First report of Betacarmovirus hibisci ( Hibiscus chlorotic ringspot virus ) and Tobamovirus fortpiercense ( Hibiscus latent Fort Pierce virus ) infecting Hibiscus rosa-sinensis in Maryland, United States

Betacarmovirus hibisci (Hibiscus chlorotic ringspot virus, HCRSV) belongs to the genus Carmovirus in the family Tombusviridae and is distributed worldwide in ornamental Hibiscus spp. Infected plants show symptoms of mottle to vein banding patterns and chlorotic ringspots. In the United States, HCRSV has been detected in Texas, Florida and Hawaii. Tobamovirus fortpiercense (Hibiscus latent Fort Pierce virus (HLFPV)) belongs to the genus Tobamovirus and the family Virgavirudae and causes symptoms such as chlorotic spots and mottling on hibiscus species. HLFPV has been detected in Florida, California, New Mexico, and Hawaii. Here, we report the full-length genome of HCRSV and a near-complete genome of HLFPV in ornamental Hibiscus rosa-sinensis plants from Maryland. In July 2022, four hibiscus plants exhibiting chlorosis, ringspot, mottle, and leaf distortion symptoms were collected from a private property in Lanham, Maryland. Total RNA was extracted from symptomatic leaf tissue and tested for HCRSV using RT-PCR as described and for HLFPV using the RT-PCR primers. Three out of four tested plants produced an expected size amplicon of 420 bp, and the amplicon sequence was determined using bi-directional sequencing. BLASTN analysis revealed ≥98.13% nt identity with HCRSV from South Korea, and 94.52% with HCRSV from Hawaii with 100% coverage. The HLFPV amplicon sequence showed 100% nt identity with HLFPV (OP572411.1). The draft genome sequence of HCRSV was obtained through high throughput sequencing. Briefly, four cDNA libraries were prepared using an optimized ribo-depleted library preparation protocol and sequenced in a 75-bp v3 Illumina NextSeq alongside alien control samples from rose and jasmine. Raw reads ranged from 20.67 million (M) to 90 M from samples H-2022-4, H-26, and H-36. After adapter trimming and filtering, clean reads were mapped to the Arabidopsis thaliana reference genome and unmapped reads were de novo assembled using Spades v3.15.2. Reads from sample H-2022-4 were assembled in two full-length viral contigs, contig-1 (3950 nt) and contig-2 (7547 nt). BLASTN search revealed that contig-1 [OR137572] shared 92.93% nt identity and 99% genome coverage with the HCRSV Brazil isolate (MK279671), and 92% nt identity and 100% genome coverage with the Hawaii OUGC isolate (MT512573.1). Contig-2 [OR137573] shared 92.43% nt identity and 98% coverage with the Hibiscus bacilliform virus (HBV) China isolate (KF875586). Sample H-26 had 1 viral contig, and BLASTN search revealed that the contig [PX431655] shared 92.17% nt identity and 98.98% genome coverage with HCRSV (NC_003608.1). Interestingly, sample H-36 had 2 viral contigs, and a BLASTN search revealed that contig-1 [PX431656] shared 98.52% nt identity and 99% genome coverage with HCRSV (PP115955), and contig-2 [PX431657] shared 99.65% nt identity and 97.56% genome coverage with HLFPV (NC_025381.1). The detection of HCRSV and HLFPV in hibiscus plants in Maryland may be associated with the expansion of tropical ornamental crops growing in temperate regions. This report is important to the multimillion-dollar ornamental industry as it continues to broaden markets across regions.