First report of Hibiscus soymovirus in Hibiscus rosa-sinensis in Colombia in mixed infection

Authors: Roy, Avijit; Grinstead, Sam; JUAN CARLOS, CAMPOS - Colombian Corporation Of Agriculture And Livestock- Agrosavia; Hammond, John; LEON, GUILLERMO - Colombian Corporation Of Agriculture And Livestock- Agrosavia

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/12/2023
Publication Date: 2/26/2024
Citation: Roy, A., Grinstead, S.C., Juan Carlos, C.P., Hammond, J., Leon, G.M. 2024. First report of Hibiscus soymovirus in Hibiscus rosa-sinensis in Colombia in mixed infection. Plant Disease. 108:826. https://doi.org/10.1094/PDIS-10-23-2153-PDN.
DOI: https://doi.org/10.1094/PDIS-10-23-2153-PDN

Interpretive Summary: The hibiscus soymovirus (HSV) is a DNA virus belonging to the family Caulimoviridae has been discovered very recently in hibiscus in Hawaii. During surveys of citrus leprosis viruses in the citrus growing regions in Colombia, several hibiscus samples were collected exhibiting green ringspots with internal chlorotic spots in senescing symptoms, mosaic, black spot, ringspot, and chlorotic spots on leaves. Samples were collected from six states in Colombia, includes Tolima, Cauca Valley, Meta, Casanare, Quindío, and Risaralda. HSV genome specific primers were designed and amplified to the expected amplicon sizes. Sanger sequencing of PCR amplicons confirmed the presence of HSV in the symptomatic hibiscus from Tolima, Meta, and Quindío. To know the other viruses infected on those five samples, virome analysis was done using High throughput metagenomic sequencing. Complete genomes of four HSV isolates were determined and assembled contigs covered 99-100% nt identity with known HSV isolate from Hawaii. Along with HSV other virus sequences (hibiscus chlorotic ringspot virus, hibiscus latent Fort Pierce virus, passion fruit green spot virus, citrus leprosis virus C2, and mycoviruses) were detected confirmed mixed infection in all 5 samples. The widespread distribution of HSV potentially threatens dispersion of HSV into a new region or neighboring countries.

Technical Abstract: Hibiscus is native to southeast Asia but well suited to Colombia’s arid soil and dry climates from the coast to the mountains of Bogotá. Study on viruses infecting hibiscus in Colombia is largely unexplored. Four viruses are known to infect hibiscus in Colombia includes hibiscus chlorotic ringspot virus (HCRSV), hibiscus latent Fort Pierce virus (HLFPV), hibiscus latent Singapore virus (HLSV), citrus leprosis virus C2 (Roy et al., 2015, 2018, Padmanabhan et al., 2023). Most of the time mixed infections between these viruses were frequently detected. Very recently virome analysis of single hibiscus infected plant in Colombia discovered the presence of four novel Carlavirus species (hibiscus carlavirus A, hibiscus carlavirus B, hibiscus carlavirus C, and hibiscus carlavirus D), one new Potexvirus (Hibiscus virus X) and a hibiscus strain of physalis vein necrosis nepovirus along with passion fruit green spot virus (PFGSV), HCRSV, HLFPV and mycoviruses. Even though high throughput sequencing (HTS) data identified two small contigs of blunervirus and soymovirus in the same plant sample but failed to validate their presence in the mixed infection (Roy et al. 2023, under review). During Brevipalpus transmitted virus (BTV) surveys, altogether two healthy and 15 hibiscus samples exhibiting green ringspots with internal chlorotic spots in senescing symptoms, mosaic, black spot, ringspot, and chlorotic spots on leaves were collected. The collection map represents six departments (states) from three geographical regions in Colombia, includes Tolima (n=4) and Cauca Valley (n=2) (Andean region) Meta (n=6) and Casanare (n=1) (Orinoquia region), and Quindío (n=1) and Risaralda (n=1) (coffee growing region). About ~100 mg of symptomatic tissue of hibiscus were excised and chopped into small pieces and processed for RNA isolation without DNase I treatment and tested for known BTVs using RT-qPCR assays (Padmanabhan et al., 2023). The hibiscus soymovirus (HSV) was very recently discovered in hibiscus in Hawaii, USA (Wang et al, 2023), which is belonging to the genus Soymovirus family Caulimoviridae. The HSV virions contain a single molecule of non-covalently closed circular dsDNA of ~8.2 Kb and the genome contains ten ORFs. To identify the potential HSV infection in these procured hibiscus samples, previously published HSV primers SVF1/SVR1 (Wang et al, 2023) and two newly designed primer pairs were used to amplify the partial transactivation (ORF-VI), replicase (REP) and coat protein gene (CPG), respectively. HSV was detected by amplifying a 631 and 401-bp fragments using primers HSV-REP-F: (5´-TAACAGCATTCAGTTGTCCACCTCA-3´), HSV-REP-R: (5´-TGAAGGATTGCTCCCCAAGAATCTT-3´) and HSV-CPG-F: (5´-TAGATCACCATCTCCAGTACCTCCA-3´), HSV-CPG-R: (5´-CTGTTTCTTGACCAGTTGTTCCCAT-3´), respectively. Out of 15, three hibiscus samples from Tolima, one each from Meta and Quindío were successfully amplified 430, 631 and 401 nts amplicons using SVF1/SVR1, HSV-REP-F/-R and HSV-CPG-F/-R primers, respectively. Bi-directional PCR amplicon sequencing followed by BlastN analysis revealed 95-98% nt identity with the CPG, REP, and ORF-VI genes of HSV (OP757659.1). An optimized Ribo-depleted library preparation protocol was utilized to prepare cDNA libraries using the total RNA extracts of five HSV PCR positive samples from Tolima, Meta and Quindío (Padmanabhan et al., 2023). HTS libraries of five samples resulted in the range of 22.7 to 29.5 million raw reads. Adapters from the raw HTS reads were trimmed and filtered with Trimmomatic v0.39 (Bolger et al. 2014) and then assembled using SPAdes v3.15.5. Contigs were searched against the proteomes of Arabidopsis genomes and in-house viral protein record and then blasted against the NCBI nr database. Assembled contigs covered 99-100% of the HSV genome, with read depths of 64,474 -119,549, respectively. Tolima and Quindío samples contigs