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

Abstract

Hibiscus chlorotic ringspot virus (HCRSV) belongs to the Betacarmovirus genus of the Tombusviridae family and is a positive-sense monopartite single-stranded RNA virus. HCRSV was first described in a hibiscus variety imported into the United States from El Salvador. The HCRSV natural host range is limited to species belonging to Malvaceae family, in particular Hibiscus rosa-sinensis, H. manihot, H. diversifolius and H. syriacus. 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, Korea), followed by cDNA library preparation with the TruSeq Stranded Total RNA (Illumina, USA). Sequencing on the Illumina NovaSeq 6000 platform (Illumina, USA) with 150-bp paired-end reads yielded 22,578,913 raw reads. 21,413,571 clean reads were obtained by quality control on the sequencing data was performed with the software FastQC (v. 0.11.5). 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 assembly viral genome by using two different algorithms (Metaspades and RNAViral) implemented in SPAdes (v. 3.15.3). 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 identify one coting of 3965 nt covering 97% to 100% of the whole genome sequence of nine HCRSV isolates, with percentage of identity of 87.8-95.2%. No other plant viruses or viroids have been identified during bioinformatic analysis. To confirm the result, a full-length genomic sequence of the Italian HCRSV isolate (named Ita-1), was obtained by reverse transcription polymerase chain reaction (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 (Acc. No. OR981792) was 3910 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, USA). 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 (OD = 1.345 ± 0.010 at 405 nm) relative to the negative (OD = 0.097) and blank (OD = 0.065) controls. Overall, the results of serological and molecular analyses supported 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 (EMDV), hibiscus latent Fort Pierce virus (HLFPV), hibiscus latent ringspot virus (HLRSV), hibiscus latent Singapore virus (HLSV). 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.