Managing Plant Viruses in Tissue-Cultured Apple and Grapevine: Strategies for Detection and Eradication

Systemic nature of viral infections in fruit crops

Plant viruses infect various tissues, including phloem, xylem, and meristematic regions, which complicates eradication efforts. Some viruses—such as ACLSV, grapevine leafroll-associated virus (GLRaV), and grapevine fanleaf virus (GFLV)—are known for their deep tissue infections, where viral particles persist despite intensive eradication strategies (Schellenberger et al., 2011). Moreover, fruit trees are frequently simultaneously infected with multiple viruses or viroids, such as mixed infection of GLRaVs and grapevine virus A (GVA) in grapevine or ASGV and ACLSV in apples. These co-infections may interfere with diagnostic accuracy, complicate symptom expression, and reduce the effectiveness of virus elimination strategies (Meng et al., 2017). Although meristem culture is widely used to generate virus-free plants, its effectiveness is limited against certain viruses, such as GVA and ASPV, which are capable of invading meristematic tissues (Bettoni et al., 2021). The widespread systemic distribution of these viruses further complicates eradication efforts, as removing visibly infected parts does not eliminate the virus from the entire plant (Meng et al., 2017).

Ineffectiveness of conventional eradication approaches

Eradicating viruses from apple and grapevine is particularly challenging owing to their perennial nature, systemic viral movement, and vegetative propagation. Various conventional eradication approaches—including thermotherapy, meristem culture, chemotherapy, and cryotherapy—have been employed to eliminate viral infections. However, these methods often have limited efficiency owing to incomplete virus elimination, reinfection risks, and various technical constraints (Bettoni et al., 2019; Wang et al., 2003). In addition to viruses, viroids—such as apple scar skin viroid (ASSVd) and hop stunt viroid (HSVd)—pose significant challenges in fruit tree propagation. Their small genome size and high sequence variability frequently lead to undetected infections, particularly in in vitro plantlets (EFSA Panel on Plant Health, 2019).

Risk of viral reinfection post-tissue culture

Even after successful virus elimination, fruit trees remain highly susceptible to reinfection by vectors such as aphids, whiteflies, and nematodes (Gaafar and Ziebell, 2020). Several apple-infecting viruses, including ACLSV and apple mosaic virus (ApMV), are transmitted by aphids in a non-persistent or semi-persistent manner (Pedrelli et al., 2024). GLRaVs—the causal agents of grapevine leafroll disease—are transmitted by mealybugs (Pseudococcus spp.) and soft-scale insects (Naidu et al., 2014). Some grapevine-infecting viruses, such as GFLV, are transmitted persistently by soil-inhabiting nematodes (Xiphinema spp.), which feed on grapevine roots and facilitate virus spread (Garcia et al., 2019).

Some viruses, including GFLV and ASPV, persist in contaminated plant debris, soil, and root remnants, enabling reinfection of new plantings. Once an orchard or vineyard is contaminated, virus particles remain viable in the soil and rootstock for several years, serving as a reservoir for future infections (Demangeat et al., 2005).

Fruit trees are commonly propagated vegetatively via grafting, budding, and tissue culture. When the parent plant is infected, viruses are transmitted to all propagated offspring, significantly contributing to the persistence and spread of viruses within orchards. ACLSV and ASGV can be transmitted via grafting, leading to long-term infections in apple trees (Pedrelli et al., 2024). Similarly, GLRaVs and grapevine leafroll disease are efficiently spread via infected propagation material (Maree et al., 2013).

Limitations of virus-free certification programs

Virus-free certification programs are essential for producing healthy planting materials for fruit crops. These programs typically involve virus indexing, sanitation measures, and certification of disease-free plant stocks. However, despite their essential roles, these programs face significant limitations. A major challenge is the difficulty in detecting viruses present at low titers or in latent states. ASGV and ACLSV can persist at undetectable levels in tissue-cultured apple plants, leading to false negatives during routine diagnostic testing (Beaver-Kanuya and Harper, 2020; Zhao et al., 2018). In grapevine, grapevine red blotch virus and GLRaVs often exhibit delayed symptom expression, complicating early detection efforts (Reynard et al., 2018). Consequently, virus-free certification programs require constant monitoring, frequent testing, and rigorous maintenance of mother plants, thereby making them expensive and limiting accessibility for many growers. In the United States and Europe, producing virus-free grapevine stock via certification programs requires multiple years of rigorous testing, thereby making the process costly and time-consuming (Maliogka et al., 2015a). Additionally, many developing countries lack the necessary infrastructure to implement effective virus certification programs, leading to the widespread use of uncertified planting materials. The criteria for virus-free certification programs vary across countries and regions, leading to inconsistencies in virus detection methods and certification standards. The EU and the US enforce stringent virus indexing and quarantine measures, whereas other regions may lack mandatory testing protocols, leading to the unintended spread of viruses via international trade (EFSA Panel on Plant Health, 2019). Successful implementation of virus-free propagation programs requires adequate infrastructure, trained personnel, and strong regulatory support. However, many developing countries face resource limitations that impede regular virus indexing, mother plant maintenance, or access to certified materials, thereby hindering sustainable virus management on a large scale (Mumford et al., 2016).

