First report of paprika as a natural host plant for tomato chlorosis virus in Korea

Paprika (Capsicum annuum var. grossum (L.) Sendtn.), also called bell pepper or sweet pepper, is a valuable greenhouse crop that is mostly consumed as fresh fruit in Korea. In 2022, it was cultivated on 726 ha in Korea and 82,042 tons were produced (KOSIS, 2023). In April 2023, interveinal chlorosis and yellowing were observed on the lower and middle leaves of greenhouse paprika in Jinju and Haman, South Korea, and many whiteflies (Bemisia tabaci) were present. The disease incidence was 30-40% on five farms (approximately 4.5 ha) in the two areas. The typical symptoms and presence of whiteflies in greenhouses indicated potential infection by the whitefly-transmitted crinivirus, tomato chlorosis virus (ToCV). Six samples collected from symptomatic plants were examined by leaf dip using transmission electron microscopy (TEM) and revealed filamentous virus particles about 800 nm long. To confirm the TEM results, six symptomatic paprika leaf samples were subject to RT-PCR using the specific primers ToCV-M-4F and ToCV-M-4R to detect ToCV (Choi, 2023). All tested samples were positive for ToCV. To confirm the presence of other viruses and obtain the complete genome sequences, one of the six ToCV-positive samples (ToCV-PAP-JJ6) was subject to high-throughput sequencing (HTS). Total RNA was extracted from symptomatic leaves using the RNeasy Plant Mini Kit (QIAGEN, Germany) and a transcriptome library was generated using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to standard protocols. HTS was performed on an Illumina NovaSeq 6000 system (Macrogen, Korea). De novo transcriptome assembly of the 85,668,854 reads with Trinity software (r20140717) yielded 817,303 contigs of 201 to 38,987 nucleotides (nt). BLASTn and BLASTx analysis of the contigs against the NCBI viral reference database showed that three large contigs were virus sequences, two of which were homologous to ToCV-RNA1 and -RNA2 and one to bell pepper alphaendornavirus (BPEV). The ToCV-RNA1 (genome 8,594 nt, 11,212 mapped reads, mean read coverage 196.2 times) and ToCV-RNA2 (genome 8,242 nt, 40,122 mapped reads, mean read coverage 734.9 times) contigs showed 100% coverage and 99% base pair matching (8,583/8,594 and 8,233/8,242, respectively) with known genome sequences of ToCV-RNA1 (MG813908) and ToCV-RNA2 (KP114534) isolated from tomato in Korea (Lee et al. 2018). The sequences were deposited in GenBank as isolates ToCV-PAP-JJ6 RNA1 (OR865222) and ToCV-PAP-JJ6 RNA2 (OR865223). The BPEV contig (genome 14,728 nt, 2,928,519 mapped reads, mean read coverage 15,012.4 times) was detected at very high read depth and RT-PCR for BPEV confirmed that all six samples were coinfected with ToCV. BPEV was recently reported in paprika and red pepper in Korea (Jo et al., 2022). The BPEV-infected pepper cultivars have been reported not to produce any symptoms (Escalante & Valverde 2019). ToCV was first reported in tomato greenhouses in Korea in 2013 and is a problematic virus in tomato crops, along with tomato yellow leaf curl virus (Jo et al. 2023; Lee et al. 2018). Due to the continuous presence of ToCV, whiteflies, and natural weed hosts (Kil et al. 2015) in tomato greenhouses, ToCV has spread to other crops, including paprika. To the best of our knowledge, this is the first report of ToCV infecting paprika in Korea. A nationwide study is needed to prevent the spread of ToCV, a new threat to paprika farms.