Molecular detection and characterization of tomato leaf curl virus in protected cultivation

Abstract:

Detecting and managing tomato leaf curl virus (ToLCV) inside greenhouses presents a particular challenge because the protected environment favours whitefly vectors while giving growers a false sense of biosecurity. This research carried out molecular detection and characterization of ToLCV in symptomatic tomato plants collected from three commercial greenhouses in Jeju and Chuncheon, South Korea, between March and November 2023. Total DNA was extracted from 96 leaf samples showing typical upward curling, vein yellowing, and stunting symptoms. PCR amplification using begomovirus-specific degenerate primers (Deng A/B) produced the expected ~520 bp fragment in 78 of 96 samples (81.3%). Rolling circle amplification (RCA) followed by restriction fragment length polymorphism (RFLP) with EcoRI and BamHI identified two distinct RFLP profiles corresponding to ToLCV and tomato yellow leaf curl virus (TYLCV). Sequencing of 12 representative isolates and BLAST alignment confirmed 94.7-97.3% nucleotide identity with ToLCV-[KR:Jeju] (GenBank accession LC385241) and 92.1-94.8% identity with TYLCV-IL[KR] (AB014346). Phylogenetic analysis placed all Jeju isolates in a distinct sub-cluster within the ToLCV clade, while Chuncheon isolates grouped with TYLCV-Israel lineage. Virus titre measured by quantitative PCR correlated strongly with symptom severity (r = 0.87). These findings confirm co-circulation of ToLCV and TYLCV in South Korean greenhouses and highlight the need for vector exclusion and resistant cultivars in protected tomato production.