Introduction

Advancements in high-throughput sequencing (HTS) technologies have significantly accelerated research on the tomato virome globally, with over 45 viruses, including newly emerging and re-emerging species, reported to infect tomato plants (Rivarez et al. 2021; Rivarez et al. 2023). These viruses can impair the host's photosynthetic capacity, disrupt essential metabolic pathways such as protein and carotenoid biosynthesis, and inhibit plant growth. As a result, they cause considerable yield losses and decline in fruit quality, leading to substantial economic damage to the global tomato industry (González-Pérez et al. 2024; Prigigallo et al. 2025). Therefore, effective management of major tomato viruses, along with the establishment of cultivation systems that utilize virus-free planting material, is crucial for ensuring stable and sustainable tomato production.

Tomato viruses are transmitted through diverse pathways, including vector-borne transmission by insects such as whiteflies, thrips, and aphids, as well as mechanical and, in some cases, seed transmission (Fiallo‐Olivé and Navas-Castillo 2019; Jones 2021). Under protected cultivation systems, these transmission pathways can be further intensified due to high planting density levels, continuous cropping, and favorable environmental conditions for vector proliferation, resulting in rapid virus spread and accumulation within greenhouses (Savary et al. 2019).

In Korea, tomatoes are a key vegetable crop, used not only for fresh consumption but also as a raw ingredient in various processed foods such as hamburgers, sauces, juices, and smoothies. As of 2024, tomatoes are cultivated on approximately 6,086 hectares, amounting to the second-largest production area among fruiting vegetable crops in the country (Park et al. 2020; Jung and Shin 2023). Despite their agricultural and economic importance, research on tomato viruses in Korea has been limited. Previous studies have mostly focused on documenting the presence of selected regulated or major viruses, such as tomato yellow leaf curl virus (TYLCV), tomato spotted wilt virus (TSWV), tomato bushy stunt virus (TBSV), and tomato chlorosis virus (ToCV), considering the specific region, cultivar, or cultivation environment (Rivarez et al. 2021; Jo et al. 2023).

Despite these studies, recent region-wide data on virus incidence across major production areas, comparative analyses among widely cultivated commercial cultivars, and systematic evaluations of mixed infection patterns under protected cultivation systems remain insufficient in Korea. In particular, there is a lack of integrated epidemiological data based on standardized diagnostic approaches that simultaneously assess multiple economically important viruses across regions and cultivars.

Therefore, to ensure the sustainability and competitiveness of the tomato industry in Korea, systematic investigations are needed to clarify the distribution and infection patterns of major tomato viruses that cause significant economic losses worldwide under local cultivation conditions. This study presents a comprehensive assessment of the occurrence and infection dynamics of ten globally prevalent tomato viruses in three commercially important cultivars—‘Daphnis,’ ‘TY Ored,’ and ‘Pink Star’—which are widely cultivated in Korea, across major tomato-producing regions using a unified molecular diagnostic approach.

Materials and Methods

Biological materials

Between September and December of 2024, a total of 342 tomato leaf samples were collected from five major tomato-producing regions in Korea: Damyang, Gimcheon, Mieyang, Buyeo, and Chuncheon. These regions were chosen due to their significant contributions to protected tomato production in Korea.

In each region, samples were obtained from multiple commercial greenhouses (3-5 farms per region) with the cooperation of local growers. Sampling sites were selected based on accessibility and grower collaboration, representing a convenience sampling approach while covering typical protected cultivation systems and regional production environments in Korea.

Leaf samples were collected prior to the appearance of visible virus symptoms in order to capture the baseline incidence of virus infections under the corresponding field conditions, including latent infections. Both asymptomatic and early-stage plants were included among the samples. All samples were collected from fully expanded mature leaves at comparable growth stages across the regions to minimize variations due to plant developmental differences.

Leaf samples were collected from three large-fruited commercial tomato cultivars—‘Daphnis’, ‘TY Ored’, and ‘Pink Star’—which are widely cultivated under protected conditions in Korea. Sampling was conducted based on cultivar availability in each region as opposed to using a fully balanced experimental design. Among these, ‘TY Ored’ is a TY-type cultivar carrying resistance genes against tomato yellow leaf curl virus (TYLCV), whereas ‘Daphnis’ and ‘Pink Star’ are commonly cultivated commercial cultivars without well-documented virus resistance traits. All samples were promptly transported to the laboratory under refrigerated conditions and stored at low temperatures until RNA extraction.

