Unraveling Shallot Viral Diversity: PCR Detection and In Vitro Culture

1. Introduction

Shallot (Allium cepa var. aggregatum), a distinct variety within the Allium cepa species commonly known as onions, is an annual herbaceous plant of the family Amaryllidaceae [1]. It is widely cultivated across the Americas, Europe, and Asia [2]. This variety, known for its vegetative reproduction via bulbs, is distinguished by its extended storage life, color, and bioactive compounds [3,4]. Shallots are well known in the culinary world as an essential ingredient in various dishes due to their unique flavor and nutritional properties [5]. They are a rich source of valuable phytochemicals, including organosulfur compounds, fructans and flavonoids [1,6]. Nevertheless, they are highly susceptible to pests and diseases, with viral infections being one of the most severe threats to yield and quality [7,8]. Viruses account for nearly half of emerging infectious diseases in various crops and are responsible for approximately 40% of total crop losses [9]. Several economically important viral pathogens belong to the Allexivirus, Carlavirus, and Potyvirus genera, which frequently lead to simultaneous infections that can further exacerbate yield reduction and crop losses [7]. The development and severity of these viral diseases depend on several factors, including the plant’s age, environmental conditions, and the vector dynamics [10].

Viral pathogens can usually be detected using serological tests, bioassays, or DNA- and RNA-based molecular techniques [11]. Bioassays involve grafting plant material or mechanical inoculation onto a viroid indicator host, which typically develops symptoms over a prolonged period of time [12]. Serological methods are based on the specific interaction between viral antigens and the plant’s antibodies. Nevertheless, the production of highly specific antisera against Allium plants is troublesome due to coinfections [13,14]. Monoclonal antibodies have been utilized to overcome this problem and differentiate between strains of potyviruses and carlaviruses by using the variability of the N-terminal viral coat protein (CP) reaction [15,16]. Although serology has been the main method for routine testing, cross-reactions can occur, and many host plants contain phenolic substances that precipitate proteins, complicating the isolation of active viruses [15,17]. In this way, molecular methods for viral nucleic acids have been optimized and developed for large-scale virus testing of shallot leaves and bulbs. Specific and accessible molecular assay development, such as Polymerase Chain Reaction (PCR) and Reverse Transcription PCR (RT-PCR), allows a specific, versatile, and sensitive diagnosis of viruses [18,19]. This methodology involves primer design based on specific conserved sequences of viral genetic material, which leads to a wide range of detection levels and specificity [7,20].

While such diagnostic tools are essential for early detection, controlling viral infection in plants requires a comprehensive approach. One widely use strategy for propagating virus-free plants in Allium crops is through meristem or shoot tip culture [21,22,23]. This method is particularly effective due to its high genetic stability and regeneration potential [7,11,21,24,25,26,27]. Shoot tip culture is a type of meristem culture in which the explant consists of the shoot tip, including the apical meristem and a small number of surrounding leaf primordia [28]. Notably, this method has been reported as an efficient tool for obtaining virus-free plants while maintaining a high regeneration rate [28,29]. For example, in Allium species, virus elimination efficiencies of approximately 50–70% and even higher have been reported depending on the virus [21,30,31]. Beyond in vitro techniques, other virus management strategies are also considered, such as the use of certified virus-free seeds, control of arthropod vectors, use of insecticides, proper field management, removal of infected plants, usage of resistant varieties, virus management during post-harvest, crop rotation, surveillance, and monitoring [7,32,33,34,35].

In Ecuador, shallots are considered an emerging crop [36]. However, the lack of effective detection and treatment options for viral infections remains a significant challenge for farmers [9]. The viral detection and its elimination are essential for sustainable crop production through vegetative propagation strategies [9,11]. The objectives of this study are (1) to molecularly detect the following five most common viruses that infect shallots: shallot latent virus (SLV), onion yellow dwarf virus (OYDV), shallot virus X (ShVX), leek yellow streak virus (LYSV), and iris yellow spot virus (IYSV) [7,37], and (2) to evaluate the effectiveness of shoot tip culture in eliminating viruses present in shallots (Allium cepa var. aggregatum) and thus propagating virus-free plants.

