The molecular characterization of the coat protein sequence and differentiation of CMV- subgroup I on tobacco from native flora in Turkey

Mustafa Usta,Abidin Günay,Abdullah Güller

doi:10.15835/nbha48211822

Abstract

Cucumber mosaic virus (CMV) has a broad plant-host range and a wide ecological zone distribution. Virus-like symptoms were observed on tobacco fields of Adiyaman province (Turkey) showing conspicuous mottling, greenish mosaic patterns and severe malformations of leaves. A total of forty tobacco samples tested positive against CMV by reverse transcription polymerase chain reaction (RT-PCR) using coat protein gene specific primers. Five randomly chosen CMV isolates were cloned into pGEM T-Easy vector and transformed into Escherichia coli JM109 strain. The recombinant bacterial clones containing insert-DNA were further purified and sequenced bidirectionally. In multiplex-RT-PCR studies carried out, it was found that all 40 CMV isolates belong to Subgroup I by resulting a 593 bp long DNA fragments. CMV subgroup IA was found to predominate in 4 out of 5 tobacco samples and CMV subgroup IB was found in 1 out of 5 CMV-positive samples by comparing the isolates with CMV reference isolates in phylogenetic tree. However, no Subgroup II sequences were found by multiplex RT-PCR using discriminating primers. The nucleic acid sequences were analyzed for the investigation of diversity of coat protein (CP) sequences of 5 CMV isolates. The sequence similarity ranged from 94.2-100% with the CMV subgroup I isolates infecting diverse plants in other regions of the world. The evolutionary tree revealed that the CMV IA Adiyaman isolates exhibited a genetic affinity with Australian and Spanish isolates. However, the CMV IB Adiyaman isolate showed a close genetic relationship with only the Australian isolates. To our knowledge, this study shows for the first time the occurrence of CMV IA and IB isolates infecting cultured tobacco plants in Adiyaman province.

Highlights

Tobacco (Nicotiana tabacum L.) is one of the substantial economic products and is grown in most countries such as Brazil, Turkey, Canada, USA, and China where it represents primary producing fields (FAO, 2013)
ELISA and RFLP assays are used (Haase et al, 1989; Shevchenko et al, 2015), Multiplex reverse transcription polymerase chain reaction (RT-PCR) is a practical method for the simultaneous differentiation of Cucumber mosaic virus (CMV) subgroup I and II (Rizos et al, 1992; Yu et al, 2005; Eyvazi et al, 2015)
Subgroup I (SI)-A, SI-B and SII groups of CMV have been reported in literature from numerous countries such as USA (D10538, IA), Japan (D16405, IA), South Korea (AJ27648, IA), Indonesia (AB042294, IB), Philippines (U20219, IB), Spain (AM183119, IA), Taiwan (D28780, IB), Italy (Y16926, IB), USA (AF127976, II), Australia (AF198103, II), South Africa (U37227, II), and Hungary (L15336, II) (Rodríguez Pardina et al, 2013; Arafati et al, 2013)

Summary

Introduction

Tobacco (Nicotiana tabacum L.) is one of the substantial economic products and is grown in most countries such as Brazil, Turkey, Canada, USA, and China where it represents primary producing fields (FAO, 2013). Virus-borne diseases frequently lead to reduced quality of product and yield. Tobacco crops are attacked by numerous viruses such as potato Y potyvirus (PVY), tobacco ringspot nepovirus (TRSV), pepper mottle potyvirus (PeMoV), cucumber mosaic cucumovirus (CMV), alfalfa mosaic alfamovirus (AMV), tobacco leaf. Received in revised form: 20 Apr 2020.

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