First Report of Tomato Necrotic Ringspot Virus and Pepper Chlorotic Spot Virus Mixed Infection in Tomato and Pepper in Laos

Tomato (Solanum lycopersicum L.) and chili pepper (Capsicum annuum L.), key Solanaceous crops, are susceptible to viral diseases impacting yield and quality (Shahriari et al., 2023). During October of 2024, tomato samples (YV39744, YV85813, YV3809, and YV32201) and pepper fruit samples (LJ001, YV33215) exhibiting yellow spot symptoms (Fig.S1) were collected from Nam Tha, Laos, approximately 3-5% plants with yellow spot symptoms were observed across the 0.7 ha pepper and 0.1 ha tomato fields, respectively. Transmission electron microscopy (TEM) revealed spherical virions (80–120 nm) resembling the members of the genus Orthotospovirus in all collected samples (Fig. S2). To further identify the viral species, total RNA extracts from all six symptomatic fruit samples were pooled together for high-throughput sequencing (Illumina HTS) at Ouyi Co., Ltd. (Shanghai, China), following previously described methods (Hu et al., 2022). The HTS generated 95.8 M clean reads with a mean length of 150 nucleotides (nt) after removing low-quality read (Grabherr et al., 2011). De novo assembly generated 34,824 contigs (≥500 bp), with 21 contigs showing viral homology. Seven contigs matched tomato necrotic ringspot virus (TNRV) and six aligned to pepper chlorotic spot virus (PCSV), and 30×coverage of TNRV and PCSV are 84.47% and 30.49%, respectively. Both the viruses belong to the members of the genus Orthotospovirus. Genome assembly successfully generated complete tripartite genomes for TNRV (The GenBank accession numbers of S, M, and L segments are PV178718, PQ872349, and PV178717, respectively), whereas only partial genomic sequences were obtained for PCSV genome. RT-PCR validation was conducted using virus-specific primers target N gene of TNRV and PCSV (Table S1). Total RNA was extracted from symptomatic tissues using the EASYSpin Plus RNA Kit (Aidlab, China). cDNA synthesis employed HiFi Script RT Master Mix (CoWin Biosciences, China) per manufacturer guidelines. PCR conditions: 94°C/4 min; 30 cycles of 94°C/30 s, 55°C/30 s, 72°C/30 s; final extension at 72°C/5 min. Amplified products were cloned into pMD18-T vector (Takara, Japan) and sequenced. RT-PCR followed by Sanger sequencing results confirmed the presence of PCSV sequence in six samples, and TNRV sequence in three tomato (YV85813, YV3809, YV32201) and one pepper (YV33215) sample (Fig. S3). BLASTn analysis showed the Laos PCSV N gene (PQ872348) shared the highest identity (96.93%) with the Chinese PCSV isolate 14YV733 (NC_033772), while Laos TNRV N gene (PV178707) exhibited the highest identity (94.56%) with the Thai TNRV isolate T1 (KM887842). Phylogenetic analysis showed Laos PCSV isolates formed a monophyletic cluster with two Chinese isolates, while the Laos TNRV isolates grouped within a clade dominated by Thai isolates (Fig. S4). To date, TNRV has only been documented in Thailand (Maneechoat et al., 2024), while PCSV was previously reported in China (Zheng et al., 2017). This study provides the first evidence of TNRV mixed infection with PCSV in solanaceous crops tomato and pepper in Laos, expanding their known geographical distribution. These findings emphasize the needs for regional surveillance and disease management.