Molecular identification of a new isolate of Hibiscus chlorotic ringspot virus in Khuzestan province, southwestern Iran

Background and Aim: Chinese hibiscus, Hibiscus rosa-sinensis L., is a common ornamental shrub in warm and humid regions of Iran including Khuzestan province. This plant is a suitable host for different RNA and DNA plant viruses. Hibiscus chlorotic ringspot virus (HCRSV) has worldwide distribution on hibiscus and it has been previously reported from Guilan province at the North of Iran. HCRSV is belonging to Betacarmovirus genus (Tombusviridae family) with icosahedral particles containing a single-stranded positive-sense RNA fragment.Methods: In our study, leaf samples of H. rosa-sinensis plants showing mottle and ring spot symptoms were collected in Ahvaz, southwestern Iran. Total RNA was extracted from symptomatic leaves by commercial RNA isolation kit (DENAzist, Iran), according to the manufacturer’s recommendation. Reverse transcription polymerase chain reaction (RT-PCR) using previously designed primer pair resulted to an amplified fragment of approximately ۱۱۰۰ bp from putative movement P۹ and coat protein genes. PCR products were sequenced in both directions and then edited and assembled using BioEdit Sequence Alignment Editor (۷.۰.۵.۳) and DNA Baser Assembler (version ۵.۱۵.۰) programs, respectively.Results: The partial consensus CP sequence was deposited in GenBank (Accession No. MN۹۰۸۲۵۸) and compared to those of known isolates by BLAST search and phylogenetic analysis. Accordingly, the newly and previously known isolates of HCRSV shared ۹۳.۸ % sequence identity in the CP gene (۵۱۶ bp) attributed to ۳۲ SNPs and without insertion/deletion. In BLASTn search, the closest hits for the CP sequence of our isolate were the sequences of the isolates SBO۱ from Brazil (MK۲۷۹۶۷۱) and XM from China (KY۹۳۳۰۶۰) with ۹۶.۹ % and ۹۶ % identical, respectively. A phylogenetic tree based on the CP sequences of all HCRSV isolates was constructed using the maximum likelihood algorithm under the K۲+G+I model of the MEGA ۶ software. Our isolate (Khuzestan) clustered distinct from the other previously known isolates and located in a large non-supported clade containing the isolates from Iran (JX۸۶۵۵۹۳), Brazil (MK۲۷۹۶۷۱), Malaysia (MN۰۸۰۵۰۰) and unknown origin (X۸۶۴۴۸ and NC_۰۰۳۶۰۸).Conclusion: To our knowledge, this is the first report of HCRSV in Khuzestan province.

Keywords:

Hibiscus , HCRSV , Phylogeny