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Nyugat-Magyarországi Egyetem UNIVERSITY OF PANNONIA Georgikon Faculty KESZTHELY Doctoral School of Crop Production and Horticultural Sciences PhD THESIS Application and development of detection methods of grape viruses in the survey of virus infection of vineyards Melinda Apró (PhD candidate) Supervisor(s): Dr. habil. András Péter Takács Associate professor Director of Institute KESZTHELY 2016 BACKGROUND AND OBJECTIVES OF THE RESEARCH In recent years not only the number of grape varieties have risen, but their parasitic pathogens have increased as well, and these pathogens have caused harm. Knowing the biology of these pathogens, the practice of modern production technologies and the use of different pesticides all enable us to defend fungous and bacterial diseases adequately. The protection against diseases caused by phytoplasma and viruses is possible with the prevention of pathogens. Up to the present more than 60 plant viruses have been found to infect grapevines. In the Hungarian vineyards 17 grape viruses have been identified so far. In Hungary the grape virological research was started by János Lehoczky and his colleagues in 1960. These researches mainly focused on the identification of pathogens and their ways of living. The aim of this work is to continue grape virological researches previously started by the Institution of Plant Protection of Pannon University. Moreover, this work aims to shed light on the spread of grapevine virus occurrence in Hungarian vineyards. Furthermore: - the molecular analysis of the Hungarian GLRaV-3 isolate, which is a worldwide grapevine virus causing leaf-roll - the diagnoses of the origin and the degree of kinship of isolates - the data of isolates in GenBanks. 2 MATERIALS AND METHODS Plant material From 2012 to 2014 grapevine leaf samples were collected from 325 different vine-stocks, which differed in age and in type as well. The leaf samples were collected from 22 wine-growing regions of the 7 wine regions in Hungary. These places were: Badacsony wine region (Csobánc), Balatonboglár wine region (Ordacsehi), Balaton-felvidék wine region (Sümeg, Szentbékkálla, Becehegy), Balatonfüred-Csopak wine region (Aszófő), Nagy-Somló wine region (Ság-hegy), Zala wine region (Zalaháshágy, Keszthely, Szalafő), Csongrád wine region (Pusztamérges), Hajós-Bajai wine region (Érsekhalma, Borota), Kunság wine region (Kiskunhalas), Bükk wine region (Miskolc), Eger wine region (Eger), Mátra wine region (Gyöngyös), Etyek-Buda wine region (Budafok), Mór wine region (Mór), Neszmély wine region (Tata), Pannonhalma wine region (Pannonhalma), Pécs wine region (Szigetvár), Szekszárd wine region (Szekszárd), Tolna wine region (Mágocs), Villány wine region (Siklós), Sopron wine region (Sopron, Kőszeg), Tokaj wine region (Tokaj). The sample collection times were taken into account carefully due to the fact that the concentration of the different viruses is not constant and it may vary depending on grape phenological condition. The best period to detect the viruses belonging to the Nepo-, Macula- and Alfamovirus stem is from grape blooming to early summer, while the 3 concentration of viruses belonging to Clostero-Ampelo- and Vitivirus stem are the highest in infected plants from the berry burgeon to early autumn. Leaf samples showing virus symptoms and apparently symptomless leaf samples were both collected. During the first sample collecting period the young leaves were collected from the top leaf floors, and during the second period the older leaves were collected from the lower leaf floors in order to find the higher virus concentration. The location of the wine-stocks were recorded. Until tests were carried out, the leaf samples were transported and stored at 4 oC. For the tests 8x12 (96 pieces) 300 μl sample places with polystyrene micro titer plates were used. The changes in colour were measured valuable at 405 nm wavelengths on Labsystem Multiscan ELISA reader. Samples were considered positive if the extinction value of the detected sample exceeded three times the extinction value of the healthy controls. Serological methods: Virus content was checked by DAS-ELISA (doubly-antybody sandwich enzyme linked immunosorbent assay) method using Loewe Biochemica (Germany), Bioreba AG (Switzerland) and Agritest S.r.l. (Italy). Twelve pathogen viruses occurrence were tested. The presence of viruses was checked for different specific antiserum: Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV) and Grapevine chrome mosaic virus (GCMV) which belongs to Nepovirus genus. Among the member of Maculavirus genus: Grapevine fleck virus (GFKV). Members 4 of Vitivirus genus: Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine leafroll-associated virus 6 (GLRaV-6) and Grapevine leafroll- associated virus 7 (GLRaV-7), Alfamovirus genius member: Alfalfa mosaic virus (AMV). Furthermore the Closterovirus genus member: Grapevine leafroll-associated virus 2 (GLRaV-2) and Ampelovirus genus members: Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine leafroll-associated virus 1 (GLRaV-1). The tests polystyrene 8x12 (96 pieces) 300 μl workplaces micro titer plates were used. Substrate absorbances were measured at 405 nm wavelengths on Labsystem Multiscan ELISA reader. Samples were considered positive if the absorbance values exceeded three times those of the healthy controls. Molecular methods: Two GLRaV-3 positive samples from North Hungary, Sopron wine region (Kőszeg), were tested by RT-PCR. The samples were red grape varieties, Blue Frankish. Aim was the characterization and sequencing the ORF7 gene of two Hungarian isolates for divergent coat protein (CPm) of GLRaV-3. The first examinations of GLRaV-3 together were done Plant protection Institute of Pannon University and Department of Plant Pathology of Corvinus University. These samples were analyzed the HSP70 gene that genes were compared other genes of the GenBank isolates. 5 These samples were analyzed previous studies. Koszeg1 isolate previous study indicated: 2.2, while Koszeg2 fell into 1.4 different groups. Total RNA was extracted and purified from leaf tissues using the Spectrum Plant Total RNA kit (Sigma - Aldrich, Germany). Primers for PCR were designed using conserved regions from alignments above and with high melting temperatures to allow for a PCR procedure. Two universal primer pairs: K12rev/for and K13rev/for (1,400 bp) were used for divergent coat protein detection. The pair: K12for.: 5’ – GAG TTT CTT AAA RTA CGT TAA GGA CGG GAC – 3’ K12.rev.: 5’ – GGG TAG ACC ACT AAC GTC CGT CGT TTG C – 3’ K13.for.: 5’ – GGA GTG GAG ATC ACC WCT GGT AAG AAY TAC – 3’ K13.rev.: 5’ – CGC GCT ATG GTC TTT ATT AAC TAA CCA CCT TA – 3’ PCR conditions were as follows: denaturation at 94oC for 5 minutes, followed by 40 cycles at 94oC for 30 seconds, 60oC for 30 seconds and 72oC for 1 minute 45 seconds. The final elongation step was done at 72oC for 7 minutes. Aliquots of PCR products were run on 1 % agar’s gels. Amplicons were purified using the Roche high pure 6 purification kit, cloned into pGEM-T Easy cloning vector and sequenced by BAY-GEN (Hungary). The phylogenetic studies were performed using multiple alignments of the amplified 1432 bp sequence but CPm was using the analysis. Construction of the evolutionary model was performed using the CLC sequence Viewer 6.5.1. using the UPGMA method and 1000 bootstrap iterations as a confidence test. In order to assess the relationship of the two Hungarian GLRaV-3 isolates, ORF7 sequences from NCBI/EMBL database were included in a phylogenetic analysis. NEW SCIENTIFIC RESULTS 1. We showed 8 viruses contaminating wine grapes in 77 samples out of 325 samples that were collected between 2012 and 2014 in Hungary’s 7 wine regions and 22 wine-growing areas. In forty six samples one virus’ presence was verifiable, and in 31 cases we identified complex contamination of two-two viruses. 2. During testing we found out that the nepoviruses that were previously considered as significant (GFLV, ArMV, TBRV and GCMV) were present individually as well as collectively in a smaller proportion than the GVA, GLRaV- 1 and GLRaV-3 serological groups. In researches of previous years the GVA was not significantly present in the samples; however, it represented the largest proportion in our job together with the Grapevine leafrolls- associated viruses. 7 3. In Hungary we tested divergent coat protein of GLRaV-3 isolates the first time. We defined the nucleotide and amino acid sequence of the ORF7 region that codes divergent coat protein (CPm) of GLRaV-3 isolates from Kőszeg that was already previously tested by molecular method (RT-PCR). Based on the filogenetical analysis the Hungarian isolates can be categorized into the already existing five groups. The Kőszeg1 isolate belongs to the group Nr. II., while the Kőszeg2 isolate belongs to the group Nr. I. The sequence data of the isolates have been registered in the GeneBank, under accession numbers of LN851187 and LN851188. 4. The sequence of the homolog protein of HSP70 isolates that are presented in the assessment got defined previously. Based on them the 2.2. isolate, in our case the Kőszeg1 isolate got categorized in to the group Nr. II. in both assessments while before the isolate Nr. I (in our test named Kőszeg2) got placed in to the group Nr. IV. based on the HSP70h sequence data, but in to the group Nr. I. based on the ORF7 protein. 8 SELECTED PUBLICATIONS Publications in Hungarian language Cseh E., Palkovics L., Apró M., Gáborjányi R. és Takács A. P. (2012): Hazai szőlő levélsodródás vírus 3 izulátumok (Grapevine leafroll- associated virus 3, GLRaV-3) molekuláris jellemzése. Növényvédelem 48 (7): 297-302. Apró M., Cseh E., Gáborjányi R., Csáky J. és Takács A. P. (2014): Magyarországon előforduló szőlővírusok 2013. évi vizsgálata. Georgikon for Agriculture 19 (1): 78-83. Apró M. és Takács A. P. (2014): A szőlővírusok által okozott élettani változások. Növényvédelem 50 (1): 27-34. Apró M., Cseh E., Gáborjányi R. és Takács A. P. (2014): Magyarországi szőlőültetvények vírusfertőzöttsége. Borászati füzetek 21 (2): 8-11. Apró M., Cseh, E.
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