Shoot tip and apical meristem culture

Shoot tip and apical meristem culture are well-established techniques for producing virus-free plants. These methods are based on the principle that viruses and viroids rarely invade the meristematic region owing to its lack of vascular tissue, high metabolic activity, and rapid cell division. Shoot tip culture typically involves excising a 0.5–5.0 mm segment of the shoot apex that includes leaf primordia, whereas apical meristem culture targets a smaller region (≤0.5 mm), excluding differentiated tissues to enhance the possibility of virus elimination (Fig. 1) (Nehra and Kartha, 1994).

Fig. 1

Schematic overview of virus elimination and regeneration of virus-free plants using shoot tip culture combined with in vitro therapies. Virus-infected fruit trees undergo shoot tip culture followed by one or a combination of virus eradication treatments, including thermotherapy, cryotherapy, electrotherapy, or combined technique. Treated shoot tips are then cultured via successive in vitro subcultures (generations) to regenerate virus-free plants. Virus-free status is confirmed before transferring the regenerated plants to in vivo conditions.

In apple, meristem-based techniques have demonstrated high efficacy in eliminating viruses such as ASGV, ACLSV, and ASPV. Kwon et al. (2024) reported that apical meristem culture using segments ≤0.3 mm yielded >50% virus-free plantlet regeneration. Although efficiency varied among cultivars, the results highlighted the robustness of this approach in apple virus eradication programs (Table 1).

Table 1

Eradication of viruses and viroids from apple plantlets using traditional eradication methods

Similarly, in grapevine, shoot tip and meristem cultures have been successfully employed to eliminate GFLV, GLRaV, and GVA. High frequencies of virus-free regenerants are achieved from meristems or shoot tips (0.1–0.3 mm) cultured under controlled in vitro conditions (Gribaudo et al., 2006; Maliogka et al., 2009), thereby demonstrating the broad applicability of this approach across diverse grapevine cultivars.

Despite their effectiveness, shoot tip and meristem culture techniques have several limitations in producing virus-free plantlets. Although smaller explants enhance virus elimination, they often lead to reduced regeneration efficiency, particularly in woody species such as apple and grapevine. Additional challenges include genotypic variability, persistence of latent virus infections, and the risk of somaclonal variation during regeneration (Li et al., 2016; Panattoni et al., 2013; Wang et al., 2016a), but shoot tip and meristem remain foundational approaches for virus eradication, valued for their chemical-free, reliable, and cost-effective attributes (Table 2).

Table 2

Eradication of viruses and viroids from grapevine plantlets using traditional eradication methods

Thermotherapy

Thermotherapy is a widely used physical method for eliminating viruses from plant tissue cultures. This technique involves exposing infected plant tissues to elevated temperatures—typically 34–42°C—for a defined period to inactivate or inhibit the replication and systemic movement of pathogens such as viruses and viroids (Fig. 1) (Wahid et al., 2007; Wang et al., 2008). Thermotherapy impedes viral invasion of meristematic tissues by inhibiting viral replication and inducing intracellular disruption of viral particles (Cooper and Walkey, 1978). Regarding the raspberry bushy dwarf virus, viral RNA within leaves and shoot tips become disorganized at 38°C, probably as a result of an increased RNA silencing mechanism (Maliogka et al., 2009; Wang et al., 2008). These findings suggest that thermotherapy can effectively inhibit viral spread into meristematic tissues by activating antiviral defense responses in plants. Thermotherapy is commonly applied to whole plants or excised shoot tips before or in combination with meristem culture, significantly increasing the possibility of obtaining virus-free plants (Díaz-Barrita et al., 2008). This technique is applied to perennial fruit crops such as apples and grapevines to eliminate systemic viral and viroid infections that negatively affect fruit quality and yield. Targeted pathogens include ASGV, ACLSV, GFLV, and GLRaVs (Hu et al., 2017; Miljanić et al., 2022). Owing to their vegetative propagation and long-life cycles, virus elimination via thermotherapy has become an essential step in producing certified virus-free plant materials. The treatment duration can vary depending on the plant species, pathogen involved, and desired outcome, typically ranging from a few days to several weeks.

In apple, combining thermotherapy with apical meristem culture has been successfully employed to eliminate several latent viruses (Table 1). A commonly used protocol involves culturing infected in vitro shoots at 38–40°C for 4–6 weeks, followed by excision and culture of the apical meristem (0.2–0.5 mm). This approach significantly enhances virus eradication while minimizing tissue damage (Wang et al., 2008). Wang et al. (2006) reported the successful elimination of ASGV and ACLSV from infected apple cultivars using thermotherapy at 38°C for 4 weeks followed by meristem culture. Khafri et al. (2022) show that thermotherapy alone, incubating apple tissue at 38°C for 28 days, successfully eliminated 60% of ACLSV infections. The effectiveness of thermotherapy appears to increase with lower initial virus titer.

