Incidence of 14 grapevine viruses in Korean vineyards

The incidence of grapevine virus infections in Korean vineyards was investigated from July to October, 2020. A total of 177 petiole samples were collected from two or three different cultivars in each of four different regions; these were examined by reverse transcription-polymerase chain reaction assay for the presence of 14 major viruses. The overall occurrence of grapevine viruses was 91.0%, and the level of incidence was high irrespective of region or cultivar. The predominant viruses were grapevine leafroll-associated virus 3 (80.2%), grapevine fleck virus (70.6%), and grapevine rupestris stem pitting-associated virus (49.2%). Most grapevines were infected with multiple viruses, suggesting that Korean vineyards are likely to suffer economic losses resulting from viral diseases. This is the first extensive survey performed in Korea to observe the outbreak status of diverse grapevine viruses; surveys of this type can provide important information for the management of grapevine viruses in Korea.


Introduction
Because they inhibit photosynthesis and anthocyanin biosynthesis, viral diseases are a major factor negatively affecting fruit yield and quality in grapevines (Martínez-Lüscher et al., 2019;Tobar et al., 2020;Lee et al., 2021). In addition, virus infection can decrease freezing tolerance, leading to increased susceptibility to winter vine damage, or even to death (Xiao et al., 2018). The impacts of viral diseases suggest the importance of establishing a system of virus-free grapevine cultivation to maintain the competitiveness of the grapevine industry.
Grapes are cultivated in Korea for the fresh fruit market, and for the production of wine and other drinks (Heo et al., 2016;Lee et al., 2019). They are the fifth most important fruit crop in terms of growing area, with a total area of 12,676 ha in 2019. Despite the crop's commercial importance, limited research regarding viral diseases of grapevines has been performed in Korea. Globally, 86 different viruses have to date been identified in grapevines (Fuchs, 2020). Some of the viruses causing significant economic loss are the grapevine fleck virus (GFkV), grapevine asteroid mosaic-associated virus (GAMaV), grapevine rupestris vein feathering virus (GRVFV), grapevine fanleaf virus (GFLV), grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4, -7, -13), grapevine virus A (GVA), grapevine virus B (GVB) and grapevine rupestris stem pitting-associated virus (GRSPaV). Although some of these viruses have been found in Korean vineyards (Jo et al., 2018;Cho et al., 2 2018), detailed information on their occurrence and distribution is not available. For this reason, we investigated the presence and distribution of the 14 most important grapevine viruses in major grape cultivars in Korea.

Materials and Methods
Biological material A total of 177 samples were collected from four major grape-growing regions in Korea from July to October 2020. In Korea, the most widely planted grape cultivars are: 'Campbell Early', 'Kyoho', and 'Shine Muscat', accounting for 38.6%, 21.4%, and 28.9% of the total grape cultivation area, respectively. These cultivars account for approximately 90% of the Korean grapevine market. The cultivation area of 'Shine Muscat', which was recently introduced to Korea, has sharply increased; it is expected to be the number one cultivar in the near future. Accordingly, most of the samples in the present study were collected from the 'Shine Muscat' cultivar.

Experimental procedures
For virus detection, two leaf petioles from each of three vines per vineyard were collected and processed within one day of collection. A reverse transcription (RT)-PCR assay was used to evaluate the presence of each of 14 viruses, with each sample being tested individually. Total RNA was extracted from 50 mg of petioles using the TaKaRa MiniBEST Plant RNA Extraction Kit (Takara Bio, Tokyo, Japan) according to the manufacturer's instructions. Final elution was performed using 30 μL of RNase-free H2O. RNA purity and yield were assessed using a Nabi NanoDrop spectrophotometer (MicroDigital, Seongnam, Korea). The first strand of cDNA was synthesized using the PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio, Tokyo, Japan). PCR amplification were performed using AccuPower HotStart PCR PreMix (Bioneer, Daejeon, Korea) with two μL of cDNA and 10 pmol of each virus-specific primer, as designed by Xiao et al. (2018), in a 20 μL final volume. Amplifications were carried out in a TP 350 thermal cycler (Takara Bio, Tokyo, Japan) under the following protocol: initial denaturation at 95 °C for 1 min, followed by 33 cycles of 30 s denaturation at 95 °C, 30 s at the annealing temperature specified for each primer, 1 min elongation at 72 °C, with a final extension at 72 °C for 5 min. PCR amplicons were checked by gel electrophoresis on 1.5% agarose gels and visualized with a GD-1000 gel documentation system (Axygen, California, USA).
Our study indicates that the degree of virus occurrence in Korean vineyards is disturbing. Commonly occurring viruses across three different grape cultivars were GFkV, GLRaV-3, and GRSPaV. GLRaV-3 is a phloem-restricted virus (Hu et al., 2020) that is predominant in vineyards worldwide, including Canada and the United States (Poojari et al., 2020;Schoelz et al., 2021). It hinders photosynthesis and carbon balance during respiration and induces a significant economic loss by reducing the vigor and yield of vines (Endeshaw et al., 2014;Montero et al., 2016). Mixed infection with other viruses also leads to commercially-significant problems in berry ripening and cluster size (Lee and Martin, 2009), and GLRaV-3 is regarded as one of the most detrimental viruses. GLRaV-3 is mainly disseminated by propagation using virus-infected vines, but subsequent spread is mediated by mealybugs or soft scale insects (Golino et al., 2008). The frequent occurrence of GFkV and GRSPaV was remarkable and believed to be explained by the use of infected cuttings in vegetative propagation or by grafting with infected rootstocks because no natural vector is known (Crnogorac et al., 2021). Although a single infection of this kind of virus does not significantly affect vine growth, it is suspected that mixed infections with other viruses could negatively affect vine vigor, fruit yield, and juice quality. Our results show that mixed infections among GFkV, GRSPaV, and other viruses frequently occur in Korean vineyards, and-to avoid economic loss-should be taken into consideration when evaluating potential hazards to productivity and grape quality in Korea.
Several viruses-such as GLRaV-3, GVA, and GVB-can be transmitted by vectors in Korean vineyards. A strong relationship between the incidence of specific viruses and the magnitude of vector populations is frequently reported (Cooper et al., 2018;Jones and Nita, 2019;Uhls et al., 2021), but there is no information on virus infection routes and management practices in Korean vineyards. These circumstances suggest that a lack of monitoring and prevention systems for viral vectors could contribute to these vineyard viruses. For this reason, it is important that efforts be made to obtain information on virus vectors in Korean vineyards.
We found differences in virus occurrence among cultivars, which might have resulted from different exposure conditions to virus infection, but equally could be due to different susceptibilities, among cultivars, to specific viruses. Along with the great success of the Shine Muscat cultivar, new grape cultivars are continuously being released in Korea (Heo et al., 2017;Roh et al., 2018;Park et al., 2020) and the diversification of grape-cultivation cultivars is expected to progress in the future. Based on our surveys, thorough virus screening should be performed before vine material is released for propagation.

Conclusions
Our study has demonstrated the local grapevine industry in Korea is exposed to a serious risk caused by viruses. In this study, GLRaV-3, GFkV and GRSPaV that can influence growth and quality of grapevine negatively are commonly found regardless of cultivars and regions, which emphasize the need to develop strategies for eliminating such viruses and preventing their spread.

Authors' Contributions
JYH conceived and designed the experiments, supervised, and revised the manuscript. SHK conducted the experiments and wrote the original manuscript. SHJ conducted parts of the experiments. All authors read and approved the final manuscript.