First report of tomato yellow leaf curl virus infecting sweet pepper in Taiwan

Tomato yellow leaf curl virus (TYLCV; species Begomovirus coheni), a member of the genus Begomovirus (family Geminiviridae), is a pathogen distributed globally that poses a severe threat to solanaceous crops (Prasad et al. 2020). In February 2025, sweet pepper (Capsicum annuum) plants exhibiting severe symptoms including leaf curling, vein yellowing, distortion, and stunting appeared in Chiayi County, Taiwan. Based on the occurrence of visual symptoms, the disease incidence reached approximately 70% in the affected greenhouse, where whitefly vectors were also prevalent. To identify the causal agent, total DNA was extracted from two symptomatic leaf tissues using a modified CTAB method (Echevarría-Machado et al. 2005). Initial screening by PCR using Begomovirus degenerate primers PAL1v1978 and PAR1c715 (Rojas et al. 1993) amplified a 1.6-kb fragment. The amplicons from the two samples were cloned into the pCRII-TOPO TA vector (Invitrogen, San Diego, CA, USA) and sequenced using an ABI3730 automatic sequencer (Applied Biosystems) at National Chung Hsing University. BLASTn analysis revealed 97.6–97.7% nucleotide identity with TYLCV isolates (Accession Nos. PP179288, PP179285, and OR644941). PCR assays using degenerate primers targeting DNA-B (DNABLC2/DNABLV2; Green et al. 2001) produced no amplification, consistent with the monopartite genome organization of TYLCV. A TYLCV-specific primer pair (FJJ2025-63/64: 5′-TAGTGATGTTACTCGTGG-3′ / 5′-ATCGACGGACGATCTGCACGTGG-3′) was designed to amplify and sequence the remaining portion of the viral genome. The assembled complete genome consisted of 2,791 nucleotides (GenBank Accession No. PX963964) and contained six typical open reading frames (V1 and V2 in the virion sense; C1 to C4 in the complementary sense), as well as the conserved nonanucleotide motif (TAATATTAC). Sequence comparison showed 99.5% nucleotide identity with TYLCV isolates ON982212, PP179280, and OR644941. Pathogenicity was confirmed by constructing an infectious clone using rolling circle amplification (RCA) with phi29 DNA polymerase (New England Biolabs). The RCA product was partially digested with EcoRI and SacI, ligated into the binary vector pCambia0380, and transformed into Agrobacterium tumefaciens strain C58. Agroinoculation was performed on C. annuum at the cotyledon stage and Nicotiana benthamiana at the 4- to 6-leaf stage. Systemic symptoms, including upward leaf curling, developed on N. benthamiana at 14 days post-inoculation (dpi), whereas C. annuum exhibited leaf curling, vein yellowing, and distortion at 70 dpi, consistent with the symptoms observed on the original diseased pepper plants in the field. The presence of TYLCV in systemic leaves was confirmed by PCR using TYLCV-specific primer pair FJJ2025-235/219 (5′-GTGGGTAAGAGGTTCTGTGTTAAATCG-3′ / 5′-AACTTGGACGACATTACAGCCTCAG-3′). To our knowledge, this is the first report of a monopartite begomovirus, TYLCV, infecting sweet pepper in nature in Taiwan. The presence of TYLCV could significantly affect the tomato and pepper industries in Taiwan.