Identification and Molecular Analysis of Sugarcane mosaic virus (SCMV) in Mazandaran Province

Abstract

Introduction: Several potyviruses infecting gramineous plants have been reported from Iran. Mosaic is the most widespread viral disease affecting sugarcane and maize production. Sugarcane mosaic virus (SCMV) is one of the causal agents of mosaic disease, which was first described in Ohio in 1963. SCMV belonging to the genus Potyvirus, family Potyviridae, is a single-stranded positive sense RNA virus which can infect gramineous plants such as sugarcane, maize and sorghum. Like all potyviruses, SCMV has non-enveloped flexuous filamentous virions of 750 nm long and 11–13 nm wide, which encapsidate a single-stranded, positive-sense RNA molecule of approximately 10,000 nt long. SCMV subgroup in the genus Potyvirus consists of seven different species viz., SCMV, Sorghum mosaic virus (SrMV), Maize dwarf mosaic virus (MDMV), Johnsongrass mosaic virus (JGMV); Sorghum mosaic virus (SrMV), Zea mosaic virus (ZeMV), Pennisetum mosaic virus (PenMV) and Cocksfoot streak virus (CSV). These viruses have been transmitted by aphids in a non-persistent manner, and cause mosaic disease and yield loss in sugarcane, maize, sorghum, and other gramineous plants. Among these viruses, only SCMV and SrMV are known to infect sugarcane under natural conditions and are considered as the causal agents of sugarcane mosaic disease. SCMV causes yield losses in many countries, and losses of maize production have been estimated at 30–80%, making it the most economically important problem in world maize production. The first report of SCMV in Iran was from Khuzestan province in 1993. There are also partial sequences, mostly from the 3'-UTR and coat protein regions, of some isolates of this virus in Iran. As Mazandaran province in North of Iran is one of the major producing areas of maize and sugarcane, identification of this virus is a concern. The purpose of this research was to study the existence of SCMV in research areas and determining of its phylogenetic relationship. Materials and Methods: From July to September 2013 and 2014, a total of 45 leaf samples of maize and sugarcane showing virus symptoms were collected from different maize and sugarcane fields in Mazandaran province. Total RNA was extracted from all samples. First-strand cDNA and PCR amplification were carried out with an SCMV-specific primer F4/R3 corresponding to the virus CP gene. Expected PCR products of 900 bp were purified from 1% agarose gels using Gel Recovery kit, then cloned into the pTG19-T vector and sequenced. Sequences were compared to data available in the GenBank. Phylogenetic tree for grouping based on nucleotide sequences was constructed by MEGA 6 software program using the neighbor-joining method. Multiple alignments of the nucleotide and amino acid sequences were carried out using the ClustalW and DNAMAN7 software. Results and Discussion: Amplification product (900 bp) was obtained from 35 infected samples, but not from healthy samples. The most typical symptoms in positive samples were mosaic, mosaic strips, and yellow chlorotic stripes. Four selected PCR positive samples were cloned into the pTG19-T vector and sequenced. BLASTn analysis of the sequenced data revealed that the PCR-amplified fragments belonged to CP gene of SCMV. Four selected isolates named Maz-SCR1 and Maz-SCR2 from sugarcane (Ghaemshahr), Maz-MR from maize (Ghaemshahr) and Maz-MB from maize (Babolsar). Phylogenetic tree based on multiple sequence alignment of CP gene divided all SCMV isolates into two groups: I and II. Members of each group were divided into two subgroups: A, B. Mazandaran isolates of SCMV were grouped with isolates of Khuzestan and Egypt in a subclade in subgroup IA. Phylogenetic analysis showed that Mazandaran isolates share 78 to 99 % nucleotide sequence identity and 79.5-100 % amino acid sequence identity with other isolates of SCMV. Also, the identity of these four isolates in the nucleotide and amino acid levels ranged between 97 to 98.9 % and 99.3 to 100 %, with each other, respectively. Isolate of Mazandaran showed the highest nucleotide sequence identity with khzL66 and khzQ86 isolates from Khuzestan (between 96.6 to 98.8 %) and EGY7-1 isolate from Egypt (between 96.7 to 99 %) and the lowest nucleotide sequence identity with BD8 isolate from China (between 78 to 78.8 %). The previously identified and conserved amino acid sequence motifs described in CP of potyviruses were present in Mazandaran isolates CP gene. Conclusion: SCMV is a widespread cereal potyvirus in the world and often occurs in a mixed infection with SrMV. In this study, for the first time, we reported the occurrence of SCMV in maize and sugarcane fields in Mazandaran province based on CP gene analyses and determined its phylogenetic relationship with other SCMV isolates available in the GenBank. Iranian isolates of SCMV were grouped with the isolate of Egypt in a subclade in subgroup IA. It is speculated that the Iranian isolates have been introduced into this country with sugarcane cuttings. The data obtained in this study will be beneficial to improve control strategies for this virus in Iran. Identification and the use of more isolates and analysis of genes in addition to CP are recommended for a better understanding of the genetic structure and variation of SCMV populations on a large geographical scale.

Keywords

• Coat protein
• Mazandaran
• Phylogenetic analysis
• Sugarcane and maize
• Sugarcane mosaic virus