2. Materials and Methods

2.1. Design and Selection of Primers

Five of the most common viruses affecting shallots were selected for molecular detection by RT-PCR: OYDV, SLV, ShVX, IYSV, and LYSV. To begin the process, nucleotide sequences of the target genes were obtained from the NCBI. The target sites selected were the coat protein for SLV, OYDV, and LYSV, and the replicase and nucleocapsid genes for ShVX and IYSV, respectively. Sequences were downloaded in FASTA format and aligned in UGENE. Primers were designed using Primer3web [38] and validated with PrimerBLAST [39] and MFEprimer [40]. Because sequence variations existed, degenerate primers were created. Two types of primers were tested: primers designed in this study and those previously reported in the literature (Table S1). Based on the analysis of the PCR results from pilot shallot samples, literature-based primers were selected for SLV and OYDV, while designed primers were chosen for ShVX, IYSV, and LYSV for molecular analysis (Table S2 and Figure S1-S2).

2.2. Synthetic DNA-Based Positive Controls for RT-PCR Detection of Plant Viruses

The target sequences for each virus (nucleocapsid for IYSV, replicase for ShVX, and coat protein for SLV, OYDV, and LYSV) were obtained from NCBI. Small fragments between 269 and 625 bp of the target sequences were selected for cloning into an ampicillin plasmid vector (pMG-Amp) and evaluated through an in silico analysis. The expected sizes of PCR products were SLV (308 bp), OYDV (625 bp), ShVX (160 bp), IYSV (189 bp), and LYSV (191 bp) (Figure S1).

2.3. Plant Material for Viral Detection and In Vitro Culture

A total of 99 shallot plants (Allium cepa var. aggregatum) were obtained from three different suppliers based on their availability: 23 from the Experimental Farm of Universidad San Francisco de Quito (EF), 26 from the Organic market (OM), and 25 from each supplier at the Iñaquito market in Quito (Iq1 and Iq2) (Figure 1). The plants were transported to the Plant Biotechnology Laboratory at USFQ. Bulbs were separated from the leaves and stored at room temperature (18 °C) for in vitro culture. Meanwhile, the leaves were stored at –20 °C for 24 hours and then at –80 °C for viral detection.

2.4. RNA Extraction from Shallot Leaves Pre- and Post-Culture

Virus detection was carried out both before and after in vitro culture. Pre-culture detection aimed to identify the viruses present in the original material, while post- in vitro culture detection assessed the effectiveness of shoot tip culture in eliminating them. Before in vitro culture, RNA was extracted from all the 99 samples of shallot leaves. After the shoot tip culture, only 31 plantlets were available for molecular analysis: 11 from EF (Assay 1), 8 from Iq1 (Assay 2), and 12 from Iq2 (Assay 3). Total RNA was extracted from leaves using the TRIzol-chloroform protocol as described by Rio et al. [41] with approximately 200–300 mg of shallot leaf powder per sample. RNA quantity and purity, as well as integrity, were checked by Nanodrop (Thermo Scientific) and Qubit (Invitrogen). The RNA was left at 4 °C for 24 hours and then stored at -20 °C.

2.5. RT-PCR

Complementary DNA (cDNA) was synthesized using the SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers (Thermo Fisher Scientific), following the manufacturer’s instructions. PCR was then performed using virus-specific primers, along with synthetic positive and negative controls. The PCR cycling conditions were 94 °C for 2 min, followed by 30 cycles of 94 °C for 30 s, 58 °C (OYDV, LYSV), 47 °C (SLV), 61.5 °C (ShVX), and 62 °C (IYSV) for 30 s, 72 °C for 1 min, and finally an extension of 72 °C for 3 min. RT-PCR products were visualized in a 1.5% agarose gel, stained with SYBR Safe, and examined under UV light using a photo-documenter (BIO-RAD).

