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]

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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 Lupinus polyphyllus AF015716 15/64 Great Britain Lupinus polyphyllus AF015717 S Great Britain Medicago sativa X00819 425_Madison USA Trifolium sp. K02703 425_Leiden USA Trifolium sp. L00162 126-A Italy Portulaca oleracea AJ130704 195-AN Italy Lycopersicon esculentum AJ130705 F-430 Italy Phaseolus vulgaris AJ130706 Danza Italy Lycopersicon esculentum Y09110 Lye-80 France Lycopersicon esculentum AJ130703 Caa-1 France Capsicum annuum AJ130707 Dac-16 France Daucus carota AJ130708 Lyh-1 France Solanum sp. AJ130709 Ca375 Canada Solanum tuberosum DQ314749 Ca175 Canada Solanum tuberosum DQ314750 Ca399 Canada Solanum tuberosum DQ314751 Ca400 Canada Solanum tuberosum DQ314752 Ca401 Canada Solanum tuberosum DQ314753 Ca508 Canada Solanum tuberosum DQ314754 Ca518 Canada Solanum tuberosum DQ314755 Ca616 Canada Solanum tuberosum DQ314756 N20 Australia / AF332998 NZ34 New Zealand Pisum sativum AF215664 KR1 Korea Solanum tuberosum AF294432 KR2 Korea Solanum tuberosum AF294433 Legend: / - no data available

RESULTS Serological analysis After visual inspection of the pepper plants in the inspected pepper production regions in R. Macedonia, plants showing general virus symptoms, such as: bright yellow to white mosaic on leaves, chlorotic line patterns and stunted growth were observed. The virus detection of AMV was determined on these plants by performing serological analysis (Table 2). K. BANDJO ORESHKOVIKJ et al.: MOLECULAR IDENTIFICATION OF AMV ON PEPPER 1051

In 2012, from 91 tested samples, 15% were infected with AMV throughout the whole vegetation. In 2013 and 2014, AMV was detected only in 2% of the inspected 84 samples.

Table 2. Virus detection of AMV on pepper cultivated in open fields in R. Macedonia for 2012-2014 Year Number of tested samples Number of infected samples with AMV 2012 91 14 (15%) 2013 84 2 (2%) 2014 84 2 (2%)

Molecular detection and identification An isolate of AMV obtained from the area around Kumanovo in 2013 (isolate KUA7-2013) that had the highest measured OD absorbance (2.575) amongst all of the tested samples was chosen for total RNA extraction and further molecular analysis. The primer set AMV-F2/R2 was used for amplification of the entire region of the CP gene, generating amplicon of 669 bp (Figure 1). In the negative control, where cDNA was omitted, no amplification product was observed.

М 1 2

669 bp

Figure 1. 1.5% agarose gel electrophoresis (AGE) analysis of the amplicon of the CP gene of Alfalfa mosaic virus (AMV) obtained by RT-PCR. M – Marker GeneRulerTM 50bp DNA Ladder (Fermentas Life Sciences GmbH, Lithuania); lane 1 – negative control; lane 2 – isolate KUA7-2013

Partial nucleotide sequence of the CP gene of the AMV isolate KUA7-2013 was generated using the forward primer AMV-F2 and was submitted in GenBank (GenBank Accession No KY549365). Sequence of the Macedonian isolate KUA7-2013 was compared with sequences of the CP gene of 29 AMV isolates from other parts of the world obtained from the GenBank database, determining nucleotide identities from 92 to 94.4%. The highest identity of 94.4% was found with the Canadian isolate Ca375 (DQ314749). The lowest nucleotide identity of 92% was determined with the isolate 15/64 from Great Britain (AF015717). 1052 GENETIKA, Vol. 49, No3, 1047-1057, 2017

The Macedonian isolate obtained from this study showed identities at the amino acid level with the other isolates from the GenBank database of 91.9 to 97.1%. The highest amino acid identity was with the isolate S from Great Britain (X00819), while the lowest was observed with the French isolate Lye-80 (AJ130703).

