First report of garlic common latent virus in elephant garlic (Allium ampeloprasum) in single and mixed infection in South Korea.

In South Korea, the cultivation area of elephant garlic (Allium ampeloprasum) is increasing as elephant garlic is milder and sweeter than garlic (A. sativum) (Kim et al., 2019; Lu et al., 2011). Viral diseases can decrease garlic productivity by up to 50% in South Korea (Nam et al., 2002). In 2022-2023, virus-like symptoms such as mosaic and yellow stripes were observed on leaves of elephant garlic in a 432㎡farm with disease incidence of approximately 40% in Yangpyeong-gun, Gyeonggi-do, South Korea. Seventy-two leaf samples were randomly collected from symptomatic plants in 2022 (n=46) and 2023 (n=26). Total RNAs were isolated from individual samples using the Total RNA Prep Kit (BioFact, Daejeon, Korea), and then two-steps RT-PCR was performed using the First Strand cDNA Synthesis kit (Thermo Fisher Scientific) and the TaKaRa TaqTM (TaKaRa Bio Inc.). These samples were tested for 13 viruses with virus-specific coat protein primers including garlic common latent virus (GarCLV) (supplementary Table 1). In 2022, GarCLV, garlic virus (GarV)-B, GarV-C, and GarV-D were detected with the expected amplicon sizes of their CP genes (960, 735, 780, and 753 bp, respectively) in four different plants. In 2023, the CP gene of GarCLV was detected in 26 samples and 4 of 26 samples were positive for GarV-B. The leaves infected with GarCLV and GarV-B in mixed infection showed synergistic effect with extended mosaic and yellow stripes than the leaves with single infection (supplementary Fig. 1). All amplicons were cloned into a pGEM-T Easy vector (Promega Co., USA), and sequenced at Bionics Co. Ltd., South Korea. The resulting nucleotide (nt) and amino acid (aa) sequences were analyzed using DNAMAN software version 5.1. Since all isolates were collected from a farm in Yangpyeong-gun, name of these isolates started with "YPG." The nt and aa sequences of the isolates were compared with those of other strains/isolates. All 27 GarCLV-YPG isolates sequences were deposited (Accessions: OP981636, and PP533185-PP533210). The GarCLV-YPG sequences shared 78.90%-94.40% nt and 92.10%-99.40% aa identities with other GarCLV strains and isolates, and they showed higher similarity (99.40% aa) to isolates produced from A. sativum in China and India (supplementary Table 2). GarV-C-YPG showed the highest similarity (99.20% aa) to isolate G81(GenBank MN059141) from A. sativum in China. GarV-D-YPG showed the highest similarity (99.20% aa) to isolates (G82, GenBank MN059388; BR, MT279193) from A. sativum in China and Brazil. Twenty-two quinoa plants (Chenopodium quinoa, local lesion host) were individually inoculated using the sap from 22 GarCLV infected plants. Chlorotic and necrotic spots appeared on inoculated leaves 12 days post-inoculation; no chlorotic and necrotic spots symptoms were observed on any other leaves except for the inoculated leaves. RT-PCR was performed and the targeted amplicon size for GarCLV was detected. In transmission electron microscope, filamentous particles of approximately 620-730 nm length and 12 nm diameter, similar to the particle description for members of the family Betaflexiviridae, were observed in the saps of symptomatic leaves of elephant garlic and quinoa plants infected with only GarCLV. To the best of our knowledge, this is the first report on GarCLV detection in elephant garlic in South Korea. We hypothesized that the presence of GarCLV in mixed infection with GarV-B might have increase the symptom severity in the elephant garlic. Further study is needed to proof the synergistic effect in mixed virus infection.