Cloning, Genetic Diversity of Coat Protein Gene and Detection of Subgroups of Cucumber mosaic virus Strains from Cantaloupe in Varamin

Varamin is one of the major producer regions of cantaloupes which is located in southern Tehran. Cucumber mosaic virus (CMV) is the type species of viral genus Cucumovirus in the family Bromoviridae and is causal agent of economically important losses in cucurbits. The CMV virions are isometric in shape and the genome is composed of three single-stranded positive sense RNAs. Each of the genomic RNAs 1 and 2 encodes a single large ORF and RNA-2 harbors an additional smaller ORF, 2b, involved in cell-to-cell movement and post-transcriptional gene silencing. The RNA-3 has two ORFs, 3a and 3b, encoding a movement protein and coat protein, respectively. Each of genomic RNAs has a Cap-like and tRNA-like structures at its 5ʹ and 3' ends, respectively. The virus is transmitted by different species of aphids in a non-persistent manner and is easily transmissible to the indicator hosts by mechanical inoculation. CMV is reported from several cucurbitaceae plants in Iran. Isolates of CMV are classified in different groups or subgroups based on different methods especially serology, RT-PCR-RFLP, nucleotide sequencing and phylogeny which are useful data for decision making or choosing the strategy during the development of CMV-resistant varieties and producing transgenic plants by RNA silencing strategy.

During 2014-2016, a total number of 132 leaf samples were collected from cantaloupes showing viral symptoms such as mosaic, mottling, leaf deformation and plant stunting. These samples were subjected to double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) with specific antibodies (DSMZ, Germany) to detect CMV based on the manufactures’ instructions. Total RNA was extracted from ELISA positive samples as explained elsewhere. Two microliters of total RNA used as template in RT-PCR to amplify a 870 base pair (bp) fragment of partial CMV genomic RNA3 including the coat protein gene and its flanking regions using specific primers CMVCPf (5'-GCTTCTCCGCGAG-3') and CMVCPr (5'-GCCGTAAGCTGGATGGAC-3') corresponded to 1149–1161 nts and 1998–2015 of CMV strain Q, respectively. In order to address the subgroups of CMV isolates, RT-PCR products were digested by MspI (HpaII) restriction enzyme and electrophoresis was done on 1.5% agarose gel. We expected the presence of the bands with 335 and 537 bp in size for the sub-group I and 393 and 559 bp for sub-group II isolates of CMV. Then, based on the geographical distance of collected CMV isolates, the CP gene from four isolates were ligated into pTG19-T, an A\T cloning vector, and the recombinant plasmids were introduced in Escherichia coli. The selected plasmids were extracted via alkaline lyses method and selected recombinant plasmids were sequenced by Macrogen, South Korea. The sequencing data were aligned with those of previously reported CMV isolates. The alignment and pairwise sequencing to estimate similarity between nucleotide sequences were done with BioEdit software. The Phylogeny Inference package (Phylip) version 3.65 was used to draw phylogeny trees.

59 out of 132 samples (%44.69) positively reacted with CMV specific antibodies in DAS-ELISA. Expected DNA fragments of about 870 bp were successfully amplified via RT-PCR from the ELISA positive samples. in RT-PCR-RFLP for addressing the CMV subgroups, all MspI digested RT-PCR products from the tested samples produced two DNA bands of 537 and 335 bp and it was concluded that these isolates belong to the CMV subgroup I. Sequencing data of selected CMV CP clones showed 99-100% and 100% similarity between tested isolates at the nucleotide and amino acid levels, respectively. These isolates shared 93-100% and 97-100% similarity with previously reported CMV isolates from Iran at nucleic acid and amino acid levels, respectively. The reconstructed phylogenetic tree, re-confirmed the results of RT-PCR-RFLP, and Varamin isolates were placed with few other Iranian CMV isolates including E12, Cues, D13, F13, Sh44, Csu and Cud which were collected from non-melon hosts. Isolate LOR-F, the only CMV isolate previously reported from melons, belongs to the IB subgroup of CMV. This is the first report of CMV subgroup I isolates from melons. 

The RT-PCR-RFLP and sequencing data confirmed the presence of subgroup IA of CMV isolates in Varamin, Pishva and Pakdasht cantaloupe fields whose express severe symptoms in melons. However, the change in Zucchini yellow mosaic virus has been reported from these area, the same isolate of CMV were distributed in the entire region. Unlike Zucchini yellow mosaic virus populations which has been changed in the Varamin, Pakdasht and Pishva regions, it seems that there is just one dominant CMV isolate widespread in these regions.