First Report of Hibiscus Chlorotic Ringspot Virus Infecting Hibiscus rosa-sinensis in Italy

Hibiscus chlorotic ringspot virus (HCRSV) belongs to the Betacarmovirus genus of the Tombusviridae family and is a positive-sense monopartite single-stranded RNA virus (Adams et al. 2016). HCRSV was first described in a hibiscus variety imported into the United States from El Salvador (Jones and Behncken 1980). The HCRSV natural host range is limited to species belonging to the Malvaceae family, in particular Hibiscus rosa-sinensis, H. manihot, H. diversifolius, and H. syriacus (CABI 2020). In September 2023, leaf mottle and chlorotic spots were observed on the leaves of two H. rosa-sinensis plants located in a public garden of Ercolano (Naples City, Southern Italy). Two leaves, one from each symptomatic plant, were pooled and submitted to double-stranded RNA extraction using a Viral Gene-Spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Korea), followed by cDNA library preparation with the TruSeq Stranded Total RNA (Illumina, U.S.A.). Sequencing on the Illumina NovaSeq 6000 platform with 150-bp paired-end reads yielded 22,578,913 raw reads. Quality control on the sequencing data was performed with the software FastQC (v. 0.11.5), providing a total of 21,413,571 clean reads. Then, low quality bases and adapters were removed with the software BBDuk in the BBTools package setting a minimum read quality of 25 and minimum read length of 35 bp. The resulted filtered reads were used to assemble viral genome by using two different algorithms (metaSPAdes and RNAViral) implemented in SPAdes (v. 3.15.3; Bankevich et al. 2012). Of the total contigs de novo assembled, the two algorithms implemented identified, respectively, 340 and 559 contigs related to viruses. BLASTn analysis of the contigs identified one contig of 3,965 nt covering 97 to 100% of the whole genome sequence of nine HCRSV isolates, with percentage of identity of 87.8 to 95.2%. No other plant viruses or viroids had been identified during bioinformatic analysis. To confirm the result, a full-length genomic sequence of the Italian HCRSV isolate (named Ita-1) was obtained from one symptomatic plant by reverse-transcription (RT)-PCR using specific primers designed on the sequence of the assembled contigs. The PCR products were sequenced using the Sanger method (Microsynth Seqlab, Germany) in both directions. The obtained full-length genomic sequence of the HCRSV isolate (accession no. OR981792) was 3,910 nt in length. Maximum-likelihood phylogenetic trees inferred from the whole genome sequence showed that Ita-1 clustered closely with HCRSV isolates. The leaf samples were further analyzed using a HCRSV ELISA kit (Agdia, U.S.A.). Healthy H. rosa-sinensis leaves were taken as a negative control and buffer solution as a blank control. The results showed a positive reaction for the two symptomatic plants (OD405 = 1.345 ± 0.010) relative to the negative (OD405 = 0.097) and blank (OD405 = 0.065) controls. Overall, the results of serological and molecular analyses suggested that symptoms observed in H. rosa-sinensis were strictly associated to HCRSV infection. Four viruses have been reported in H. rosa-sinensis in Italy so far: eggplant mottled dwarf nucleorhabdovirus (De Stradis et al. 2008), hibiscus latent Fort Pierce virus (Adkins et al. 2003), hibiscus latent ringspot virus (Brunt et al. 1980), and hibiscus latent Singapore virus (Srinivasan et al. 2005). To our knowledge, this is the first report of HCRSV in H. rosa-sinensis in Italy, where probably infected plants were accidentally introduced. At present, HCRSV has been reported in three continents (Asia, Oceania, and North America), and the number of countries where the virus has been detected is likely to increase rapidly as a result of increased surveillance and availability of diagnostic methods. This study will help the management of viral diseases on H. rosa-sinensis in Italy.