xia C., Matsuda T., Taneda A., Murosaki F., Hou W., Owens R. A., Li S., Sano T.. 2020;Effects of host-adaptive mutations on hop stunt viroid pathogenicity and small RNA biogenesis. Int. J. Mol. Sci. 21:7383.

Article information Continued

Fig. 1

Detection of hop stunt viroid (HSVd) infection in grapevine plantlets in vitro using nanopore sequencing. (A) Grapevine plantlets grown in vitro. (B) HSVd reads mapped to the HSVd reference genome. (C) Reverse transcription polymerase chain reaction validation using HSVd-specific diagnostic primers.

Fig. 2

Optimization of garlic extract concentration for hop stunt viroid (HSVd) inhibition. (A) Reverse transcription polymerase chain reaction analysis confirmed the inhibitory effect of garlic extract diluted 10-, 100-, and 1,000-fold. At 0 days post-treatment (dpt): plants before garlic extract treatment; 28 dpt: plants at 28 dpt with garlic extract. (B) Grapevine plantlets were treated with 10-fold, 100-fold, and 1,000-fold dilutions of garlic extract.

Fig. 3

Comparison of the growth of grapevine plantlets treated with garlic extract and DEPC. (A) Growth of grapevine plantlets. Distilled water: plantlets treated with DEPC; garlic extract: plantlets treated with garlic extract. (B) Comparison of shoot and root lengths. *P < 0.05, **P < 0.01.

Fig. 4

Inhibitory effect of 1,000-fold diluted garlic extract on hop stunt viroid (HSVd). (A) Grapevine plantlets were treated with the diluted garlic extract. (B) Reverse transcription polymerase chain reaction results following treatment with the garlic extract. dpt, days post-treatment.

Fig. 5

Verification of the inhibitory effect of garlic extract using digital PCR (dPCR). (A) Grapevine plantlets treated with garlic extract. (B) Quantification of hop stunt viroid (HSVd) in garlic extract-treated grapevine plantlets using dPCR. dpt, days post-treatment.

Fig. 6

Detection of hop stunt viroid (HSVd) before and after garlic extract treatment using nanopore sequencing. (A) Mapping of HSVd reads to the reference genome. (B) The number of HSVd reads was mapped before and after garlic treatment. dpt, days post-treatment.

Fig. 7

Inhibition effect of garlic extract against hop stunt viroid (HSVd) in pot-grown cucumber plants. (A) Comparison of cucumber plant growth and symptoms at 25 days post-inoculation. Healthy: untreated plants; garlic extract: plants inoculated with HSVd treated with garlic extract; Positive: plants inoculated with HSVd only. (B) Detection of HSVd in cucumber plants using reverse transcription polymerase chain reaction.

Fig. 8

Sustained hop stunt viroid (HSVd) inhibition was confirmed in grapevine plantlets treated with garlic extract across four generations of regenerated plants. (A) Detection of HSVd by reverse transcription polymerase chain reaction. At 0 days post-treatment (dpt): plants before garlic extract treatment; 2nd generation: second regeneration of grapevine plantlets with confirmed HSVd inhibition; 4th generation: fourth regeneration of grapevine plantlets with confirmed HSVd inhibition. (B) Quantification of HSVd by digital PCR. (C) Fourth-generation regenerated plants showing sustained HSVd inhibition following garlic extract treatment.