UDC 575.630 https://doi.org/10.2298/GENSR1703047B Original scientific paper MOLECULAR DETECTION AND IDENTIFICATION OF ALFALFA MOSAIC VIRUS (AMV) ON PEPPER CULTIVATED IN OPEN FIELDS IN R. MACEDONIA Katerina BANDJO ORESHKOVIKJ1,a, Rade RUSEVSKI2, Biljana KUZMANOVSKA2, Mirjana JANKULOVSKA3, Zoran T. POPOVSKI4 1 Department of Plant Protection, Institute of Agriculture, University “Ss. Cyril and Methodius” Skopje, R. Macedonia 2 Department of Phytopathology, Faculty of Agricultural Sciences and Food, University “Ss. Cyril and Methodius” Skopje, R. Macedonia 3 Department of Genetics and Plant Breeding, Faculty of Agricultural Sciences and Food, University “Ss. Cyril and Methodius” Skopje, R. Macedonia 4 Department of Biochemistry and Genetic Engineering, Faculty of Agricultural Sciences and Food, University “Ss. Cyril and Methodius” Skopje, R. Macedonia Bandjo Oreshkovikj K., R. Rusevski, B. Kuzmanovska, M. Jankulovska, Z. T. Popovski (2017): Molecular detection and identification of alfalfa mosaic virus (AMV) on pepper cultivated in open fields in R. Macedonia.- Genetika, Vol 49, No.3, 1047-1057. Alfalfa mosaic virus (AMV) is one of the most distributed and economically important plant viruses in pepper in R. Macedonia. Serological detection of AMV in eight important pepper production regions in R. Macedonia and molecular identification of a representative isolate were performed. The virus detection of AMV was conducted using DAS-ELISA method. In order to make molecular detection, RT-PCR was performed. Phylogenetic analysis was conducted, based on the partial sequences of the coat protein gene. A genetic relationship of the Macedonian isolate KUA7-2013 gained in this study was compared with 29 AMV isolates from other parts of the world. High level of nucleotide (92 – 94.4%) and amino acid identities (91.9 – 97.1%) were determined. In the constructed phylogenetic tree, the Macedonian AMV isolate was clustered in group II together with isolates from France and Great Britain. In this ___________________________ Corresponding author: Katerina Bandjo Oreshkovikj, Institute of Agriculture, blvd.”16-ta Makedonska Brigada” no.3A, 1000 Skopje, Republic of Macedonia, tel: +389 2 3230910, fax: +389 2 3114283, e-mail: [email protected] 1048 GENETIKA, Vol. 49, No3, 1047-1057, 2017 study, for the first time in R. Macedonia, an isolate of AMV was identified at the molecular level. Key words: Alfalfa mosaic virus, pepper, molecular detection, phylogenetic analysis, coat protein gene sequences. INTRODUCTION Alfalfa mosaic virus (AMV) is one of the most spread viruses on pepper cultivated in open fields in R. Macedonia (JOVANCHEV et al., 1996; RUSEVSKI and BANDZO, 1998; RUSEVSKI et al., 2010; 2013). Due to its high incidence in the infected fields, AMV can cause significant damage on pepper yield, which can be reduced up to 65% (ŠUTIĆ, 1995). AMV was described for the first time by WEIMER (1931) as alfalfa pathogen. It belongs to the family Bromoviridae (BÜCHEN-OSMOND, 2006) as a sole representative of the genus Alfamovirus (BOL, 2003; BÜCHEN-OSMOND, 2006). The virus is distributed worldwide (ŠUTIĆ, 1995) affecting more than 400 plant species from over 50 families (BÜCHEN-OSMOND, 2006). АМV has a tripartite genome, consisted of single-stranded, linear, infective RNA (MURPHY et al., 1995; BOL, 2003; BÜCHEN-OSMOND, 2006; RUSEVSKI and KUZMANOVSKA, 2014), where RNA1 is the largest and RNA3 is the smallest macromolecule (MURPHY et al., 1995). Beside the genomic RNA molecules, AMV also contains and sub-genomic RNA4 molecule which serves as messenger RNA for the coat protein (CP) (MURPHY et al., 1995; BOL, 2003). The coat protein participates in the virus movement (HERRANZ et al., 2012), genome activation and virus expression (NEELEMAN et al., 1993; HOUWING et al., 1998; HOUWING and JASPARS, 2000; JASPARS and HOUWING, 2002). The CP also partakes in virus transmission by insect vectors in non-persistent manner (ŠUTIĆ, 1995; ORMEÑO et al., 2006; BÜCHEN-OSMOND, 2006). In the family Bromoviridae it was observed that various mutations of the CP gene, which reflect on the amino acid sequence of the CP, could alter aphid transmissibility (SMITH et al., 2000; NG and PERRY, 2004; GARCIA-ARENAL and PALUKAITIS, 2008), even if the change occurs only in one amino acid (PERRY et al., 1998). By determining the nucleotide sequences of the CP-gene, AMV isolates have been clustered in various groups by different authors (PARRELLA et al., 2000; 2010; 2011; XU and NIE, 2006; MILOŠEVIĆ, 2013; STANKOVIĆ et al., 2014). Following previous findings of AMV on pepper in open fields in R. Macedonia (RUSEVSKI et al., 2009; 2010; 2011; 2013), and aiming to underline AMV influence on pepper production, a study was conducted in order to detect occurrence of AMV and to determine