Experimental procedures

For virus detection, petiole tissues were collected from fully expanded mature leaves of each tomato sample for the subsequent analyses. Immediately after collection, the samples were placed in insulated ice boxes, transported to the laboratory, and stored at −80 °C until RNA extraction. Total RNA was extracted from approximately 50 mg of petiole tissue using the MiniBEST Plant RNA Extraction Kit (TaKaRa Bio, Tokyo, Japan), following the manufacturer's instructions. The extracted RNA was eluted in 30 µL of RNase-free water, and its concentration and purity were assessed using a NanoDrop spectrophotometer (MicroDigital, Seongnam, Korea). First-strand cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa Bio, Tokyo, Japan). RT-PCR assays were performed to detect ten tomato viruses: tomato yellow leaf curl virus (TYLCV), tomato chlorosis virus (ToCV), tomato spotted wilt virus (TSWV), tobacco mosaic virus (tomato strain; ToMV), cucumber mosaic virus (CMV), pepper mottle virus (PepMoV), southern tomato virus (STV), tomato bushy stunt virus (TBSV), pepino mosaic virus (PepMV), and tomato brown rugose fruit virus (ToBRFV). Virus-specific primer sets previously reported in the literature were used for amplification.

The PCR reactions in this study were conducted using AccuPower HotStart PCR Premix (Bioneer, Daejeon, Korea) in a total reaction volume of 20 µL, which included 2 µL of cDNA template. The thermal cycling conditions, in this case the initial denaturation, annealing temperature, number of cycles, and final extension, varied among the viruses and are summarized in Table 1. PCR products were separated by electrophoresis on 1.5% agarose gels and visualized using a GD-1000 gel documentation system (Axygen, CA, USA). Selected positive amplicons were additionally confirmed by Sanger sequencing when necessary.

Definition of incidence and mixed infection

Virus incidence was defined as the plant-level detection frequency among the tested plants, calculated as the proportion of plants in which a given virus was detected.

In cases of mixed infection, a plant in which multiple viruses were detected was counted as a single infected plant for incidence calculation, while each virus was recorded separately for virus-specific detection frequency analyses.

Results and Discussion

A total of 342 tomato samples collected from five major tomato-producing regions in Korea (Damyang, Gimcheon, Mieyang, Buyeo, and Chuncheon) were analyzed to determine the incidence of ten globally significant tomato viruses (Table 2). Among the detected viruses, TSWV was most prevalent, occurring in 48 samples (14.0%), followed by CMV in 40 samples (11.7%), TYLCV in 31 samples (9.1%), and ToCV in 11 samples (3.2%). In contrast, ToMV (1.8%), PepMoV (1.5%), STV (0.9%), and TBSV (0.6%) were detected at low frequencies, while PepMV and ToBRFV were not found in any of the analyzed samples. However, the absence of PepMV and ToBRFV in this survey should be interpreted with caution, as virus detection may be influenced by the sampling period (September-December), regional coverage, and assay sensitivity. Therefore, non-detection for these viruses does not necessarily indicate their absence in Korean tomato production systems, and they may still be present at low prevalence or outside the surveyed temporal and spatial scope.

Overall, virus infections were detected in 15.5% of the surveyed plants (53 out of 342 samples), indicating a moderate level of virus occurrence under commercial greenhouse conditions in Korea. Among the ten tested viruses, four (TSWV, CMV, TYLCV, and ToCV) accounted for the majority of detections, while the remaining viruses were detected at low frequencies or were not detected. Detailed virus-specific detection frequencies are presented in Table 2.

These results indicate that TSWV, CMV, and TYLCV were the most frequently detected viruses in this survey. Notably, the high incidence of TSWV is closely linked to its transmission by thrips, consistent with previous reports indicating that virus spread is accelerated under protected cultivation systems as vector populations increase (Zhang et al. 2023; Bupi et al. 2024). Overall, this survey provides empirical evidence with which to identify the key tomato viruses that should be prioritized in disease management strategies for Korean tomato production systems.

