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The Nucleotide Sequence of Barley Strain of Wheat Dwarf Virus Isolated in Hungary

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The Nucleotide Sequence of Barley Strain of Wheat Dwarf Virus Isolated in Hungary Cereal Research Communications 38(1), pp. 67–74 (2010) DOI: 10.1556/CRC.38.2010.1.7 The Nucleotide Sequence of Barley Strain of Wheat Dwarf Virus Isolated in Hungary I. TÓBIÁS1*, B. KISS1,K.SALÁNKI2 and L. PALKOVICS3 1Plant Protection Institute, Hungarian Academy of Sciences, P.O. Box 102, H-1525 Budapest, Hungary 2Agricultural Biotechnology Center, Szent-Györgyi A. út 4, H-2100 Gödöllõ, Hungary 3Department of Plant Pathology, Corvinus University of Budapest, Ménesi út 44, H-1118 Budapest, Hungary (Received 20 February 2009; accepted 17 September 2009) Barley-infecting isolates of Wheat dwarf virus (WDV) were collected in the field in the vi- cinity of the cities Dunakiliti, Heves and Siófok, in Hungary. Viral genomic DNA was ampli- fied by the rolling circle amplification technique, digested with HindIII, cloned into pBSK+ plasmid and sequenced. The clones were of the same size and showed above 99% identity to each other. Based on DNA sequences WDV-D01, WDV-H1 and WDV-H07 isolates showed high identity (94–99%) to isolates of WDV barley strain and Barley dwarf virus and lower identity to Oat dwarf virus (71% identity) and WDV wheat strains (85% identity). Keywords: Wheat dwarf virus, Barley dwarf virus, Oat dwarf virus, barley-infecting iso- lates, sequence analysis Introduction Wheat dwarf virus (WDV) is a frequent causal agent of dwarfing, mottling, yellowing or reddening in cereals. Suppressed heading and root growth in infected plants can drasti- cally reduce yield. WDV was first described by Vacke (1961) in former Czechoslovakia and later it has been found in Sweden (Lindsten et al. 1970), Bulgaria (Stephanov and Dimov 1981), Hungary (Bisztray et al. 1989), France (Lindsten and Lindsten 1993), Ger- many (Huth 2000), Poland (Jezewska 2001), Finland (Lemmetty and Huusela-Veistola 2005), Romania (Jilaveanu and Vacke 1995), Spain (Achon et al. 2006), Tunisia (Najar et al. 2000), Turkey (Köklü et al. 2007), Zambia (Kapooria and Ndunguru 2004) and China (Xie et al. 2007). WDV is transmitted by the leafhopper Psammotettix alienus (Vacke 1961), therefore the occurrence of diseased plants in the field depends on the presence of the vector. The incidence of WDV in crops differs greatly from year to year and among fields. Several important factors of the epidemiology of WDV are still poorly understood, such as alternative hosts in the field and virus variability. Two different forms of WDV ex- ist: a wheat-adapted form (WDV wheat strain) and a barley-adapted form (WDV barley strain) (Lindsten and Vacke 1991; Bendahmane et al. 1995; Kvarnheden et al. 2002). More recently Schubert et al. (2007) surveyed cereal samples and based on DNA sequence * Corresponding author; E-mail: [email protected] 0133-3720/$20.00 © 2010 Akadémiai Kiadó, Budapest 68 TÓBIÁS et al.: Sequence Comparison of Barley Strains of WDV differences they proposed the new mastrevirus species Barley dwarf virus (BDV) and Oat dwarf virus (ODV). WDV belongs to the genus Mastrevirus (family Geminiviridae) and infects monocoty- ledonous plants. Mastreviruses have a monopartite single-stranded genome of circular DNA and the genome encodes four different proteins: movement protein (MP) and coat protein (CP) on the viral-sense strand, while two replication-associated proteins (Rep and RepA) on the complementary-sense strand (Gutierrez 1999). The presence of an intron in the rep gene makes it possible for WDV to produce the two different forms of the replica- tion protein. The non-coding long intergenic region (LIR) and short intergenic region (SIR) contain sequence elements necessary for viral replication and transcription. The LIR comprises the origin of rolling circle replication of the virus (Heyraud et al. 1993). The SIR contains polyadenylation signals, and a region to which a short complementary primer for the second strand synthesis binds (Kammann et al. 1991). In the last ten years WDV has been found to be the most frequent causal agent of cereal viral diseases in Hungary (Mesterházy et al. 2002; Szunics et al. 2003). For this reason an etiological study and molecular characterisation has been started. The complete genome is known for two isolates of the wheat strain of WDV in Hungary (Tóbiás et al. 2006) but no complete genome of the barley strain has been published yet. The aim of this study was to characterise some isolates of the barley strain of WDV occurring in Hungary and compare them with the previously sequenced barley strain isolates of WDV originating from other regions. Hungarian barley strain isolates collected by us are designated as WDV with an isolate code, for the other barley isolates we use the name indicated in the literature. Materials and Methods Virus isolates The symptoms of viral infection