First report of tobacco ringspot virus infecting Hibiscus sabdariffa (roselle) in Oklahoma, USA

In October 2025, Hibiscus sabdariffa plants exhibiting leaf distortion, mottling, and mosaic symptoms were observed in Oklahoma, USA (Figure 1). Disease incidence was approximately 45% in the affected field. Symptomatic leaf samples were submitted to the Plant Disease and Insect Diagnostic Laboratory at Oklahoma State University for analysis. Initial testing with a polyclonal Agdia ImmunoStrip® lateral flow device assay (Cat. No. ISK 64001/0005) was positive for tobacco ringspot virus (TRSV). Because TRSV has not previously been reported infecting H. sabdariffa or any other Hibiscus species in the United States, the detection was further validated by RT-PCR and sequencing. Total RNA was extracted from symptomatic leaf tissue using the Invitrogen PureLink Viral RNA/DNA Mini Kit (12280050) and subjected to RT-PCR using TRSV-specific primers described by Digiaro et al. (2007). The assays produced the expected 480 bp and 354 bp amplicons, respectively. Amplicons were purified and Sanger sequenced (GenBank Acc. No. PZ527703-4). BLASTn analysis revealed 98.01% nucleotide identity to tobacco ringspot virus isolate TRSV-EC3 RNA1 (ON751941) and 99.57% identity to TRSV isolate TRSV-EC6 RNA2 (ON751948). To further characterize the virus associated with symptomatic tissue, total RNA from symptomatic plants was analyzed by Oxford Nanopore Technologies (ONT) sequencing on a MinION platform using the cDNA-PCR Barcoding Kit V14 (SQK-PCB114.24) and a DNA Flow Cell (FLO-MIN114). A total of 6,217,995 reads were generated (2.5 Gb total data), of which 3,753,042 non-host reads remained after quality filtering and host subtraction. Kraken2 analysis (Wood et al. 2019) classified 2,091 reads as TRSV and 1,245 reads as TRSV satellite RNA. No additional viruses were detected in the sequencing data. Reference mapping against TRSV RNA1 (NC_005097), RNA2 (NC_005096), and satellite RNA (NC_003889) produced near-complete genome coverage, with 99.99% breadth of coverage and 45.1× mean depth for RNA1 (PZ507414), 99.69% breadth of coverage and 165.3× mean depth for RNA2 (PZ507415), and 100% breadth of coverage and 785.9× mean depth for TRSV satellite RNA (PZ507416). BLASTn analysis (Altschul et al. 1990) of the ONT-derived consensus sequences showed 95.6%, 96.9%, and 97.8% nucleotide identity to ON751941, ON751948, and NC_003889, respectively. The combined serological, RT-PCR, Sanger sequencing, and ONT metagenomic results confirmed TRSV infection in symptomatic H. sabdariffa plants. TRSV is a bipartite nepovirus with a broad host range and can be transmitted by nematodes, seeds, and pollen. To our knowledge, this is the first report of TRSV infecting H. sabdariffa in the United States, expanding the known host range of this economically important virus.