The three tomato cultivars examined—‘Daphnis’, ‘TY Ored’, and ‘Pink Star’—exhibited distinct infection patterns despite being surveyed under identical diagnostic conditions (Tables 2 and 3). Among them, ‘Daphnis’ had the highest overall infection rate at 37.7%, with relatively high detection frequencies for TSWV (26.1%), TYLCV (15.9%), and CMV (15.9%). Thus, the detection frequency differed among the cultivars in this survey, and this variation may reflect differences in cultivation environments, vector exposure levels, or virus accumulation dynamics rather than inherent susceptibility (Mirzayeva et al. 2023; Koeda et al. 2025).

‘Pink Star’ demonstrated a lower overall infection rate of 31.9% than ‘Daphnis’; however, TSWV (14.0%), CMV (11.7%), and TYLCV (9.1%) were the predominant detected viruses, indicating a broadly similar composition of viral infections. In contrast, ‘TY Ored’ exhibited the lowest overall infection rate at 15.5%, with notably low detection frequencies of TYLCV (3.9%) and TSWV (7.8%). These results may be associated with the presence of resistance genes or may reflect cultivar-specific traits, including potential resistance mechanisms, under commercial cultivation conditions (Cabrera et al. 2025; Prabhakar et al. 2025).

These cultivar-dependent differences are unlikely to stem from a single factor, and they should be understood as the combined result of multiple ecological and physiological determinants. These include genetic background, the capacity for virus accumulation within host tissues, and the feeding and settlement preferences of vectors (Kim et al. 2021). These differences should be interpreted with caution, as environmental and management factors were not fully controlled in this study. Given that symptom expression and viral titers can vary significantly among cultivars, even for the same virus, cultivar selection is a crucial element to consider when developing effective virus management strategies at the field level.

Clear regional differences in virus incidence were observed (Table 4), attributed to variations in climatic conditions, cultivation systems, cropping patterns, and vector population dynamics. Among the surveyed regions, Mieyang had the highest overall infection rate at 22.2%, with both TSWV and TYLCV consistently detected across all three cultivars. This prevalence is likely linked to intensive protected cultivation, continuous cropping practices, and localized climatic conditions that favor insect vector proliferation and activity, thereby enhancing virus transmission (Bupi et al. 2024; Tiberini et al. 2025).

Damyang recorded the second highest infection rate at 15.6%, with TSWV, TYLCV, and ToMV consistently found across the cultivars. The prevalence of these viruses may be related to the regional production system, where mixed cropping practices and diverse cultivation types facilitate vector overwintering and seasonal transmission. Furthermore, the relatively high connectivity among greenhouses in this area may increase the likelihood of virus spread through the inter-farm movement of insect vectors (Ontiveros et al. 2022; Chinnaiah et al. 2023).

In Gimcheon, the detection frequencies of CMV and TSWV were notably high. This region features extensive protected cultivation and diverse cropping systems, which contribute to significant seasonal fluctuations in vector population densities. These cultivation practices align with the observed infection pattern, where a non-vector-borne virus (CMV) and a vector-dependent virus (TSWV) coexist at elevated frequencies (Tatineni and Hein 2023).

Buyeo displayed the most diverse virus spectrum among the surveyed areas, being the only location where low-incidence viruses such as STV and TBSV were sporadically detected. This diversity likely results from the regional coexistence of various cultivars and cultivation systems combined with an open pathway for virus introduction via the movement of infected seedlings or highly mobile insect vectors. Such conditions may enable the introduction and local persistence of a wider range of viruses within the production system (Singh et al. 2023).

Chuncheon generally exhibited a low intensity of virus occurrence, with infected plants rarely detected, particularly in the cultivar ‘TY Ored’. This pattern suggests that lower temperatures and humidity levels, a production system dominated by a single cropping type, and a limited density of insect vectors may have collectively suppressed virus transmission in this region (Skendžić et al. 2021; Fanourakis et al. 2025). Although Chuncheon did not have the lowest overall infection rate among the surveyed regions, the notably low detection frequencies of major vector-borne viruses, in this case TSWV and TYLCV, indicate that region-specific cultivation environments can significantly influence the intensity and spectrum of viral infections.

The observed differences in virus incidence among the regions and cultivars here should be interpreted with caution, as cultivation environment factors such as previous infection history, vector population dynamics, and management practices were not quantitatively assessed in this study. These factors may have contributed to the variability in the virus occurrence patterns observed across the regions.

Future studies should incorporate detailed environmental data, including vector monitoring, cropping history, and greenhouse management practices, to gain a better understanding of the interactions between the cultivation environment and virus epidemiology.