Phylogenetic analysis A phylogenetic tree was constructed based on the partial sequences of the CP gene of the Macedonian AMV isolate and 29 other isolates from the GenBank database using the Maximum parsimony method (Figure 2). All of the AMV isolates were clustered in four groups: group I, II, III and IV. The reliability of the second group, where the isolate KUA7-2013 generated from this study was situated, was evaluated with a high bootstrap value of 92%.

Figure 2. Phylogenetic tree constructed with the use of Maximum parsimony method (number of bootstrap trials: 1000; bootstrap values >50% are shown next to relevant branches), based on the partial nucleotide sequences of the CP gene of 30 AMV isolates. The Macedonian AMV isolate is underlined and bolded.

The average genetic diversity of the AMV sequences in the phylogenetic tree was 0.038±0.004. The genetic diversity between the four groups ranged from 0,023±0,004 to 0,062±0,009. The diversity within the groups was: 0,020±0,004 for group I, 0,035±0,005 for II, 0,017±0,004 for III and 0,019±0,004 for group IV. K. BANDJO ORESHKOVIKJ et al.: MOLECULAR IDENTIFICATION OF AMV ON PEPPER 1053

The AMV isolates from the phylogenetic tree were clustered in four groups, regardless of their geographic origin or plant host. In group I, two isolates from Korea, two from USA and one isolate from Canada, Great Britain, Croatia, Italy and Australia were clustered. In the second group, the isolate KUA7-2013 from R. Macedonia was situated, together with isolates from France and Great Britain. Group III was formed with two isolates from Croatia and one isolate from Serbia and New Zealand and in the group IV, isolates from Italy and Canada were clustered.

DISCUSSION In this three-year study, AMV was observed in pepper plants cultivated in open fields in R. Macedonia during the whole trial. During 2012, AMV was detected in 15% of the tested samples, later on to show a decrease in the distribution (only 2% of the examined pepper plants in 2013 and 2014). Fluctuations in the dispersal of AMV, ranging from several tested samples to widespread distribution, were also observed by other authors (CHATZIVASSILIOU et al., 2004; MASSUMI and POUR, 2007; WANG et al., 2012; MILOŠEVIĆ, 2013; RUSEVSKI et al., 2009; 2010; 2011; 2013; STANKOVIĆ et al., 2014). Total RNA extraction and amplification of the CP gene, generating amplicon of 669 bp were performed on a chosen KUA7-2013 isolate. The obtained CP gene partial sequence of the Macedonian AMV isolate was compared with CP gene sequences from other parts of the world, determining nucleotide identities from 92 to 94.4% and identities at the amino acid level of 91.9 to 97.1%. A high degree of nucleotide (93.7 – 99.5%) and amino acid identity (92 – 100%) between sequences of various AMV isolates were observed in other studies (FINETTI SIALER et al., 1997; PARRELLA et al., 2000; XU and NIE, 2006; MILOŠEVIĆ, 2013). Since AMV is the sole representative of the genus Alfamovirus, there are no demarcation criteria in determining different species in this genus (PLYUSNIN et al., 2011) and that is why no “outgroup” was used in creating of the phylogenetic tree. After performing phylogenetic analysis based on the CP gene partial sequence, it was observed that the Macedonian isolate KUA7-2013 and the other 29 AMV isolates from different parts of the world have been clustered in 4 groups (I, II, III and IV). The reliability of the second group, where the isolate KUA7-2013 generated from this study was situated, was evaluated with a high bootstrap value of 92%. Clustering of AMV isolates in groups varied according to different authors. According to PARRELLA et al. (2000) and PARRELLA et al. (2010), AMV isolates were divided in two distinct groups: I and II. In later examinations, PARRELLA et al. (2011) fragmented the second group on subgroups AMV IIA and AMV IIB. These authors based the division of groups on the assumption of different evolution between the groups as a result of geographical distance. Our findings correspond to those of XU and NIE (2006) and STANKOVIĆ et al. (2014), who have clustered AMV isolates in four or more different groups, which grouping wasn’t based on geographical distribution. As stated above, various authors performed clustering of AMV isolates on different divisions. Mutual for all of these divisions was the clustering of AMV isolates within group II, where the Macedonian isolate KUA7-2013 was situated. This group could be observed in all types of divisions (PARRELLA et al., 2000; XU and NIE, 2006; PARRELLA et al., 2010; PARRELLA et al., 2011; MILOŠEVIĆ, 2013; STANKOVIĆ et al., 2014). Within the deduced amino acid sequences of the AMV isolates from group II, 4 conservative positions could be observed: at 1054 GENETIKA, Vol. 49, No3, 1047-1057, 2017 position 67 the amino acid Serine (Ser; S) was observed, at position 84 Alanine (Ala; A), at position 176 Leucine (Leu; L) and at position 214 Aspartic acid (Asp; D) was observed (PARRELLA et al., 2000). From these 4 conservative positions, the Macedonian isolate KUA7- 2013 generated in this study, within its amino acid sequence had only one conservative position, at 84 (Alanine). That is why, a mild separation of the isolate KUA7-2013 from the other isolates within group II was observed.

