Phylogenetic Analysis of New Isolates of Cucumber mosaic virus from Iran on the Basis of Different Genomic Regions

Abstract

Plant Protection Department, University of Tabriz, 29 Bahman Blvd., Tabriz 51664 Iran(Received on June 10, 2012; Revised on July 31, 2012; Accepted on August 5, 2012)Molecular characterization of Cucumber mosaic virus(CMV) was done by using samples from tomato andcucurbitaceous plants collected from different locationsin the northwest region of Iran. After screening byenzyme-linked immunosorbent assay, 91 CMV-infectedsamples were identified. Biological properties of eightrepresentative isolates were compared with each otherrevealing two distinct phenotypes on squash and tomatoplants. Phylogenetic analyses based on nucleotide sequencesof the coat protein (CP), movement protein (MP) and 2bof the new isolates, together with that of previouslyreported isolates, led to the placement of the Iranianisolates in subgroups IA and IB according to CP andMP genes, but in subgroup IA according to the 2b gene.These data suggest that reassortment may have been amajor event in the evolution of CMV in Iran, and thatthe Iranian isolates are derived from a common recentancestor that had passed through a bottleneck event. Keywords :CMV, CP, genotype, MP, phylogenetic analysis,subgroup, 2bCucumber mosaic virus (CMV) is the type member of thegenus Cucumovirus in the family Bromoviridae. CMV hasa wide host range in 85 distinct families of dicotyledonousand monocotyledonous angiosperms (Bujarski et al., 2012).The virus is transmitted by some 80 aphid species belong-ing to 33 genera in a non- persistent non- circulative manner(Palukaitis and Garcia-Arenal, 2003). CMV causes diseaseoutbreaks in tomato, pepper or cucurbits which result ingreat economic losses for many countries (Gallitelli, 2000).CMV has three single-stranded plus-sense RNAs (RNA1-3) that code for five functional proteins [1a, 2a, 2b, 3a andcoat protein (CP)]. Proteins 1a and 2a, encoded by RNA1and RNA2, respectively, are components of the replicasecomplex (Hayes and Buck, 1990). The RNA3- encodedproteins, 3a [movement protein (MP)] and CP are involvedin viral movement. The CP is translated from the sub-genomic RNA4 and required for encapsidation, long-distance movement and transmission by aphids (Boccardand Baulcombe, 1993; Perry et al., 1994; Shi et al., 2002).Protein 2b is expressed from RNA 4A, a subgenomic RNAderived from RNA2, is involved in the long-distancemovement of CMV in the plant (Soards et al., 2002) andfunctions as a suppressor of RNA silencing (Ding et al.,1994; Li et al., 1999). Many strains and isolates of CMVhave been characterized on the basis of symptoms and hostrange (Choi et al., 2005; Huppert et al., 2002; Palukaitis andGarcia-Arenal, 2003) and often, differences in such pheno-types were mapped to the genomic RNAs that code for theCP (Shintaku et al., 1992; Sugiyama et al., 2000; Suzuki etal., 1995), MP (Choi et al., 2005) or 2b protein (Du et al.,2007; Shi et al., 2002; Lewsey et al., 2009, 2010). Phylogenetic studies have resulted in classification ofCMV isolates to three subgroups designated subgroup IA(S-IA), subgroup IB (S-IB) and subgroup II (S-II) (Palukaitiset al., 1992; Roossinck et al., 1999). Subgroup I (S-I) and S-II isolates have 75% nucleotide (nt) sequence identitywhereas S-IA and S-IB are more closely related and share92−95% identity (Roossinck, 2001). In a previous study wedetected members of S-IA in cucurbits in Iran and analyzedtheir phylogenetic positions based on the CP sequences(Bashir et al., 2008). Here, we studied some of the bio-logical properties of eight representative CMV isolates ondifferent hosts. In addition to amplification of the MP andCP genes from the collected samples, a specific pair ofprimers was designed to amplify the 2b gene from Iranianisolates of CMV. The three amplified genomic regions werecloned, sequenced and their phylogenetic relationshipswere determined. Also, the genotypes of eight representativeisolates were investigated in relation to their symptoms onexperimental host plants.Leaves of tomato and cucurbitaceous plants showingsymptoms similar to those caused by CMV, were collectedfrom 17 locations in the Ardabil, East Azarbaijan, and WestAzarbaijan provinces during 2009−2010. Eight representa-tive CMV isolates from distinct geographical locations