In addition to single-virus infections, the surveyed samples also revealed cases of mixed infections involving two or more viruses (Table 5). The incidence of mixed infections varied among the cultivars, likely reflecting differences in virus exposure and infection pressure experienced in their respective cultivation environments. Mixed infections may arise from vector co-transmission, sequential infections under greenhouse conditions, and interactions among viruses, including potential synergistic or antagonistic effects. Such mixed infections can lead to complex virus-virus interactions, including synergistic or antagonistic effects, which may alter viral accumulation, symptom expression, and disease progression. In particular, synergistic interactions between viruses such as TYLCV and other co-infecting viruses have been reported to exacerbate symptom severity and result in greater yield losses under greenhouse conditions.

In the 'Daphnis' cultivar, mixed-virus infections were found in 13 out of 69 samples (18.8%), including two samples (2.9%) with simultaneous infections from three different viruses. This was the highest frequency of mixed infections among the cultivars studied here, indicating that multiple viruses were co-circulating in the same production fields. Such infection structures increase the risk of symptom exacerbation, leading to greater potential yield and quality losses compared to single-virus infections. Notably, the presence of latent viruses such as STV and TBSV— which typically do not cause obvious symptoms in single infections—alongside other viruses aligns with previous findings suggesting that these viruses may affect symptom expression or infection persistence in mixed-infection scenarios (Elvira-González et al. 2021; Hussain et al. 2022).

The mixed-infection rate in ‘Pink Star’ was 13.2% (19 out of 144), a rate lower than that observed in ‘Daphnis.’ Most cases involved co-infections with two virus species, and only two plants (1.4%) were simultaneously infected with three viruses. The combinations of viruses in mixed infections were relatively limited. This pattern indicates that even within the same cultivation areas, single-virus infections were generally predominant in ‘Pink Star,’ while multiple-virus infections appeared to be restricted to specific time periods or particular fields (Temple et al. 2023).

In the cultivar ‘TY Ored,’ mixed infections with two viruses were found in only 4 out of 129 samples (3.1%), and there were no instances of triple or higher-order virus co-infections. Given the overall low rate of single-virus infections in this cultivar, ‘TY Ored’ exhibited a relatively low frequency of mixed-virus infections in this survey.

Overall, the frequency of mixed-virus infections ranked as follows: ‘Daphnis’ > ‘Pink Star’ > ‘TY Ored’. Although mixed infections were observed in only a subset of samples, plants with multiple-virus infections are inherently at a greater structural risk for symptom exacerbation, leading to reductions in yield and fruit quality. Therefore, alongside proper cultivar selection, fields with recurrent mixed infections necessitate integrated management strategies. These strategies should include effective vector control, the use of virus-free seedlings, and measures to prevent both internal and external virus introductions within production systems.

This study provides essential epidemiological data for understanding tomato virus dynamics in Korea by presenting the occurrence patterns of ten major viruses along with their cultivar- and region-specific infection structures. This was achieved through a unified molecular diagnostic protocol applied across key tomato-producing areas. By identifying the structurally dominant viral threats—specifically TSWV, CMV, and TYLCV—and highlighting heterogeneous infection patterns among cultivars and regions, the study outlines priority target viruses and vulnerable infection combinations to address in future disease management strategies. Further research should focus on the quantitative monitoring of vector populations and seasonal dynamics, HTS-based virome analyses to detect latent and emerging viruses, the identification of cultivar-specific resistance QTLs and candidate genes, and the establishment of standardized diagnostic systems at the seed and seedling stages. Collectively, these efforts are expected to mitigate the risks of viral diseases significantly in the Korean tomato production system.

Conclusion

This study provides a quantitative analysis of the infection patterns of ten tomato viruses across different cultivars, regions, and mixed infection scenarios using a unified molecular diagnostic protocol applied to major tomato-producing areas in Korea. The findings contribute to an improved understanding of tomato virus occurrence under the specific temporal and spatial conditions of this survey, rather than representing a comprehensive national assessment. Based on these results, future research should focus on year-round monitoring of virus incidence, the inclusion of seedlings and seed lots to track early-stage infections, the integration of high-throughput sequencing (HTS) approaches for detecting emerging or latent viruses, systematic vector population monitoring, and the development of cultivar-specific disease management strategies. These efforts will be essential for establishing more robust and proactive virus management systems in tomato production.