the genetic relationship of the Macedonian AMV isolate gained in this study with isolates from other parts of the world. MATERIAL AND METHODS Plant samples and serological testing A total of 259 pepper plant samples (91 sample in 2012, 84 samples in 2013 and 84 samples in 2014) were collected after visual inspection at 13 different localities from eight pepper production regions in R. Macedonia (areas around Skopje, Kumanovo, Sveti Nikole, Kochani, Strumica, Radovish, Prilep and Bitola). Samples were collected from symptomatic plants showing general virus symptoms, such as: bright yellow to white mosaic on leaves, chlorotic line patterns and stunted pepper plants. In order to perform isolation and testing of AMV, pepper leaves were K. BANDJO ORESHKOVIKJ et al.: MOLECULAR IDENTIFICATION OF AMV ON PEPPER 1049 collected from the upper parts of the plant. The samples were brought to the laboratory by placing on ice and stored at -20ºC until tested. Serological testing was performed utilizing double antibody sandwich (DAS) ELISA kit with commercial antisera specific for detection of AMV, as described by CLARK and ADAMS (1977) and modified and proposed by BIOREBA - AG (WERNLI, 1999) using commercial polyclonal antiserum. Plant tissue samples were homogenized in extraction buffer (1:10 w/v). Commercial positive and negative controls produced from the same manufacturer were included on each plate. The tested samples were considered to be positive if the average optical density (OD) value after incubation of one hour at room temperature in the dark was higher at least twice than the average OD of the negative control, measured with an ELISA microplate reader MULTISCAN ASCENT at absorbance of 405 nm. RNA extraction and RT-PCR of the coat protein gene Total RNA was extracted using TRIzol® Reagent (Ambion, Life Technologies) according to manufacturer’s instructions (XU et al., 2004; XU and NIE, 2006; CHEN et al., 2011; WANG et al., 2012). Homogenization of the plant material was performed in liquid nitrogen, in order to prevent RNA degradation (BERTOLINI et al., 2003). Reverse transcription (RT) was performed in a total volume of 20 μl reaction mixture using 3μl of total RNA, which was added to 2μl 10xPCR Buffer Gold, 4μl MgCl2 (25mM), 8μl dNTP’s (2.5mM), 1μl (50pM/μl) of reverse primer AMV-R2 (5’- TCAATGACGATCAAGATCGTC-3’), 1μl RNase Inhibitor and 1μl of MuLV Reverse Transcriptase (Applied Biosystems, USA). The RT was performed according to VAN DONGEN et al. (1999). Polymerase chain reaction (PCR) of the coat protein gene was done in 25μl mixture volume, which contained 5μl cDNA, 2.5μl 10xPCR Buffer II, 2.5μl MgCl2 (25mM), 2μl dNTP’s (2.5mM), 0.5μl (100pM/μl) of forward primer AMV-F2 (5’- ATCATGAGTTCTTCACAAAAGAA-3’), 0.5μl (100pM/μl) of reverse primer AMV-R2 and 0.25μl of Taq DNA Polymerase (Sigma-Aldrich, USA). The AMV primers and PCR protocol were according to XU and NIE (2006). RT-PCR was performed on thermocycler Techne, TC – 512 (Fisher Scientific, USA). In the negative control, cDNA was omitted. Amplified products were analyzed by 1.5% agarose gel electrophoresis, in 1xTBE buffer, stained with ethidium bromide and visualized under a UV transilluminator (POPOVSKI et al., 2013). Sequencing and phylogenetic analysis PCR product of KUA7-2013 isolate was purified using a BigDye® XTerminator Purification Kit (Applied Biosystems, USA) and performed by DNA-analyzer (Genetic Analyzer 3500, Applied Biosystems), using AMV-F2 primer. The nucleotide sequence of the amplification product was deposited in GenBank database and it was assigned an accession number. The AMV sequence generated in this study was compared with previously reported AMV isolates available in GenBank (http://www.ncbi.nlm.nih.gov/BLAST/), using the ClustalW program (THOMPSON et al., 1994) and MEGA6 software (TAMURA et al., 2013). A p-distance model was applied for nucleotide (nt) and deduced amino acid (aa) sequence analyses. A phylogenetic tree was created using the AMV CP gene partial sequence generated in this study and 29 CP gene sequences of AMV retrieved from GenBank (Table 1) by the 1050 GENETIKA, Vol. 49, No3, 1047-1057, 2017 bootstrap Maximum parsimony method (number of bootstrap trials: 1000; bootstrap values <50% were omitted). Intra- and inter-group diversity values were calculated as the average genetic distance using Kimura 2-parameter model Gamma distributed (K2+G) (KIMURA, 1980). Table 1. Coat protein gene sequences of Alfalfa mosaic virus isolates from GenBank database used in the phylogenetic analysis Isolate Country Plant host Accession number 70-12 Croatia Lavandula x intermedia JX996119 371-13 Croatia Lavandula x intermedia KJ504107 373-13 Croatia Lavandula x intermedia KJ504108 196-08 Serbia Nicotiana tabacum FJ527749 VRU Great Britain