First report of tomato leaf curl Laos virus causing leaf curl disease in tomato in Vietnam

Tomato (S. lycopersicum) cultivation has been hampered by the emergence of begomovirus causing leaf curl diseases, resulting in significant yield reduction (Charoenvilaisiri et al., 2020; Lai et al., 2024). During field surveys between December 2024 and May 2025 in Hue city, Vietnam, we surveyed five tomato gardens (n = 100 plants, 100-200 m2 each garden) but found the occurrence of leaf curl disease symptoms only in two plants. Disease symptoms included upward leaf curling, stunted and chlorotic leaves. Whiteflies (Bemisia tabaci) were observed on the abaxial leaf surface of symptomatic plants. Total DNA was isolated from two plants using a cetyltrimethylammonium bromide-based DNA extraction protocol (Doyle and Doyle, 1987). The presence of begomovirus was confirmed by polymerase chain reaction (PCR), using degenerate begomovirus-specific primers SPG1/SPG2 (Li et al., 2004). The SPG1/SPG2 amplified products (from two plants) were sequenced using Sanger sequencing method (Apical Scientific, Selangor, Malaysia) and the subsequent BLASTn analysis of nucleotide sequencing results returned tomato leaf curl Laos virus (ToLCLV) as the closest match (percent identity of 97.46% and 97.57%; e-value of 0.0). Taxonomically, ToLCLV (Begomovirus solanumlaosense) belongs to the genus Begomovirus under the family Geminiviridae. Due to the limited publicly available sequences of ToLCLV genomes, we sequenced the ToLCLV genome using primer walking approach. Based on SPG1/SPG2 sequences, ToLCLV_A_F3 (5’-GCAGATCTTCCATCGATCTG-3’) and ToLCLV_A_R3 (5’-GGAGCCACTGTACTCAGGT-3’) were designed to amplify the rest of the ToLCLV circular DNA-A and the 2120-bp PCR product was sequenced by Sanger sequencing. In addition, primer pair ToLCLV_A_F4 (5’-AGCCTATTTAGTGTGTGGTC-3’) and ToLCLV_A_R1 (5’-CATCACTGCAACTCAAGCAGA-3’) were used for PCR diagnostic and sequencing. The complete DNA-A sequence was assembled based on read overlaps to obtain the full 2758-nt sequence (GenBank accession PX020954). It shared 91.41 percent identity to the nucleotide sequence of ToLCLV in Laos (GenBank accession NC_004613). The phylogenetic tree was constructed in MEGA12 using the neighbor-joining method with a bootstrap of 1000 replicates. The analysis displayed ToLCLV Vietnam isolate ‘Hue01’ with the Laos isolate of ToLCLV (NC_004613) in the same clade. Both the ToLCLV isolates are most closely related to the tomato leaf curl Malaysia virus (NC_004648). Based on the International Committee on Taxonomy of Viruses (ICTV) species and strain demarcation thresholds for begomoviruses (Brown et al., 2015), this begomovirus species was designated as ToLCLV strain ‘Hue01’. This is the first report of ToLCLV in Vietnam. Further investigations are needed to determine the distribution of tomato leaf curl Laos virus in the broader Southeast Asia geographic region, their infectivity, and effects on tomato cultivation.