2.6. Bulb Sterilization

The protocol of in vitro culture used in this study was standardized and taken from previously developed protocols [36,42], with some modifications introduced as detailed in Table S3 and Figure S3. A total of 73 fresh shallot bulb samples were analyzed: 23 from EF (Assay 1), 25 from Eq1 (Assay 2), and 25 from Eq2 (Assay 3). The OM samples were excluded from the in vitro assays due to fungal contamination. The disinfection protocol began with a 10-minute pre-wash of the bulbs in tap water to remove soil residues and eliminate dry outer layers. The bulbs were then immersed in Mertect fungicide (Syngenta; 3.5 g/L) and agitated at 420 rpm for 15 minutes.

Afterwards, in a laminar flow chamber (LABCONCO Purifier Clean Bench), bulbs were immersed in ethanol (70%) for 10 minutes. Then the bulbs were submerged in sodium hypochlorite (4.5%) with three drops of Tween 20 and agitated for 35 minutes. The bulbs were then rinsed with sterile distilled water. One-third of the length of the bulb was excised with a sterile scalpel, retaining the basal portion and removing external cataphylls to obtain a basal disc explant of about 1.5 cm in diameter and 1 cm in height. Each explant was cultured on M1 initial medium in a glass bottle (Table S4).

2.7. Shoot Tip Culture

After 15 days in culture, plantlets regenerated from basal discs and shoot tips (6 to 10 mm long) were excised. Shoot tip refers to the apical portion of the plant shoot, which includes the apical meristem (0.1 to 0.5 mm) and surrounding leaf primordia [28,43,44]. The excised shoot tips were cultured on M1 regeneration medium to induce the formation of plantlets (Figure 2). After one month of shoot tip culture, young leaves were collected from each plantlet for the post-culture viral detection. All plantlets were subcultured on M2 rooting medium (Table S4). After four weeks, when plantlets developed roots, they were transferred to M3 medium and then subcultured every 30 days (Table S4). All plant material was kept at 25 ± 1 °C with a photoperiod of 16 hours in white light.

2.8. Acclimatization

When plants showed roots and bulbs, acclimatization took place. First, shallot plants were carefully removed from the 25 mL culture tubes, keeping the plants and their roots intact, and then rinsed with distilled water to remove the residual agar. They were then transferred to clay plots with autoclaved black soil inside 1,900-mL glass jars. Each jar was covered with plastic wrap and maintained at 25 °C with a 16-hour photoperiod. Twice a week, plants were watered with sterile distilled water, and two holes were made in the plastic wrap. After one month, the plastic was completely removed. All plants received Hoagland fertilizer [45] every 15 days.

2.9. Data Collection and Statistical Analysis

The efficiency of the disinfection protocol was assessed by measuring the percentage of sterility after 15 days of culture. We also determined the sprouting rate of basal discs after 15 days of culture, as well as the percentage of shoot tip explants that successfully regenerated into plants. The efficiency of the in vitro culture system was evaluated over a 360-day period based on sprouting response, plantlet development, rooting capacity, and bulb formation. Differences in sterility, plantlet growth, rooting, and bulb formation were analyzed using Chi-squared test (χ²). One-way Analysis of Variance (ANOVA) was used to determine differences in the number of shoot tips excised per assay. Two-way ANOVA was applied to evaluate differences in rooting and bulb formation over the time and by assay. All statistical analyses were conducted using GraphPad Prism version 8.0.1 (GraphPad Software, San Diego, CA, USA), and statistical significance was established at p < 0.05. Additionally, the effectiveness of the elimination of viruses was determined by the presence or absence of them after shoot tip culture.

3. Results

3.1. Viral Detection

3.1.1. RNA Extraction Before and After Shoot Tip Culture

Total RNA extracted from the 99 shallot leaf samples before in vitro culture showed concentration averages that varied between 261.4 and 552.2 ng/µL (Table S5). After in vitro culture, the average RNA concentration from the 31 leaf samples of regenerated shallot plantlets ranged from 691.3 to 910.5 ng/µL (Table S6).