Grapevines are susceptible to multiple virus infections, many of which are localized in the shoots and challenging to eliminate using conventional treatments (Table 2). Previous studies show that thermotherapy has the potential to eliminate specific viruses under optimized conditions. Panattoni et al. (2013) conducted thermotherapy on grapevine shoots infected with GVA, exposing them to 38°C for 57 days under a 16-h photoperiod. The virus was detected in approximately 60% of the treated plantlets, indicating that thermotherapy alone can be utilized for virus sterilization in grapevines. This study highlighted the advantages of thermotherapy in mass propagation systems, including reduced technical complexity and lower cost. However, the successful application of thermotherapy requires careful environmental management. Precise control of humidity and light is essential during grapevine thermotherapy to prevent stress-induced necrosis and maintain tissue viability throughout the treatment process.

Despite the potential of thermotherapy, prolonged exposure to high temperatures or excessive heat can adversely affect plant viability, leading to tissue necrosis or growth inhibition. Furthermore, the effectiveness of thermotherapy varies with virus type, plant variety, and initial virus load, necessitating careful optimization. Therefore, precise regulation of temperature and treatment duration is essential to balance effective pathogen elimination with the maintenance of plant tissue health.

Chemotherapy

Chemotherapy involves applying antiviral chemicals to eliminate plant viruses in vitro. This application is especially effective against systemic viruses that are challenging to eradicate using thermotherapy or meristem culture alone. Antiviral agents such as ribavirin, acyclovir, and amantadine have been commonly applied individually to suppress viral replication in cultured tissues (Fig. 1) (Panattoni et al., 2013).

In apple and grapevine tissue cultures, ribavirin has been extensively used as a chemotherapy agent to eliminate viruses such as ASGV, ACLSV, and grapevine fleck virus (GFkV) (Komínek et al., 2016; Kwon et al., 2024). Depending on the virus strain and concentration applied, ribavirin can significantly reduce viral titers or achieve complete eradication of the infection. Ribavirin exerts its antiviral activity through various mechanisms, including inhibiting inosine monophosphate dehydrogenase, inducing mutagenesis through the interaction of its active triphosphate form with viral RNA polymerase, or inhibiting viral RNA synthesis (Table 1) (Parker, 2005).

Hu et al. (2012) reported successful elimination of ASGV and ACLSV from infected apple shoot tips, with treatment durations of 4–6 weeks leading to virus removal rates > 70%, depending on the concentration and cultivar. Similarly, Komínek et al. (2016) treated virus-infected grapevine shoot cultures with ribavirin at concentrations of 10–25 mg/L and observed a significant reduction in virus incidence after 4–5 weeks.

Chemotherapy can be readily standardized by adjusting the concentration of antiviral chemicals in the culture medium. Nevertheless, chemotherapy has several limitations. Phytotoxic effects of these antiviral agents can cause growth inhibition, leaf chlorosis, and even plant death, especially at high concentrations or in susceptible cultivars (Magyar-Tábori et al., 2021; Mazeikiene et al., 2019). The effects can also vary significantly depending on the virus type and host genotype, with some viruses developing resistance or recovering after treatment cessation (Mazeikiene et al., 2019). Prolonged exposure to antiviral agents can induce somatic mutations or epigenetic changes, affecting crop growth in regenerated plants (Špak et al., 2010). Therefore, careful optimization of the drug type and treatment duration is essential to maximize efficacy while minimizing adverse effects on plant viability.

Cryotherapy

Cryotherapy is an advanced physical method that involves rapid freezing of plant explants in liquid nitrogen (–196°C), followed by rapid thawing, to selectively eliminate virus-infected cells while preserving meristematic tissues. This approach leverages the high freeze tolerance of meristem cells—attributable to their small size and low vacuolation—compared to that of the susceptibility of differentiated, virus-infected cells. Owing to its broad applicability, cost-effectiveness, and ability to rapidly regenerate virus-free plants, cryotherapy has emerged as a promising strategy for restoring virus-infected, high-value cultivars (Fig. 1) (Bettoni et al., 2016; Wang et al., 2009).

Bettoni et al. (2016) show that cryotherapy applied to in vitro cultured apple shoot tips infected with common viruses yielded an average of 37.5% virus-free regenerants. Beyond effective virus elimination, the method also enhanced healthy plant regeneration while reducing symptom recurrence. In grapevine, Marković et al. (2015) reported virus-free regeneration rates of >70% following cryotherapy of vitrified shoot tips infected with GLRaV-3 and GVA, highlighting the high efficiency of this technique in woody species (Table 1).

While cryotherapy has demonstrated significant efficacy in virus elimination, comparative studies with other sanitation methods remain scarce, and comprehensive agronomic evaluations of treated plants are limited. Its effectiveness may also be restricted by genotype-dependent responses within and across species. Future research should prioritize developing genotype-independent protocols and assessing the genetic stability and agronomic performance of cryo-derived plants (Bettoni et al., 2016, 2022; Wang et al., 2009).

