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International audience; Although poleroviruses are known to infect a broad range of higher plants, carnivorous plants have not yet been reported as hosts. Here, we describe the first polerovirus naturally infecting the pitcher plant Nepenthes mirabilis. The virus was identified through bioinformatic analysis of NGS transcriptome data. The complete viral genome sequence was assembled from overlapping PCR fragments and shown to share 91.1 % nucleotide sequence identity with the US isolate of beet western yellows virus (BWYV). Further analysis of other N. mirabilis plants revealed the presence of additional BWYV isolates differing by several insertion/deletion mutations in ORF5.

Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD.

Kanola (Brassica napus var. napus) Dünya’da bitkisel yağ kaynağı olarak önemli bir kültür bitkisidir. AB ülkeleri gıda ve gıda sanayi hammaddesi olarak kanola yağını kullanırken ABD, Güney Amerika ve Uzak Doğu ülkeleri soya yağını değerlendirmektedirler. Türkiye’de ise yetersiz üretime ve ithalata dayalı ayçiçeği yağı kullanılmaktadır. Türkiye’nin bitkisel yağ açığını gidermek için Trakya Bölgesi’nde kanola üretimi başlatılmıştır. Bölgede kanola tohum verimini ve kalitesini olumsuz yönde etkileyen abiyotik, sistemik ve viral hastalıklar bu çalışma ile saptanmış bulunmaktadır. Bu amaçla Edirne, Kırklareli ve Tekirdağ illerinin 7 ilçesinde, 2013-2014 sezonunda sürveyler gerçekleştirilmiştir. Yapılan bu çalışma sonucu üreticilerin geç ekim, sıralı ekimi göz ardı etmeleri ve gübreleme hataları gözlenmiştir. Böylece körpe kanola bitkilerinde soğuk ve don zararları, bitki besin elementlerinden N, P, K, Ca, S, Fe, Cu ve Mg noksanlıkları kanola tohum verimini olumsuz yönde etkileyen hatalı tarımsal uygulamalar olarak saptanmıştır. Kanola bitkilerinde nadiren ve sporadik olarak gözlenmiş olan yassılaşma (Fasiasyon) simptomları ise fitoplazmik stolbur hastalığı ile ilişkilendirilebilir. Sürveylerde karakteristik sarılık ve mozayik belirtileri sergileyen 73 adet kanola yaprak örneği toplanmıştır. Double Antibody Sandwich EnzymeLinked Immunosorbent Assay (DAS-ELISA) testleri için kanolada en sık karşılaşılan Beet western yellows virus (BWYV) ve Turnip mosaic virus (TuMV)’lerine karşı hazırlanmış test kitleri kullanılmıştır. DAS-ELISA testleri sonucu 73 örnekten 10 adedinde ve % 13,73 oranında Beet western yellows virus (BWYV) bulunduğu kesin olarak saptanmıştır. Test sonuçlarına göre örneklerde Turnip mosaic virus (TuMV)’e ise rastlanmamıştır. Bu güne kadar Türkiye’de kolza ve kanolada herhangi bir virüs araştırması yapılmamış olduğu için yapılan çalışmada BWYV’ünün bulunmuş olması bu konudaki ilk bulgudur. Canola (Brassica napus var. napus) has been one of the most important cultivated plants as the source of vegetable oil in the world. Canola oil has been consumed as food and used in food industry in EU counties. Soy bean oil however has been preferred by USA, South American and Far Eastern countries. In spite of limited production and depending on importation, sun flower oil has been consumed as vegetable oil in Turkey. In order to meet the demand of vegetable oil, canola seed production has been initiated in the Trakya region of Turkey. Abiotic and systemic virus diseases, reducing yield and quality of canola seed were determined in this study. A survey study was implemented in the 7 districts of Edirne, Kırklareli and Tekirdağ provinces during the 2013 – 2014 Season. Some improper agricultural practices were observed like late sowing, to neglect rotation and the fertilization mistakes. So because of the late sowing frost and chilling damages on young and tender canola seedlings as fertilization mistakes revealed the nutritional deficiencies of N, P, K, Ca, S, Cu and Mg. Some hyperplastic fasiation symptoms were also observed sporadically which related to phytoplasmic stolbur disease. During the surveys at least 73 leaf samples were collected from canola plants exhibiting characteristic yellowing and mosaic symptoms. In order to search the most common viruses occurring on canola antisera against Beet western yellows virus (BWYV) and Turnip mosaic virus (TuMV) with serological test kits were used. As a result of Double Antibody Sandwich Enzyme – Linked Immunosorbent Assay (DAS– ELISA) tests 10 out of 73 samples with the rate of 13.73 % were found infected with BWYV. According to DAS-ELISA test results TuMV was not present in canola samples. So any study has not been implemented on canola viruses in Turkey our findings may be considered first report about the presence of BWYV on canola in Turkey.