3.1.2. Virus Detection Before Shoot Tip Culture

From the 99 plants evaluated before in vitro culture, a total of 87 samples were positive for SLV, 29 samples were positive for IYSV, 27 samples presented both viruses (co-infection), and 10 samples were negative for both viruses. Of the 27 samples coinfected, 72% belonged to Iq2 and 16% to Iq1 (Table 1). All samples analyzed tested negative for OYDV, ShVX, and LYSV viruses (Table 1).

3.2. Disinfection of Plant Material

The disinfection protocol of basal discs resulted in a sterility rate of 47.8% for Assay 1, 32% for Assays 2 and 48% for Assay 3 (Table 2). Statistical analysis showed no significant difference in the sterility of the basal discs among the three assays (Chi-square test, p = 0.7899), indicating the reproducibility of the sterilization process.

3.3. Shoot Regeneration from Basal Discs

Sprouting of basal discs was 100% in all assays and controls after 15 days of culture.

3.4. Shoot Tip In Vitro Culture

The mean of shoot tips obtained per basal disc was 6.5 for Assay 1, 5.5 for Assay 2, and 4.8 for Assay 3 (Figure S4). These differences were not statistically significant (one-way ANOVA, p = 0.2466). The percentage of plantlet development from shoot tips was highest in Assay 1 (88.7%) and lowest in Assay 2 (59.1%) (Table 3), with the difference being statistically significant (Chi-square test, p = 0.0084). In all assays, the regenerated plantlets developed into complete plants and successfully rooted (Figure 3A). Regarding bulb formation, it was faster in Assay 1, in which plants began to develop bulbs after 90 days of culture. In contrast, bulb development in Assays 2 and 3 was observed only after 210 days. By 300 days of culture, 100% of the plants in Assay 1 formed bulbs, whereas plants in Assays 2 and 3 required an additional 60 days to reach full bulb formation (Figure 3B). These differences were statistically significant (Two-way ANOVA, p = 0.0004).

3.5. Detection of Plant Viruses After In Vitro Culture

After shoot tip culture, IYSV was not detected in any sample, indicating its successful elimination. However, SLV remained present in all shallot samples that had previously tested positive (Table 4, Table 5 and Table 6). Moreover, four samples that tested negative for SLV before in vitro culture were found to be positive afterward (Table 5 and Table 6).

4. Discussion

4.1. Effectiveness of Viral Detection

The results of this study revealed that our virus detection protocol using RT-PCR, with positive controls and a combination of specific and degenerate primers, was effective. The primers amplified the expected target region of the virus, enabling positive detection of SLV and IYSV in shallot samples. The analyzed shallots tested negative for OYDV, ShVX, and LYSV. OYDV has previously been reported in shallots and garlic in Ecuador [42,46], whereas LYSV and ShVX have been detected exclusively in garlic [47,48]. The absence of these viruses in our samples may be influenced by seasonal factors [49] or may reflect that they remain restricted to other Allium species, such as garlic, and have not yet spread to shallots. Future studies could consider the use of more sensitive techniques, such as nested RT-PCR [50] or digital PCR (ddPCR) [51], which can detect very low levels of viral nucleic acids and help to confirm the presence and distribution of these viruses in shallot crops across Ecuador. Moreover, techniques such as multiplex RT-qPCR or high-throughput sequencing (including RNA-seq) [52,53,54,55] could be employed for quantitative detection and characterization of known and unknown viral variants.

The presence of SLV in 87.9% of the analyzed shallot plants indicates that this virus is widely distributed within the sampled population. IYSV was present in 29.3% of shallot samples, with 27.3% showing co-infection with both viruses. SLV, a member of the Carlavirus genus and Betaflexiviridae family, is easily transmitted through sap and by aphid vectors such as Myzus ascalonicus and possibly Aphis fabae in a non-persistent manner [7]. Its presence is well-documented, having been detected in shallots [42] and garlic (Allium sativum) [48] in Ecuador, with reported SLV detection rates of 100% in shallots and approximately 88.7% in garlic. This virus has also been reported in other American countries such as Brazil, Mexico, and Argentina [56,57,58], as well as in Europe and Asia, including Hungary, India, and China [59,60,61].