Electrotherapy

Electrotherapy is an emerging, non-chemical technique that involves short-term plant tissue exposure to low-voltage electric fields (typically 5–20 V) to inactivate viruses or disrupt their replication. The applied electric fields may damage viral RNA or interfere with membrane-associated replication complexes (Fig. 1) (González et al., 2006; Hu et al., 2024b).

Hu et al. (2024b) achieved a 16.7% virus elimination rate from ASPV-infected apple stem tips using 50 V for 30 min, with moderate survival rates; however, excessive voltage caused browning and necrosis. Although electrotherapy has been less explored in grapevine, some in vitro studies show reduced viral titers following electric pulse treatments (Table 1) (Adil et al., 2022; Bayati et al., 2011; Hu et al., 2024b).

Major limitations of electrotherapy include limited standardization, incomplete understanding of its underlying mechanisms, and high variability in responses depending on species, cultivar, and graft type (Hu et al., 2024b; Sastry and Zitter, 2014). Precise control over equipment settings also remains underdeveloped, thereby compromising reliability. Nevertheless, electrotherapy is valued for its rapid application and chemical-free nature, and continued refinement may enhance its effectiveness in virus sanitation protocols (Khafri et al., 2022; Magyar-Tábori et al., 2021).

Combined virus elimination techniques

Producing virus-free plantlets in apple (M. domestica) and grapevine (V. vinifera) often requires integrating multiple elimination techniques to overcome challenges posed by systemic and persistent infections. While individual approaches such as thermotherapy, chemotherapy, or shoot tip culture can be effective, their combined application significantly enhances virus elimination efficiency (Tables 1 and 2).

A widely adopted combination strategy involves thermotherapy followed by meristem culture. This approach has achieved virus elimination rates of approximately 80–90% for ASPV, ACLSV, and ASGV using thermotherapy at 37–40°C for 4 weeks, followed by excision of 0.3–0.6 mm meristems (Vivek and Modgil, 2018). Similarly, GFLV, grapevine rupestris stem pitting-associated virus (GRSPaV), and GLRaV-3 were effectively eliminated from infected V. vinifera via heat treatment at 37°C for 6 weeks, followed by culture of 0.1–0.3 mm shoot tips (Maliogka et al., 2009). In this method, sublethal high temperatures inhibit viral replication, while subsequent meristem excision eliminates residual infected cells.

The synergistic application of thermotherapy and cryotherapy has also been investigated. Zhao et al. (2018) show that applying cryotherapy to thermotherapy-pretreated apple shoots significantly increases the recovery of virus-free plantlets, especially for viruses resistant to conventional treatments. Integrating thermotherapy with advanced tissue culture techniques continues to expand the applicability and efficiency of virus elimination protocols.

Combining thermotherapy with chemotherapy or cryotherapy has also been investigated to enhance virus elimination efficiency. Antiviral agents such as ribavirin—applied during or after heat treatment—can inhibit residual viral replication and increase the possibility of obtaining virus-free regenerants. Li et al. (2016) and Hu et al. (2021) show that integrating thermotherapy with ribavirin treatment enhanced virus inhibition in infected apple rootstocks and grapevine plantlets.

Combining virus elimination techniques helps overcome the inherent limitations of individual methods. Thermotherapy alone may be ineffective against heat-tolerant viruses, while meristem tip culture may fail to eliminate viruses that have penetrated deeper tissues. Cryotherapy selectively destroys virus-infected differentiated cells, but its regeneration efficiency can be genotype-dependent (Bettoni et al., 2016; Bhojwani and Dantu, 2013; Díaz-Barrita et al., 2008). Integrated approaches can enhance virus elimination, enhance plant survival, and increase regeneration success across diverse cultivars.

Several challenges remain despite these advantages. The effectiveness of combined treatments depends heavily on the precise optimization of parameters such as treatment temperature, duration, explant size, cryoprotectant exposure, and culture medium composition. Moreover, genotypic variation in thermal and freezing tolerance requires the refinement of species- and cultivar-specific protocols (Faccioli and Marani, 1998; Meng and Rowhani, 2017; Wang et al., 2014).

Difference in eradication efficiency reported among studies may be attributed to factors such as virus species (heat-tolerant vs. heat-sensitive), host cultivars and their regeneration capacity, treatment parameters (temperature regimes, duration, explant size, and chemical concentrations), and initial virus titer. For example, Panattoni et al. (2013) and Wang et al. (2008) highlighted that even under similar thermotherapy or cryotherapy protocols, success rates varied significantly across cultivars and virus types. Likewise, Hu et al. (2012) observed that efficiency in apple and grapevine eradication depended strongly on cultivar genotype and treatment duration.

Conventional virus diagnostic methods

Visual symptom observation remains the initial step in diagnosing virus infection and serves as a primary approach for identifying infected fruit plants. In grapevines, some viruses induce characteristic symptoms, enabling preliminary diagnosis through visual inspection. For instance, GLRaV infection typically induces reddening or yellowing along leaf margins, while GFLV induces leaf malformations, such as twisting or fan-shaped malformations (Naidu et al., 2014; Zherdev et al., 2018). In such cases, visual diagnosis has been widely used as an initial screening tool before confirmatory diagnostic assays (Table 3).

