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.

Identification of viruses infecting cucurbits and determination of genetic diversity of Cucumber mosaic virus in Lorestan province, Iran

“…Mgh91 and Jol186 isolates were placed in subgroup IA by Nematollahi et al (2012) in their study to assess CP and MP genes, and draw a phylogenetic tree. LORA, LORB, LORD, LORE and LORH were placed in subgroup IA close to S337 and E5 isolates (Bashir et al 2006). Khn1 and Esf172 isolates from East Azerbaijan province of Iran, by assessing CP and MP genes, were placed in subgroup IB and LARC, LORF and LORG isolates in our research were grouped with Khn1 and Esf172 isolates (Nematollahi et al 2012).…”

“…It has been shown that 80% of the known isolates belong to subgroup I and the frequency of subgroup IA is more than that of IB (Gallitelli 2000). Group II is more common in cold climates (Roossinck 2002) and since Iran is a subtropical country, it was expected that more Iranian isolates would belong to group I, although group II has been reported from Iran (Bashir et al 2006;Sokhandan et al 2007;Rasoulpour and Izadpanah 2008;Nematollahi et al 2012). In our research, we detected IB subgroup strains from melon in Iran for the fi rst time.…”

“…It is therefore concluded that Lorestan province isolates were phylogenetically close to East Azerbaijan province isolates. In previous studies, it has been demonstrated that Jol186, S337, E5 and Mgh91 isolates were members of subgroup IA (Bashir et al 2006;Nematollahi et al 2012). It is evident that LORA, LORB, LORD, LORE and LORH isolates of Lorestan province can be placed in subgroup IA, while other isolates, including LARC, LORF and LORG belong to a separate group adjacent to subgroup IB isolates.…”

Partial characterization and development of sensitive and reliablediagnostic for the detection of cucumber mosaic virus

“…Banana, cucurbit, tomato, and pepper are the crops most commonly affected by CMV (Palukaitis and GarciaArenal, 2003;Bashir et al, 2006;Dubey et al, 2010). A total of 300 samples were collected from three banana growing states in different geographical areas in India.…”

Serological Methods to Confirm Expression of Coat Protein Gene From an Iranian Isolate of Cucumber Mosaic Virus in Escherichia coli

“…The CMVCP gene was previously amplified from an Iranian CMV isolate (B13) by PCR, cloned in pTZ57R/T and sequenced with the following accession number AY871070 (13). Here, the CMV CP gene was subcloned from pTZ57C The diagram was generated by the use of the plasmid processor (15).…”