Interestingly, 10.1% of samples tested negative for SLV, suggesting that although SLV is widespread in shallot plants, it is not always present in all samples. These negative cases were detected in the Iq1 and Iq2 markets, with Iq2 also showing a higher incidence of IYSV infection. This variation in viral presence could be related to differences in shallot crop management practices, including water management, crop rotation, fertilization, and pest and disease control measures, such as the use of chemical pesticides [62,63,64]. Other studies have reported different viral profiles in shallots, including cases where SLV was less dominant than other viruses. For example, a study from Indonesia reported SLV infection rates ranging from 60% to 78%, indicating that 22% to 40% of the samples were free of SLV [65]. Furthermore, another study in France reported some shallot samples negative for SLV but positive for ShVX and newly identified carlaviruses and potyviruses [66]. Collectively, these findings suggest that shallots are susceptible to diverse viral infections and that SLV is not consistently present in all populations.

The other virus detected in our samples was IYSV, which belongs to the Tospovirus genus and Bunyaviridae family and represents an emerging threat to onion bulb and seed production [67]. Symptoms produced by IYSV include diamond-shaped lesions of 1–5 cm limited by a chlorotic zone resulting from chloroplast malformation [68,69,70,71]. These symptoms were observed on some leaves of shallot plants from OM, Iq1, and Iq2, but not in EF. Subsequent molecular analyses revealed a correlation between the observed symptoms and the presence of the viruses. Although IYSV had not been previously reported in shallots or garlic in Ecuador, it has been detected in onions [72]. To our knowledge, this is the first report of IYSV infecting shallots in Ecuador. Given that IYSV infects a wide range of Allium species worldwide—including onion, shallot, leek, garlic, and chive—in Europe (the Netherlands, Germany, Spain, France, and Austria), the Americas (the United States, Mexico, Peru, Brazil, and Uruguay), Africa (South Africa), Asia (Japan and India), Oceania (Australia), and the Mediterranean region [7,73,74,75,76,77,78,79,80,81,82,83,84,85,86], these findings are particularly noteworthy. Considering the agricultural importance of Allium crops in Ecuador, the implementation of early preventive measures against IYSV could be highly beneficial in the long term.

The transmission of IYSV is due to vectors and virus reservoirs in plants [87]. The main vector, Thrips tabaci Lindeman, commonly known as “onion trips”, transmits the virus in a persistent and propagative manner [88,89]. The detection of IYSV in shallots and onions in Ecuador probably indicates the presence of the vector. A recent study confirmed the presence of Thrips palmi and Thrips tabaci in Ecuador [90]. Although there is limited evidence regarding the crops affected by T. tabaci in the country, this warrants further investigation, as it is the main vector of IYSV. Moreover, this virus can infect other Allium species, such as garlic and onion, which are also widely cultivated in Ecuador [47,72,91]. The infection with IYSV also makes the plant more vulnerable to environmental stresses such as drought, high temperatures, and excessive irrigation [7,89]. Moreover, some secondary pathogens, such as Alternaria spp. and Stemphylum spp., can colonize those necrotic viral lesions [73], complicating the diagnosis. Therefore, early IYSV detection is critical and could help to implement suitable control and prevention methods to limit viral spread and reduce potential economic crop losses in the future. In addition, accurate identification of the vector and study of its biology could provide valuable insights to help growers manage this disease, particularly in countries like Ecuador, where Allium crops are commonly used and cultivated.

Several viruses are capable of simultaneously infecting shallots, resulting in mixed infections within a single plant [66]. Such coinfections were observed in samples OM, Iq1, and Iq2. Notably, infections of IYSV with Carlavirus and Allexivirus have been documented, and their concurrent presence often produces a synergistic effect that intensifies symptoms and causes severe yield losses [92]. This poses particular challenges for farmers in rural areas, who may lack the necessary information and resources to manage these complex infections effectively. In addition, virus monitoring in shallots and other Allium crops within the country remains limited and is not as extensively documented as in other countries. These findings underscore the importance of government agricultural agencies establishing routine screening programs, not only for the viruses identified in this study, but also for additional viruses that may threaten crop production.