Table 3

Diagnostic methods for verifying virus-free plantlets

Another conventionally employed method is biological testing, which involves inoculating suspected virus-infected plant tissue onto a susceptible indicator plant, followed by monitoring for viral replication and symptom expression over a defined period (Legrand, 2015). This approach has been effectively applied in tissue cultured-based virus-free plant selection for fruit crops such as grapevine and apple, as it enables infection assessment based on the response of the host to specific viruses. For instance, GFLV infection in grapevine has been evaluated by grafting onto a susceptible grapevine cultivar and monitoring symptom development (Demangeat et al., 2005). Similarly, biological indexing in apples is utilized to evaluate the pathogenicity of apple viruses using indicator plants such as Chenopodium quinoa and C. amaranticolor (Brakta et al., 2013).

However, visual symptom observation and biological testing have inherent limitations, as their effectiveness can vary with virus species and environmental conditions, and they may fail to detect latent infections (Soltani et al., 2021; Weber et al., 2002). Consequently, these methods alone are insufficient for accurate and reliable virus detection. Biological indexing, in particular, requires a prolonged incubation period for symptom development, and its reliability can be compromised by variability in indicator plant susceptibility and changing environmental conditions, leading to inconsistent outcomes.

Commonly used virus diagnostic methods

Enzyme-linked immunosorbent assay (ELISA) is a widely employed immunological technique for detecting viral infections by utilizing the specific binding interactions between viral antigens and corresponding antibodies (Table 3). As a protein-based detection method, ELISA complements nucleic acid-based diagnostic approaches (Torre et al., 2020). ELISA is employed in apple tissue culture to detect several viruses, including ACLSV, ApMV, ASGV, and ASPV (Hu et al., 2015b; Khafri et al., 2022). Similarly, ELISA is widely applied in grapevine to confirm infections caused by GFLV and GLRaVs (Gambino et al., 2006).

Reverse transcription polymerase chain reaction (RT-PCR) is a highly reliable molecular diagnostic technique used to detect RNA viruses by converting viral RNA into complementary DNA (cDNA), followed by amplification of target gene sequences to confirm viral presence (Table 3) (Bachman, 2013). In apple and grapevine tissue culture studies, RT-PCR is commonly employed in virus-free plant production to detect viral and viroid infections and to evaluate the effectiveness of virus elimination techniques. For instance, studies targeting the elimination of ACLSV, ASGV, ApMV, and ASPV using cryotherapy and thermotherapy combined with shoot culture have employed RT-PCR to assess virus eradication efficiency (Liu et al., 2021; Romadanova et al., 2016). A similar approach has been applied in grapes, where plants infected with GFLV, GVA, GLRaV-1, GLRaV-3, and GFkV underwent somatic embryogenesis subculturing. RT-PCR was used to verify the rate of virus reduction across successive generations (Gambino et al., 2006).

Quantitative RT-PCR (qRT-PCR) is a molecular diagnostic technique related to RT-PCR but incorporates real-time fluorescence detection to simultaneously detect and quantify viral RNA (Malan, 2009). By detecting fluorescence signals during amplification, qRT-PCR provides precise, real-time quantification of viral load. This technique is widely used alongside RT-PCR to verify virus-free status in tissue culture-based plant production. A key advantage of qRT-PCR is its ability to deliver more precise quantification of virus elimination efficiency (Hu et al., 2024a). For instance, in apple tissue culture research, qRT-PCR has been employed to evaluate the elimination efficiency of ASGV, ACLSV, ASPV, and ApMV (Beaver-Kanuya and Harper, 2020; Chen et al., 2014; Nabi et al., 2023). Similarly, in grapevine tissue culture, qRT-PCR has been employed to quantify the reduction of GLRaVs, GFkV, and GRSPaV (Hu et al., 2021). By integrating qRT-PCR, the effectiveness of virus elimination methods—such as thermotherapy and meristem culture—can be quantitatively analyzed, making it an essential tool in tissue culture-based virus-free plant production.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that enables rapid and specific detection of target DNA or RNA sequences under isothermal conditions. Contrary to conventional PCR, which requires thermal cycling, LAMP utilizes a strand-displacing DNA polymerase along with four to six specifically designed primers to initiate and accelerate amplification at a constant temperature (typically 60–65°C). The LAMP reaction proceeds via self-priming loop structures, producing large quantities of amplified DNA with a characteristic ladder-like pattern. This method generates turbidity or colorimetric changes from accumulated reaction byproducts, enabling visual detection without requiring electrophoresis or complex instrumentation. Owing to its high specificity, efficiency, and rapid reaction time, LAMP is widely adopted in virus diagnostics. For virus-free plant screening, LAMP offers several advantages over RT-PCR and qRT-PCR, including reduced reliance on specialized equipment and lower experimental costs, making it especially well-suited for field diagnostics (Zherdev et al., 2018). In apple and grapevine tissue culture studies, LAMP has been successfully applied to detect ASGV and GLRaV-3, demonstrating its efficacy as a practical and efficient virus detection method (Table 3) (Walsh and Pietersen, 2013; Zhao et al., 2014).

Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique designed to overcome the limitations of conventional PCR methods, especially the need for thermal cycling. RPA enables rapid detection of viral DNA or RNA (via cDNA) from infected tissues at a constant temperature range of 25–42°C, with amplification completed in as little as 15 min (Table 3) (Bakheit et al., 2008). RPA relies on recombinase proteins (usvX and usvY) and single-strand DNA-binding proteins (gp32) to facilitate primer binding and homologous sequence recognition on the target DNA. Subsequently, a mesophilic DNA polymerase—typically Bacillus subtilis DNA polymerase I—extends the primers, synthesizing new DNA strands complementary to the target sequence (Piepenburg et al., 2006). Recent studies have demonstrated the successful application of RPA for diagnosing viral infections in apple and grapevine trees. RPA assays have been developed for detecting ASPV and ASSVd in apple trees. Additionally, in grapevine, RPA assays have been developed and successfully applied to field samples, offering a rapid and sensitive alternative to conventional diagnostic methods (Kim et al., 2019, 2021). These findings indicate that RPA is an effective diagnostic tool in virus-free plant production.

Antiviral agents for virus eradication

RNA interference (RNAi) is a sequence-specific gene silencing mechanism mediated by double-stranded RNA (dsRNA) that directs the degradation of target messenger RNAs, thereby suppressing gene expression. In plants, RNAi is extensively applied to control viral, fungal, and insect threats through transgenic approaches and the exogenous application of synthetic dsRNA (Germing et al., 2025). Owing to its high specificity and eco-friendly profile, RNAi is increasingly recognized as a next-generation strategy for managing viruses in horticultural crops such as grapevine and apple.

Recent advancements in RNAi technology have demonstrated its potential for controlling fungal pathogens in grapevine. For example, Qiao et al. (2023) employed spray-induced gene silencing (SIGS) to deliver dsRNA targeting Botrytis cinerea via artificial vesicles, thereby enhancing dsRNA uptake and stability in grape leaves and berries. Although this study targeted a fungal pathogen, the compatibility of SIGS platforms with grapevine tissues suggests their potential adaptation for virus suppression within in vitro system, supporting future virus-free plant production strategies (Fig. 3).

Fig. 3

Schematic representation of virus elimination in fruit tree tissue culture using RNAi and antiviral agents. Applying exogenous dsRNA or antiviral compounds to in vitro-grown fruit tree plantlets triggers multiple antiviral defense mechanisms. dsRNA is processed via Dicer-like enzymes into siRNA, which are loaded into the RISC to guide sequence-specific degradation of viral or viroid RNAs. Concurrently, antiviral agents may inhibit viral infection by disrupting the viral capsid, preventing viral uncoating, denaturing viral proteins, or activating plant immune response, including PR gene expression and hypersensitive response. These combined mechanisms contribute to the effective selection and regeneration of virus-free plantlets during tissue culture. dsRNA, double-stranded RNA; RNAi, RNA interference; siRNA, small interference RNA; RISC, RNA-induced silencing complex.

In apples, RNAi-based studies show that exogenously applied dsRNA can be systemically delivered and stably maintained in woody perennial species (Wise et al., 2022). Trunk injection of dsRNA into mature apple trees leads to its persistence in leaf tissue for up to 141 days under field conditions. Although these RNAi approaches have not been applied in tissue culture for virus elimination, current findings indicate their potential to enhance virus-free plant production when combined with in vitro propagation techniques.

Garlic (Allium sativum) is renowned for its broad-spectrum therapeutic properties, including antiviral activity against animal and plant viruses (Tesfaye, 2021; Wang et al., 2020). In in vitro-grown grapevine plantlets (“Summer Black”) naturally infected with GLRaV-2 and GFkV, treatment with diluted garlic extract reduced viral RNA levels to 22–27% for GLRaV-2 and 20–30% for GFkV at 10 days post-treatment, as measured by RT-PCR. While the inhibitory effect diminished over time, it persisted up to 20 days (Wang et al., 2020). More recently, treatment of grapevine plantlets infected with HSVd using garlic extract results in a viroid reduction rate of up to 74.45%. Nanopore sequencing revealed structural alternations in the HSVd genome, suggesting that garlic-derived bioactive compounds may directly disrupt viroid integrity (Fig. 3) (Kang and Jeong, 2025).

Glycyrrhizin—a bioactive compound derived from licorice (Glycyrrhiza spp.)—has demonstrated antiviral activity against various human and animal viruses (Zuo et al., 2023). Glycyrrhizin combined with quercetin (0.1 mM) was evaluated for its antiviral effect against ASGV in apple tissue culture. Although initial RT-PCR results showed virus absence in two of eight explants, subsequent immunocapture (IC)/RT-PCR shows that all explants remained infected, suggesting that glycyrrhizin alone is insufficient for effective ASGV elimination in apple tissues (James et al., 1997).

