First report of Hibiscus chlorotic ringspot virus in Turkey

Hibiscus rosa-sinensis (rose of China) is an ornamental plant grown throughout the tropics and subtropics, and under glass in more temperate areas. It is commonly used in landscape design; however, pathogens causing diseases on H. rosa-sinensis are not well described, especially in Turkey. Hibiscus chlorotic ringspot virus (HCRSV) is one of the pathogens causing diseases on H. rosa-sinensis. The virus belongs to the genus Betacarmovirus in the family Tombusviridae. Symptoms of HCRSV vary from vein banding to chlorotic ringspots on leaves of H. rosa-sinensis (Waterworth et al., 1976; Luria et al., 2013). In September 2016, similar symptoms were observed on leaves of H. rosa-sinensis in the Mugla province of Turkey (Figs. 1-2).

Figure 1: Ringspot symptoms on leaves of Hibiscus rosa-sinensis caused by Hibiscus chlorotic ringspot virus.

Infected leaves were collected from eight different plants and stored at -80°C until further analyses. For RT-PCR, a pair of primers specific to parts of the coat protein (CP) gene of HCRSV were designed (AK-1 HCRSV-F 5′-AAGAGAGCAGCCAATAGA-3′ and AK-2 HCRSV-R 5′-GAAGAAGAACAAGAAGCGA-3′), based on the complete genome sequence of HCRSV (GenBank Accession No. NC_003608; Huang et al., 2000). Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, Canada) and RT-PCR was done using the PrimeScript RT-PCR kit (Takara, Japan) according to the manufacturer's instructions. As expected, a 759 bp DNA fragment corresponding to the partial CP gene was amplified from all samples. Two PCR products, designated MGL1 and MGL2, were selected randomly, purified and sequenced bi-directionally using primers AK-1 HCRSV-F and AK-2 HCRSV-R. The sequences of isolates MGL1 and MGL2 were deposited in GenBank with the accession numbers KY420907 and KY420908, respectively BLAST analysis of these sequences confirmed similarity to HCRSV sequences.

Figure 2: Ringspot symptoms on leaves of Hibiscus rosa-sinensis caused by Hibiscus chlorotic ringspot virus.

Different HCRSV sequences from various regions of the world were used for sequence analysis. The identities at nucleotide and amino acid level between Turkish and other HCRSV isolates were determined using a partial 517 bp sequence of the CP gene after alignment with ClustalW, and ranged from 94-98% and 86-97% identity, respectively. The phylogenetic relationship of different HCRSV isolates was determined by using the neighbor-joining-method and the Turkish isolates clustered closely with isolates from Iran and Israel (Fig. 3). To the knowledge of the researchers, this is the first report of HCRSV in Turkey.

Figure 3: Phylogenetic relationship between Turkish (MGL1 and MGL2) and other Hibiscus chlorotic ringspot virus isolates (labels show GenBank Accession Nos.). Phylogenetic tree based on partial sequence (517 bp) of the coat protein gene and created by neighbor-joining method applying Kimura80 parameters with 1000 bootstrap replication after ClustalW alignment in CLC Main Workbench v7.7.3. Cardamine chlorotic fleck virus (CCFV) was used as an outgroup.

Source: New Disease Reports