4.2. In Vitro Shoot Tip Culture of Shallot

Our standardized disinfection protocol achieved sterility in 32% to 48% of shallot basal disc assays. Although these percentages were lower than expected, they provided us enough sterile plant material for subsequent in vitro assays. Fungi are the most common pests that attack shallots, especially Fusarium oxysporum, Spodoptera exigua, Alternaria porri; each causing a different disease in the plant [93]. Therefore, selecting an effective fungicide to obtain sterile basal discs was essential. We used Mertect, which contains thiabendazole, a compound known for its effectiveness and broad spectrum of activity against various pathogenic fungi [94]. Benomyl is often recommended and has been widely reported to inhibit most fungal taxa [95,96], and to achieve 100% sterility in shallots [42]; however it is banned in Ecuador [97] due to its teratogenic and toxic effects [98].

In all assays, 100% basal disc sprouting was achieved after the sterilization process. This should be highlighted because, unlike previous studies by Vega et al. [36] and Ramírez [42], we did not use a drying or cooling treatment for the shallot bulbs [99,100]; all the bulbs were fresh. Our results showed that all discs sprouted after only 15 days of culture, demonstrating the efficiency of our protocol.

More than 59.1% of shoot tips developed into plantlets, 100% of shallots developed roots, and over 83.1% of plantlets produced bulbs after up to 300 days of culture. Rapid root system development is a key strategy for plant survival and growth [101], and these regeneration rates are notably higher than those reported in previous studies by Vega et al. [36] and Ramírez [42], which achieved only 30% to 40% and 68%, respectively. In this study, adding ancymidol (an inhibitor of gibberellin biosynthesis) to the M3 medium significantly increased the percentage of bulb formation. Ancymidol promotes bulbing and rhizogenesis by reducing sucrose levels at the leaf bases and increasing the contents of glucose, fructan, and fructose [102]. The observed bulb formation rate surpassed the 77% previously reported [36], underscoring the effectiveness of this approach.

All rooted and bulb-forming plants were successfully acclimatized, representing a clear improvement over previous studies in which acclimatization was either not achieved [42] or only partially successful [36]. Further research is needed to clarify all the factors that influence shallot development, particularly bulb formation, and to enhance the production and selection of high-quality plants for in vitro propagation. Overall, shoot tip culture proved effective, yielding high regeneration rates and efficient bulb formation, making it a promising tool for in vitro research on shallots.

To explore the virus elimination potential of shoot tip culture, we evaluated its effectiveness against both viruses. The shoot tip culture effectively removed IYSV, resulting in 100% virus-free plants, regardless of the source (Iq1, Iq2). Since 1999, IYSV has been included in the European and Mediterranean Plant Protection Organization (EPPO) alert list due to its potential hazard to onion crops [103,104]. It is an industry concern due to the limited availability of effective treatment options to control the virus and its vector [105]. Unfortunately, until 2023, no chemical and biological methods were available to eliminate the virus within the plant [67]. Our study demonstrates the effectiveness of shoot tip culture in eliminating IYSV, revealing its potential for future applications.

Coinfections are more challenging to eliminate than infections caused by a single virus [106,107], and virus-host interactions likely affect eradication success [108,109]. However, the precise reasons behind this remained unclear due to its complexity [108,110]. SLV is generally asymptomatic and, on its own, is not considered to cause significant economic losses in shallot crops. However, coinfections with other viruses, such as IYSV, may intensify the disease symptoms and reduce plant vigor and yield [7,66]. In this case, although SLV could not be eliminated, the elimination of IYSV may benefit co-infected plants by reducing the overall viral load and improving plant health, thereby allowing continued growth and development.

