Distribution and molecular detection of apple mosaic virus in apple and hazelnut in Turkey

Abstract

Apple mosaic virus (ApMV) is one of the most important diseases limiting the production of hazelnut and apple in Turkey and the objectives of this research were to determine the convenient and reliable method for RNA isolation and also to determine primer pair for real time polymerase chain reaction (RT-PCR) detection of coat protein gene for Turkish ApMV isolates. Apple mosaic virus isolates were collected in 2007 to 2010 and the presence of the pathogen was detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and RT-PCR tests. Six different RNA extraction protocols and three primer pairs were applied in RT-PCR amplifications and 44 hazelnut and 15 apple ApMV isolates were obtained. All of the amplicons were subjected to enzymatic digestion with restriction endonuclease enzymes and phylogenetic analysis were performed according to the digestion profiles.

Is It Possible to Produce Certified Hazelnut Plant Material in Sicily? Identification and Recovery of Nebrodi Genetic Resources, in vitro Establishment, and Innovative Sanitation Technique From Apple Mosaic Virus

“…The amplified products of RT-PCR were 262 bp long fragments. Although, some amplified RNAs showed clear bands on electrophoresis gel, tissues of the bark of young branches gave a satisfactory result for total RNA extraction and reliable amplification of ApMV in one step RT-PCR, confirming the previous findings by Ertunc et al (2014) . The eight hazelnut samples from “Curcia,” “Nociara Collica,” “Panottara Collica,” “Panottara Galati Grande,” “Parrinara”, “Panottara Baratta Piccola,” “Enzo,” and “Rossa Galvagno” genotypes resulted positive to ApMV ( Supplementary Figure 1 ).…”

“…The obtained results confirmed the detection of the most economically damaging Ilarvirus affecting hazelnut by one step RT-PCR applied on a set of local Sicilian cultivars using dormant tissues, as described by Ertunc et al (2014) , overcoming the problem of having high amount of phenolic compounds, tannins in particular. The latter are considered a problem for the extraction of good quality RNA by several researchers that tried the herbaceous host inoculation ( Sokmen, 2003 ) and flower tissues extraction ( Akbas and Degirmenci, 2010 ).…”

“…Reverse transcriptase (RT)-PCR tests were made on total nucleic acids (TNAs) extracted from bark scraps of young branches since they showed to give better results in terms of RNA extract quality and quantity if compared with leaf tissues ( Ertunc et al, 2014 ) and using the silica capture method as described by Foissac et al (2001) . Bark scraps were frozen in liquid nitrogen and ground using mortar and pestle; the obtained powder was macerated with 1 mL of buffer (6 M guanidine thiocyanate containing 0.2 M sodium acetate, 25 mM EDTA, 1 M potassium acetate, 2.5% PVP-40 and 1% β-mercaptoethanol).…”

“…Following centrifugation at 13,000 rpm for 3 min, 150 μL of the supernatant was transferred into a new eppendorf tube and stored at –20°C. One step RT-PCR was performed using the primer pair designed for the detection of ApMV by Menzel et al (2002) , since no amplification was obtained with other primer sets previously tested for ApMV ( Ertunc et al, 2014 ). The PCR mixture contained virus-specific forward and reverse primers (0.2 μM each) ( Table 1 ), 1.5 mM dNTPs mix (Thermofisher), 2.5 μL 10 X Taq buffer (Thermofisher), 8 U MMLV_RT (Invitrogen), 1.2 U RNase inhibitor (Promega), 0.6 μM MgCl 2 , 1 U Taq DNA Polymerase (Thermofisher), 2 μL RNA template in a final volume of 25 μL adjusted with sterile distilled water.…”