First Report of Cyrtanthus Elatus Virus A Infection in Daffodils in Korea

In Korea, the limited production and distribution of virus-free bulbs necessitate reliance on imported bulbs for daffodil (Narcissus pseudonarcissus) cultivation. Three viruses have been reported in daffodils in Korea, and quarantine measures target six viruses in imported bulbs (Kim and Jeong 2024; Kim et al. 2024). However, with 26 viruses reported globally in daffodils (Probowati et al. 2022), the risk of introducing novel viral pathogens remains a concern. In this study, we report the detection of cyrtanthus elatus virus A (CyEVA, Potyvirus cyrtanthi) in daffodils in Korea, through high-throughput sequencing (HTS). In May 2021, 75 daffodils were collected from four regions (Yesan, Wonju, Geoje, Shinan), including symptomatic and asymptomatic plants. Total RNA was extracted from pooled leaf tissues (10 mg each) using the Maxwell 16 LEV Plant RNA Kit (Promega, USA) and subjected to HTS on the Illumina NovaSeq 6000 platform (Macrogen, Korea). A cDNA library was constructed using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina, USA), yielding 656,983,464 reads. De novo transcriptome assembly, performed using Trinity software, produced 96,215 contigs. BLASTn and BLASTx analyses identified five contigs mapped to CyEVA, with lengths of 860, 932, 1,713, 2,901, 3,646 bp. These contigs shared 92.06% to 98.72% nucleotide (nt) identity with the Marijiniup 7 isolate (NC_017977). The complete genome sequence of CyEVA was determined using 18 primer pairs, with the 5' and 3' terminal sequences obtained via RACE-PCR, following the previous method (Bak et al. 2024). All amplicons were cloned and sequenced (Bioneer, Korea), and the resulting sequences were assembled into the complete genome using DNAMAN software version 7.0. The sequence was deposited in GenBank (LC847160). It revealed a typical Potyvirus genome structure, measuring 10,024 nt long, and shared 96.47% nt identity with the Marijiniup 7. To confirm the presence of CyEVA, RT-PCR was individually performed on 75 samples using a primer pair (5'-CTCAGAGGGATGTTGAAGGT / GAAGCGTGCACCCTGTAAG-3'; amplicon size: 711 bp), and CyEVA was detected in only 4 samples collected from Yesan. Electron microscopy of infected leaves revealed filamentous rod-shaped particles about 800 nm long (Supplementary Fig. 1). The infected plants showed symptoms such as chlorotic stripes, stunting, and leaf distortion (Supplementary Fig. 2), which were similar to those described in previous studies on CyEVA (Kumar et al. 2015; Raj et al. 2018; Wylie et al. 2010). On May 12, 2022, 8 daffodils collected from Shinan were tested using RT-PCR, and 2 samples exhibiting symptoms similar to those of infected plants in 2021 were positive for CyEVA. Sap inoculation was performed using infected leaves on 19 indicator plants and CyEVA-uninfected daffodils (Supplementary Fig. 3); no CyEVA infection was detected via RT-PCR. As mentioned in previous literature (Ohshima et al. 2016; Probowati et al. 2022), the host range of Narcissus viruses generally seems to be narrow or limited to Narcissus in nature. As a Potyvirus, CyEVA may be transmitted to daffodils by aphids; but, further studies are needed to understand its transmission and epidemiology. CyEVA is presumed to cause poor growth in daffodils due to the described symptoms, and considering its consecutive detection, ongoing monitoring is thought to be needed. This study is the first report of CyEVA in Korea and is expected to contribute to the management of viral diseases in daffodils.