A factor that may contribute to SLV persistence is explant size, which is critical for successful virus eradication, particularly for latent viruses [109]. In our study, we selected shoot tips measuring 6 to 10 mm, as smaller explants failed to regenerate in pilot experiments. While shoot tips contain the meristem, they also include leaf primordia tissue, and some viruses are known to migrate through the vascular elements [111]. Meristem culture using explants of 0.5–0.8 mm has produced 25% to 50% virus-free garlic and shallot plants for SLV and OYDV [112], while other studies report higher eradication rates even with smaller meristem sizes [30,108,113]. For example, Verbeek et al. [30] obtained 91 to 100% OYDV-G-free garlic plants using meristems of 0.21 to 0.33 mm, while cultures of 0.21 mm meristems produced 62-65% virus-free garlic plants for OYDV, LYSV, and GCLV [113]. In shallots, Wang et al. [108] found that 0.5 mm meristem culture resulted in only 10% OYDV and 15% SLV-free plants. Conversely, larger explants such as shoot tips produce plants with a viral elimination rate of 96.2% for SLV and OYDV by the third generation [114]. Generally, smaller explants improve virus elimination but reduce survival and regeneration rates [28,115,116]. Since one of the main objectives of in vitro culture is to generate new plants, selecting an appropriate explant size requires balancing these factors. The size of shoot tips used in our study allowed both a high rate of regeneration and viral elimination of more symptomatic viruses.

SLV is a complex virus that is difficult to eliminate fully with a single treatment. Bradamante et al. [117] suggest that some viruses can also evade the antiviral barriers of the meristem. Supporting this, Magyar-Tábori [118] found that only 20% to 27% of the 0.5 mm shallot meristematic region was free of SLV, demonstrating that combined methods may be necessary for higher or complete elimination. This study serves as a preliminary screening before testing other or integrated treatments. Therefore, future efforts should focus on integrated approaches, combining methods like thermotherapy, chemotherapy, and shoot tip culture for SLV higher eradication rates [21,108].

Unexpectedly, four shallot samples from assays 2 and 3 tested negative for SLV before in vitro culture and positive after shoot tip culture. This result may be explained by low initial viral loads below RT-PCR detection thresholds. SLV levels appeared to fluctuate across plant developmental stages [119]. Velásquez-Valle [120] also showed that virus concentrations tend to increase after an ineffective eradication treatment, suggesting that the virus may have replicated and accumulated during shoot tip culture, making it detectable later. Studies on SLV mechanism are limited, but in a few cases latent virus titers may vary under certain conditions, such as those induced by environmental stress (produced by the in vitro culture process itself) or during specific developmental stages of the host plant [121,122,123].

5. Conclusions

This study reports an efficient protocol for the molecular detection of two of the most common viruses in shallot crops, SLV and IYSV. To our knowledge, this is the first report of the presence of IYSV in shallots in Ecuador. This detection may support the implementation of control, monitoring, and prevention methods to limit the spread of the virus and reduce significant economic and crop losses in shallot or other Allium crops. The presence of the virus implies the presence of its vector [88,91]. Now that Thrips tabaci has been reported in Ecuador [90], understanding its life cycle and interactions with IYSV could help growers to control the disease. The results of our in vitro shoot tip culture proved to be effective in eliminating IYSV, which in other countries has caused significant economic losses by impacting crop quality and yield, and no chemical and biological methods were available to eliminate the virus within the plant [67]. Although our study focused on the detection of five viruses, other viruses may be present in shallots, so it would be interesting in further studies to use third-generation sequencing to understand the complete shallot virome. The in vitro culture was efficient and allowed a high percentage of plant development. Therefore, in vitro shoot tip culture could be a valuable tool for future research and an effective method to eradicate symptomatic viruses in shallots. Performing multiple rounds of shoot tip culture could further ensure the production of truly virus-free plants. Good-quality plants (free of IYSV and effectively acclimatized) obtained in this study were provided to local farmers, thereby enabling them to obtain high-quality plant material for propagation in the field. Research of this kind not only advances scientific knowledge but also provides valuable inputs for the improvement of agricultural practices.