Study on the molecular and biological properties of Tomato ringspot virus isolates infecting pepper plants in Tehran province

Abstract

Tomato ring spot virus (ToRSV) is one of the most important viruses threatening vegetable production worldwide. During the years 2010 to 2012, a total of 110 asymptomatic and symptomatic pepper leaf samples with viral affecting like symptoms were randomly collected from fields and greenhouses of Tehran province. Plant samples were tested for the presence of ToRSV using Double Antibody Sandwich Elisa (DAS-ELISA) and Dot immunobinding assay (DBIA). Results indicated that 22% of the samples were infected with ToRSV. Bioassay was performed with the mechanical inoculation of herbaceous indicator plants to evaluate the biological properties of the detected isolates. The morphological features of the virus isolates were studied using Immunosorbent electron microscopy (ISEM). Specific pair of primers designed on the basis of a portion of the RdRP gene sequence and the presence of the ToRSV was confirmed with the reverse transcription-polymerase chain reaction (RT-PCR) for serologically detected samples. An PCR amplicon of a sequence related to the RdRP gene from a representative isolate, was selected and cloned into the pTZ57R/T plasmid vector. Recombinant plasmid contained ToRSV-RdRP gene was transformed into Escherichia coli DH5α. Recombinanat plasmid was isolated and the inserted DNA fragment was sequenced and submitted to NCBI with accession number (JQ972695). At the deduced amino acid and nucleotide sequence levels of the RdRP gene, Iranian ToRSV isolate showed 94-98% and 87-94% identity to corresponded American isolates respectively. Results of this study could be useful for ToRSV detection, host range determination, distribution condition and designing of control management strategies against ToRSV in pepper fields of Iran.

Keywords

• torsv
• cloning
• phylogenetic analysis
• rt-pcr
• sequencing