were found during autumn observations carried out in winter barley crops in Dunakiliti (northwestern Hungary), Heves (Northeastern Hungary) and Siófok (Southern Hungary). Plants manifesting yellowing of leaves were placed in an insect-proof greenhouse and were tested for WDV with ELISA using a WDV kit (Bio-Rad No. 31202). The collected WDV-infected plants were replanted into clay pots and placed in an insect-proof isolation net. For use as a vector organism, thirty individuals of vi- rus-free Psammotettix alienus (Dahlb.) were placed underneath each net. One week later the leafhoppers were transferred to young seedlings (10 cm long) of barley (Hordeum vulgare) cv. Botond or cv. Jubilant. Six weeks later the plants were tested again for WDV with ELISA. Three isolates WDV-Bar[HU:D01:08] (collected in 2008 from Dunakiliti), WDV-Bar[HU:H1:08] (collected in 2008 from Heves) and WDV-Bar[HU:H07:04] (col- lected in 2004 from Siófok and maintained in our greenhouse by subsequent transmission) were selected for further studies. Cereal Research Communications 38, 2010 TÓBIÁS et al.: Sequence Comparison of Barley Strains of WDV 69 Isolation of virus DNA, cloning and sequence analysis of the WDV isolates DNA extraction and amplification were done according to Shepherd et al. (2008). A small leaf sample (approximately 40 mg) from WDV-positive barley plants was placed into an Eppendorf tube covered with 50 μl extraction solution (Extract-n-AmpTM Plant PCR kit, Sigma) and heated at 95 ºC for 10 min. Lastly 50 μl of dilution solution (also supplied with the kit) was added and the sample was then stored at –20 ºC or used directly as a template for rolling circle amplification (RCA) of the WDV genome (Haible et al. 2006). One microlitre of the final Extract-n-Amp DNA solution was mixed with 4 μl of Templi PhiTM sample buffer (TempliPhiTM, Amersham Biosciences), heated for 2 min at 94 ºC, and then brought to room temperature. Five μl of reaction buffer and 0.2 μl of enzyme mix were added to the cooled mixture and the Templi PhiTM extension reaction was run at 30 ºC for 18–20 h. WDV genome concatemers generated during Phi29 DNA polymerase amplifica- tion were digested with HindIII to release unit-length genomes. After digestion genomic DNA was separated in 1% agarose gels and extracted by a DNA purification kit (Fer- mentas DNA Extraction Kit). The WDV genome was inserted into a HindIII digested pBSK + plasmid (Stratagene). The recombinant plasmids were transformed into Esche- richia coli DH5a (Sambrook et al. 1989). Clones containing inserts with the expected size of 2.7 kb were sequenced with the DyeDeoxyTerminator Kit (Applied Biosystems) using reverse, universal (–20) and inter- nal primers. Sequence analysis was performed using University of Wisconsin Genetics Computer Groups (GCG) sequence analysis software package version 9.1. In order to determine the phylogenetic relationships between different barley isolates complete genomes were analysed. Sequence alignment, tree formation, and bootstrap analysis were done with the help of the software Clustal X 1.83. Results Virus isolates The collected barley plants were WDV infected as confirmed by ELISA tests. The virus was transmitted by P. alienus and maintained on barley plants. Nucleic acids were isolated from plants infected with WDV-Bar[HU:D01:08], WDV-Bar[HU:H1:08] and WDV-Bar[HU:H07:04]. Virus DNA isolation, cloning and sequence analysis of the WDV strains The Extraction-n-Amp DNA extraction method was very rapid and simple in order to iso- late the full length WDV genomes. Combined with the RCA method a vast number of vi- rus genome concameters were produced, which were digested with HindIII and released as unit-length genomes (Fig. 1) with sticky ends. The size of the full length genome prepared from WDV-D01, WDV-H07 and WDV-H1 was the same, exactly 2734 nucleotides. The genome contained all the four ex- pressed mastrevirus ORFs (MP, CP, Rep, RepA), and the intergenic regions LIR and SIR. Cereal Research Communications 38, 2010 70 TÓBIÁS et al.: Sequence Comparison of Barley Strains of WDV 1M2 Figure 1. Separation of rolling circle amplification DNA product of WDV-Bar[HU:H07:04] isolate on a 1% agarose gel stained with ethidium bromide. M: DNA length marker Pst I digested lDNA (11501, 5077, 4749, 4507, 2838, 2556, 2443, 2140, 1986, 1700, 1159, 1093, 805, 514, 468, 448, 339, 264 and 247 bp), Lane 1: Phi29 polymerase amplified DNA product (WDV-H07) digested with Hind III, Lane 2: Undigested DNA product (WDV-H07) The nucleotide sequences were deposited into GenBank as WDV-Bar[HU:D01:08] (FM 999832), WDV-Bar[HU:H07:04] (FM210034) and WDV-Bar[HU:H1:08] (FM 999833). The nucleotide sequences of WDV-Bar[HU:D01:08], WDV-Bar[HU:H1:08) and WDV-Bar[HU:H07:04] were compared to those of known isolates of WDV barley strain (Köklü et al. 2007; Tóbiás et al. 2009), BDV (Schubert et al. 2007), ODV (Schubert et al. 2007) and WDV wheat strain (Tóbiás et al.
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