Detection, Differentiation and Phylogenetic Analysis of Cucumber Mosaic Virus Isolates from Cucurbits in the Northwest Region of Iran

Abstract

One hundred and twenty three cucurbit samples with one or more symptoms of leaf mosaic, leaf distortion, fruit mosaic, stunting, mottling and yellowing were collected from several locations in the northwest region of Iran. Screening by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with a cucumber mosaic virus (CMV) polyclonal antibody, produced positive reactions from 13 samples. However, none of these positive samples reacted with a CMV subgroup-II (S-II)-specific monoclonal antibody in a triple antibody sandwich (TAS)-ELSIA. When total RNA from the CMV-infected samples was subjected to reverse transcription polymerase chain reaction (RT-PCR) with a pair of primers corresponding to the flanking regions of the virus coat protein (CP) gene, an expected DNA fragment of about 872 bp was amplified from 10 of the 13 isolates. This fragment covered the CP open reading frame (ORF) plus 92 and 123 bp of the 5′ and 3′ flanking regions, respectively. Restriction analysis with MspI (HpaII) was done on 9 of the PCR products and revealed a previously described CMV subgroup I (S-I) specific profile (537 and 335 bp fragments) for the isolates B13, B23, B5, SH5, SH17, S342 and S337, and an additional fragment, suggestive of combined profiles, was present for B13, SH5 and S342. Two other isolates, SH12 and B7 had a CMV S-II MspI profile (four visible fragments and a predicted non-visible 28-bp fragment on 2% agarose). Also, BsuRI (HaeIII) did not cut the PCR products characteristic of the CMV S-I specific MspI profile, whereas for the S-II isolates, BsuRI gave two fragments with sizes of approximately 559 and 313 bp. Nucleotide (nt) sequences of clones from the isolates B13, B23, SH5, SH17, S337 and SH12 were determined and aligned with those of previously published CMV strains and isolates. Consensus parsimonious trees constructed on the basis of the whole amplified region (841 nt excluding the primer sequences), CP ORF (nt or deduced amino acid data), or either of the flanking regions confirmed the RFLP data so that B13, B23, S337, SH5 and SH17 were placed in the CMV S-IA subclade, and SH12 in the S-II Clade. These analyses showed that both CMV S-I and S-II variants occur in the northwest region of Iran although S-I variants appeared to have a higher incidence.