ÖZETYüksek Lisans TeziTRAKYA BÖLGESİ ŞEKER PANCARI ÜRETİM ALANLARINDA BEET NECROTIC YELLOW VEIN VIRUS (BNYVV), BEET WESTERN YELLOWS VIRUS (BWYV) VE BEET YELLOWS VIRUS (BYV) HASTALIKLARININ SAPTANMASI ÜZERİNE ARAŞTIRMALARHarun ÖZDEMİRNamık Kemal ÜniversitesiFen Bilimleri EnstitüsüBitki Koruma Anabilim DalıDanışman: Doç. Dr. Havva İLBAĞITrakya Bölgesi'nin Edirne, Kırklareli ve Tekirdağ illerindeki şeker pancarı üretim alanlarında 2013 yılı üretim döneminde gerçekleştirilen sürvey çalışmalarında, verim kayıplarına neden olan virüs hastalıklarını saptamak amacıyla 126 yaprak örneği toplanmıştır. Şeker pancarı üretim alanlarında yapraklarda sararma, nekroz, mozayik ve şekil bozukluğu simptomları gösteren bu yaprak örneklerinde Beet necrotic yellow vein virus (BNYVV), Beet western yellows virus (BWYV), Beet yellows virus (BYV) hastalıklarını saptamak üzere Double Antibody Sandwich Enzyme-linked Immunosorbent Assay (DAS-ELISA) testi gerçekleştirilmiştir. Test edilen toplam 126 yaprak örneğinden 25 adedinde BNYVV, 6 adedinde BWYV ve 5 adedinde ise BYV bulunduğu saptanmıştır. 8 yaprak örneği BNYVV+BYV, 1 örnek BNYVV+BWYV, 1 örnek BWYV+BYV ile enfekteli iken 14 adet örneğin ise BNYVV+BWYV+BYV ile enfekteli oldukları tespit edilmiştir. DAS-ELISA testi sonucunda Trakya Bölgesi'ndeki illerden alınan 126 adet yaprak örneğinin 60 adedinde tekli ve karışık enfeksiyonlar saptanmıştır. Tek enfeksiyon halinde BNYVV'nin % 19.84, karışık enfeksiyonlar halinde % 38.09 olduğu tespit edilmiştir. BWYV'nin tek enfeksiyon halinde % 4.76, karışık enfeksiyon halinde % 12.69, BYV'nin ise tek enfeksiyon halinde % 3.97, karışık enfeksiyon olarak ise % 18.25 olduğu belirlenmiştir. Söz konusu virüslerin illere göre dağılımının ise Edirne ilinde % 50, Kırklareli ilinde % 41.51, Tekirdağ ilinde ise % 53.33 düzeyinde olduğu saptanmıştır.Anahtar Kelimeler: Şeker Pancarı, BNYVV, BWYV, BYV2014, 42 sayfa ABSTRACTMSc. ThesisINVESTIGATIONS ON SUGAR BEET VIRUS DISEASES CAUSED BY BEET NECROTIC YELLOW VEIN VIRUS (BNYVV), BEET WESTERN YELLOWS VIRUS (BWYV) AND BEET YELLOWS VIRUS (BYV) IN THE SUGAR BEET GROWING AREAS IN THE TRAKYA REGION OF TURKEYHarun ÖZDEMİRNamık Kemal UniversityGraduate School of Natural and Applied SciencesDepartment of Plant ProtectionSupervisor: Assoc. Prof. Dr. Havva İLBAĞIIn order to determine yield reducing beet virus diseases in the sugar beet growing areas of Edirne, Kırklareli and Tekirdağ provinces in the Trakya region of Turkey. 126 symptomatic sugar beet leaf samples were collected in 2013. For the identification of Beet necrotic yellow vein virus (BNYVV), Beet western yellows virus (BNYVV) and Beet yellows virus (BYV) Double Antibody Sandwich Enzyme Linked Immunosorbent Assay (DAS-ELISA) tests were implemented to sugar beet leaf samples which were exhibited yellowing, necrosis, mosaic and leaf distortions. As a result of DAS-ELISA tests 25out of 126 leaf samples were found infected with BNYVV, 6 of the them had BWYV and 5 leaf samples revealed the presence of BYV. Beside these individually infected samples 8 out of 126 leaf samples had BNYVV+BYV, 1 of them infected with the mixture of BNYVV+BWYV, another one of them had BWYV+BYV and 14 out of 126 leaf samples however were found infected with those three viruses as individually and as their mixed infections. Individually BNYVV was found 19.84 %, as mixed it was 38.09 %. In the case of BWYV individually 4.76 % as mixed infection 12.69 % rate of disease were determined. Individually infection of BYV was 3.97 % as the mixed infection rate of this virus was found 18.25 %. Those sugar beet virus infections occurred 50 % in Edirne, 41.51 % in Kırklareli and 53.33 % in Tekirdağ province.Key words: Sugar beet, BNYVV, BWYV, BYV2014, 42 pages 51