While glycyrrhizin showed only partial suppression of ASGV without achieving complete virus elimination, recent research has identified more effective herbal extracts. Extracts of Hypericum perforatum (HPR) and Pelargonium sidoides were evaluated for their antiviral efficacy against ACLSV and ASGV in apple explants. Treatment with HPR reduced viral load by up to 33.5% in certain cultivars, as confirmed by ELISA and RT-PCR analyses. Although higher concentrations induced phytotoxic effects, lower concentrations preserved explant viability while providing partial suppression. These findings suggest that selected plant-derived extracts may offer more effective alternatives to earlier natural compounds for managing viruses in apple tissue culture (Masoudi et al., 2024).

Toward a next-generation diagnostic framework

HTS—commonly referred to as next-generation sequencing—has emerged as a powerful tool for detecting viral pathogens in tissue-cultured apple and grapevine plants. HTS enables comprehensive nucleic acid analysis, enabling the identification of known and novel viruses with high sensitivity and specificity. By simultaneously sequencing millions of DNA or RNA fragments in parallel, HTS generates vast datasets that can be assembled and analyzed to detect the presence of viral genomes. This technology bypasses the need for prior knowledge of target sequences, making it especially valuable for identifying unexpected or novel pathogens (Liu et al., 2018; Maliogka et al., 2018).

In apple tissue cultures, HTS has been employed to detect various viral infections. For instance, in a study where high-throughput RNA sequencing was utilized, multiple viruses and viroids were identified across apple cultivars, including previously undetected pathogens. This comprehensive detection capability highlights the potential of HTS in producing virus-free apple plants (Ben Mansour et al., 2025; Liu et al., 2018). Similarly, HTS is effectively applied to grapevine tissue cultures. Several studies demonstrated the use of HTS to analyze the virome of grapevine samples, revealing a complex assemblage of viral pathogens. The sensitivity of HTS in detecting these viruses highlights its value for monitoring and managing viral infections in grapevine propagation materials (Turcsan et al., 2020). While HTS offers numerous advantages, factors such as cost, data analysis complexity, as well as the need for specialized equipment and expertise may influence its adoption in routine diagnostics. However, as HTS technology becomes more accessible and affordable, its integration into virus-free certification programs for apple and grapevine tissue cultures is expected to increase.

Nanopore sequencing has emerged as a transformative technology in genomics, offering real-time, long-read sequencing capabilities. Its application in plant virology, especially for diagnosing virus-like symptoms in plants, offers clear advantages over conventional diagnostic approaches (Lee et al., 2022a). This technology functions by translocating nucleic acid molecules via a biological or synthetic nanopore embedded in a membrane. As individual nucleotides pass through the pore, they induce characteristic disruptions in the ionic current, which are measured and decoded to determine the nucleotide sequence. This approach enables direct sequencing of native DNA or RNA strands without requiring amplification or chemical labeling, thereby preserving genetic integrity and enabling the detection of base modifications (Wang et al., 2021).

Nanopore sequencing offers several advantages that make it a powerful tool for detecting viruses in tissue-cultured plants. A major benefit is its real-time data acquisition capability, which enables the immediate generation of sequencing data. This enables rapid detection of viral pathogens and supports timely decision-making in plant health management (Marcolungo et al., 2022; Sun et al., 2022). Additionally, its long-read capability enables the resolution of complex genomic regions, structural variants, and full-length viral genomes, thereby enhancing the precision of virus identification. Another significant advantage is its portability—compact devices such as Oxford Nanopore Technologies' MinION enable on-site sequencing in field settings—enabling real-time monitoring and rapid decision-making (Liefting et al., 2021; Sun et al., 2022). Furthermore, nanopore sequencing requires minimal sample preparation, as it allows the direct sequencing of nucleic acids without the need for extensive processing or amplification. This reduces biases introduced during amplification (Zheng et al., 2023). Collectively, these features will enhance the efficiency, accessibility, and accuracy of virus detection, making nanopore sequencing a valuable tool for virus-free plant production in tissue culture systems. Despite its numerous advantages, nanopore sequencing also presents certain limitations that warrant consideration. A key challenge is its higher raw read error rates than that of other sequencing platforms, necessitating the application of robust bioinformatics pipelines and, in some cases, integrating complementary sequencing technologies to enhance accuracy. Throughput limitations also persist, as the sequencing capacity of current nanopore technology is generally lower than that of HTS platforms, potentially limiting its application in large-scale studies. Another key limitation is the complexity of data analysis—interpreting nanopore sequencing data requires advanced bioinformatics tools and expertise—posing challenges for laboratories with limited computational resources (Delahaye and Nicolas, 2021). While ongoing technological advancements are addressing these issues, researchers should carefully consider these limitations when integrating nanopore sequencing into virus detection and plant health monitoring workflows.