CONCLUSIONS Alfalfa mosaic virus was detected during the whole three-year trial on pepper plants cultivated in open fields. The Macedonian isolate KUA7-2013 showed high nucleotide (92 – 94.4%) and amino acid identities (91.9 – 97.1%) with AMV isolates from other parts of the world. In the phylogenetic tree created in this study, KUA7-2013 was situated in group II, together with isolates from France and Great Britain. This is the first time a Macedonian isolate of AMV to be identified at the molecular level. The information obtained from these investigations will contribute for further analysis of AMV on pepper and other host plants in R. Macedonia. Received, March 22th, 2017 Accepted July 18th, 2017

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MOLEKULARNA DETEKCIJA I IDENTIFIKACIJA ALFALFA MOSAIC VIRUS (AMV) KOD PAPRIKE GAJENE U R. MAKEDONIJI

Katerina BANDJO ORESHKOVIKJ1,a, Rade RUSEVSKI2, Biljana KUZMANOVSKA2, Mirjana JANKULOVSKA3, Zoran T. POPOVSKI4

1 Odeljenje za zaštitu bilja, Poljoprivredni institut, Univerzitet “Sv. Ćiril i Metodije” Skoplje, R. Makedonija 2 Odeljenje za fitopatologiju, Fakultet za poljoprivredne nauke i hranu, Univerzitet “Sv. Ćiril i Metodije” Skoplje, R. Makedonija 3 Odeljenje za genetiku i oplemenjivanje biljaka Fakultet za poljoprivredne nauke i hranu, Univerzitet “Sv. Ćiril i Metodije” Skoplje, R. Makedonija 4 Odeljenje za biohemiju i genetički inžinjering, Fakultet za poljoprivredne nauke i hranu, Univerzitet “Sv. Ćiril i Metodije” Skoplje, R. Makedonija

Izvod Alfalfa mosaic virus (AMV) je jedan od najrasprostranjenijih i ekonomski veoma značajnih biljnih virusa na paprici u R. Makedoniji. Izvršena je serološka detekcija AMV u osam proizvodnih područja u R.Makedoniji, kao i molekularna identifikacija reprezentativnih izolata. Detekcija AMV urađena je DAS-ELISA metodom, a za molekularnu detekciju korišćen je i RT- PCR. Na osnovu parcijalnih sekvenci urađena je filogenetska analiza. Makedonski izolat KUA7- 2013, dobijen u ovom radu, upoređen je sa 29 AMV izolata iz drugih delova sveta. Utvrđen je visok nivo nukleotidnog (92 – 94.4%) i aminokiselinskog idenditeta (91.9 – 97.1%). Na filogenetskom stablu makedonski izolat je bio u klasteru II zajedno sa izolatima iz Francuske i Velike Britanije. U ovom radu, po prvi put u R.Makedoniji, jedan izolat AMV je identifikovan na molekularnom nivou.

Primljeno 22.III.2017. Odobreno 18. VII. 2017.