The aim of this survey was to identify viruses infecting spinach (Spinacia oleracea L.) in the most important spinach-producing areas in Greece. A total of 1074 spinach samples were collected from eleven districts belonging to the prefectures of Thessaloniki, Chalkidiki and Imathia in northern Greece, and Evia in central Greece. Samples were tested by ELISA, mechanical inoculation onto indicator plants and immunoelectron microscopy. Beet western yellows virus (BWYV), Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV) were identified in 13.5%, 7% and 5.4% of samples, respectively, infected samples being detected in all regions examined. This is the first record of CMV and TuMV infecting spinach in Greece, and the first report of BWYV occurrence in any crop nationwide. Surveys were also conducted to assess the potential reservoir hosts of BWYV, CMV and TuMV in weeds collected from spinach fields. All three viruses were detected among 125 samples tested by ELISA. TuMV prevailed as it occurred in 14.4% of all weed samples

National audience; Le Beet western yellows virus (BWYV) est un polerovirus (Luteoviridae) qui est transmis par Myzus persicae de manière circulante non-multipliante. Une fois ingérées, les particules virales doivent obligatoirement franchir l’épithélium de l’intestin moyen puis celui des glandes salivaires accessoires afin d’être transmises à une nouvelle plante hôte. Ces deux étapes de transcytose (endocytose/exocytose) des virions au travers du puceron-vecteur requièrent des interactions spécifiques entre les particules virales et des récepteurs localisés au niveau des deux épithéliums (intestin et glandes salivaires accessoires). Des expériences de far-Western blot ont été réalisées à partir de protéines (totales, membranaires et/ou solubles) extraites de Myzus persicae [1] et ont permis, après séquençage par spectrométrie de masse, l’identification de plusieurs candidats homologues à certaines protéines de Drosophila melanogaster dont Rack1 (Receptor for Activated C Kinase 1) et Gapdh3 (GlycerAldehyde-3-Phosphate DesHydrogenase). Bien qu’aucune de ces deux protéines ne soit probablement le récepteur membranaire (extracellulaire) du BWYV, elles interviendraient plus ou moins indirectement dans les mécanismes de transcytose et pourraient ainsi participer activement au passage du BWYV dans le corps du puceron-vecteur. Les tests d’interaction réalisés avec différents mutants du BWYV suggère que Rack1 interagit avec un motif spécifique de la protéine mineure de capside (la RT : ReadThrough) qui est impliquée dans l’efficacité d’endocytose des virions au niveau intestinal. Gapdh3, à l’inverse, interagit avec tous les virus mutés Ŕ même ceux délétés du domaine de readthrough (DRT) Ŕ vraisemblablement par le biais de la protéine majeure de capside (la CP). Afin de confirmer ces interactions observées par far-Western blot, nous avons utilisé un système split-ubiquitin de double hybride de levure, le « mbSUS ». Les protéines appâts (CP ou DRT) ont été fusionnées à la partie C-ter de l’ubiquitine (Cub), elle-même en fusion avec un facteur de transcription synthétique (PLV). Celui-ci est uniquement libéré par clivage lorsqu’il y a interaction de l’appât avec une proie fusionné à la partie N-ter de l’ubiquitine (Nub) ce qui permet l’action de protéases spécifiques reconnaissant l’ubiquitine (entière). La libération du facteur de transcription induit l’activation des gènes rapporteurs du système permettant, entre autre, la croissance des levures sur un milieu spécifique. Le génome d’Acyrthosiphon pisum étant maintenant disponible et les EST pour Myzus persicae assez nombreux, nous avons pu identifier chez M. persicae les gènes codant pour les protéines Rack1 et Gapdh3. Les séquences de ces deux gènes ont été fusionnées à la partie N-ter de l’ubiquitine (Nub) et serviront de proie dans les expériences de double hybride. Les expériences sont en cours, résultats sur le poster !

International audience; Beet western yellows virus (BWYV) is a Polerovirus that relies on the aphid Myzus persicae for its transmission, in a persistent-circulative mode. To be transmitted, the virus must cross the midgut and the accessory salivary glands (ASG) epithelial barriers in a transcytosis mechanism where vector receptors interact with virions. In this paper, we report in vitro interaction experiments between BWYV and aphid components. Using the M. persicae clone from Colmar, we showed that a set of aphid polypeptides, separated by SDS-PAGE or 2D electrophoresis (2DE), can bind in vitro to purified wild type or mutant particles. Using subcellular fractionation, we showed that the 65-kDa polypeptide identified as symbionin is a soluble protein whereas the other polypeptides seem to be associated more or less strongly to the membrane. We hypothesize that three polypeptides, identified by mass spectrometry as Rack-1, GAPDH3, and actin, may be involved in the epithelial transcytosis of virus particles in the aphid vector.