Recent studies have demonstrated the potential of nanopore sequencing for detecting viruses in apple and grapevine plants, although its application in tissue-cultured plantlets remains an emerging area of interest. Regarding virus-free plant production, nanopore sequencing has been employed to evaluate the effectiveness of garlic extract treatment in grapevines infected with HSVd. This approach enables whole-genome sequencing and detailed mutation analysis, demonstrating its value in assessing the effectiveness of virus elimination during the virus-free plant production process (Kang and Jeong, 2025). Adopting nanopore sequencing in virus-free certification programs holds the potential to enhance the health and quality of tissue-cultured plants, thereby supporting sustainable practices in apple and grapevine cultivation.

Digital PCR (dPCR) is a highly sensitive molecular technique that partitions nucleic acid samples into thousands of individual reaction compartments, each undergoing PCR amplification. This partitioning ensures that each compartment contains zero or one target molecule, enabling absolute quantification of viral nucleic acids without reliance on standard curves. Following amplification, the presence or absence of fluorescence signals in each compartment is analyzed to determine the precise concentration of the target virus (Tan et al., 2023). dPCR offers several key advantages, including absolute quantification that enables precise viral load measurements without relying on external calibration curves; high sensitivity and accuracy, facilitating the detection of low-abundance viral genomes and identification of latent infections and low-titer viruses; and increased resistance to PCR inhibitors, reducing the effect of sample impurities and ensuring reliable results even in complex plant matrices. However, PCR also has certain limitations, such as higher costs, longer processing times, and increased technical complexity than that of qPCR.

While studies on dPCR-based virus detection in tissue-cultured apple and grapevine plants are still emerging, the superior sensitivity and precision of this technique position it as a highly valuable tool in this field. dPCR is especially advantageous for virus-free plant certification programs, as it can enable the accurate detection of low viral loads in tissue-cultured plantlets before commercialization. For instance, dPCR has been successfully employed to detect and quantify viruses in woody perennial crops. It has enabled absolute quantification of ACLSV, ASGV, and ASPV, thereby enhancing the accuracy of virus detection in propagation materials (Kim et al., 2022, 2023). Within virus-free plant production, dPCR has been employed to evaluate HSVd inhibition following garlic extract treatment in micropropagated grapevine plants (Kang and Jeong, 2025).

CRISPR-Cas12a—also known as Cpf1—has emerged as a transformative tool in molecular diagnostics, especially for detecting plant viruses across various crops, including fruit trees. This system offers distinct advantages over conventional diagnostic methods owing to its unique mechanism and high adaptability. CRISPR-Cas12a functions as an RNA-guided endonuclease that specifically recognizes and binds to DNA sequences complementary to its CRISPR RNA. Upon target recognition, Cas12a cleaves the target DNA sequence and simultaneously exhibits nonspecific single-stranded DNase activity, enabling collateral cleavage of nearby single-stranded DNA molecules. This collateral activity forms the basis for sensitive and specific detection assays, where the cleavage of labeled reporter molecules generates a detectable signal, indicating the presence of the target viral DNA or RNA (Aman et al., 2020). The CRISPR-Cas12a-based detection platform offers several advantages, establishing it as a powerful tool for virus diagnostics in tissue-cultured apple and grapevine plants. It delivers high sensitivity and specificity through precise recognition of target nucleotide sequences, thereby minimizing false positives and enhancing diagnostic accuracy (Ali and Mahfouz, 2021; Sharma et al., 2021). Additionally, when combined with isothermal amplification techniques such as RPA or LAMP, it enables rapid virus identification within 20–60 min. Another key advantage of this platform is its minimal equipment requirements; the isothermal nature of the amplification process eliminates the need for sophisticated thermal cyclers, making it highly suitable for point-of-care testing and field diagnostics (Mahas et al., 2021). However, certain limitations should be acknowledged. The technique typically requires a pre-amplification step to enhance sensitivity, which can add complexity to assay design. Temperature sensitivity remains a challenge, as the enzymatic activity of Cas12a is influenced by reaction conditions, necessitating precise temperature control to maintain accuracy (Bhat et al., 2022). Despite these challenges, the distinctive benefits of CRISPR-Cas12a highlight its potential as a highly sensitive, rapid, and field-deployable tool for detecting viruses in tissue-cultured fruit plants. Within the tissue-cultured apple plants, CRISPR-Cas12a has been effectively employed to detect multiple RNA viruses and viroids. Jiao et al. (2021) developed a visual assay that integrates CRISPR-Cas12a with RPA, enabling the simultaneous detection of several pathogens directly in field conditions. This method demonstrated high sensitivity and specificity, producing results consistent with that of conventional RT-PCR, thereby validating its effectiveness for in-field diagnostics (Sharma et al., 2021). In grapevine, applying CRISPR-Cas12a for viral detection has been explored. A CRISPR-Cas12a-based detection assay has been developed for grapevine red-blotch virus, enabling rapid and sensitive identification of viral infections in vineyards (Li et al., 2019). Integrating CRISPR-Cas12a into virus detection workflows for tissue-cultured apple and grapevine plants holds significant promise. Its adaptability, combined with advancements in isothermal amplification techniques, supports the development of portable, user-friendly diagnostic platforms. These innovations are poised to enhance early virus detection and management, thereby enhancing the quality and yield of these economically important crops.