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Molecular Characterization of Capsicum Chlorosis Virus, Tomato

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Molecular characterization of Capsicum chlorosis virus, Tomato yellow leaf curl Thailand virus, Tobacco leaf curl Thailand virus and RNA-mediated virus resistance in Nicotiana benthamiana Domin. Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des akademischen Grades eines Doktors der Gartenbauwissenschaften -Dr. rer. hort.- genehmigte Dissertation von Dipl.-Ing.agr. Dennis Knierim geboren am 21.11.1974 in Lich Angefertigt am Institut für Pflanzenkrankheiten und Pflanzenschutz Hannover, Februar 2007 Referent: Prof. Dr. Edgar Maiß Koreferent: Prof. Dr. Günter Adam Tag der Promotion: 05.02.2007 Abstract Abstract Production of vegetables in the tropics and subtropics demands special requirements for cultivation. One reason is the occurrence of pests like insects, mites and nematodes, whereas the direct damage caused by pathogens is often not a factor for yield reduction. More serious is the possibility of virus transmission and the quantitative and qualitative yield losses triggered by virus infection. The cultivation of virus resistant plants offers the possibility to circumvent frequent pesticide applications. However, not for every crop and for every viral disease adequate natural resistant sources are available and therefore, the application of pathogen-mediated transgenic resistant may be an appropriate alternative. As a part of a larger project in Thailand tomato plants were grown under protected cultivation. Here, disease symptoms occurred on tomato plants, which were partly caused by phytopathogenic viruses. This study presented here the molecular characterization of these tomato infecting viruses and the production of transgenic virus resistance plants by using the strategy of RNA-silencing. By using different antisera one virus was identified as a Tospovirus with relationship to Watermelon silver mottle virus and Groundnut bud necrosis virus. For the molecular characterization, the complete genome sequence was determined. Phylogenetic analysis revealed the highest identity to Capsicum chlorosis virus (CaCV) isolates described in Australia. Beside the CaCV isolate collected at the Asian Institute of Technology in Bangkok [CaCV-(AIT)], several other Thai tospovirus isolates were identified after multiple sequence analyses as CaCV isolates. For a biological characterization, the host range of CaCV-(AIT) revealed difference to the host range described for the Australian CaCV isolates. Moreover, two begomoviruses were identified as causal agents in diseased tomato plants. The complete genome sequences of these both viruses were determined by applying rolling circle amplification (RCA) with Phi29 DNA polymerase. Sequence analysis exhibited the presence of the bipartite Tomato yellow leaf curl Thailand virus (THLCTHV), as well as the infection with an unknown monopartite recombinant begomovirus, tentatively named as Tobacco leaf curl Thailand virus (TbLCTHV). The infectivity of TYLCTHV and TbLCTHV full-length clones was verified by inoculation of N. benthamian plants. For this the cloned DNA-A and DNA-B Abstract components of TYLCTHV were excised by restriction enzyme digest from the respective plasmids, recircularized by ligation and subsequently amplified by Phi29 DNA polymerase. The concatameric viral copies were inoculated by particle bombardment on Nicotiana benthamiana plants and proved to be at least as effective as partial repeat construct inoculation, but being less laborious. For generation of transgenic virus resistance a post-transcriptional gene silencing (PTGS) approach was used. For this purpose viral CP or N gene sequences of 423 to 548 nts were arranged as inverted repeats joined by the ST-LS1 intron. The sequences were chosen from four RNA viruses, which can infect tomato plants in South East Asia: CaCV, Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV) und Tomato mosaic virus (ToMV). For all four constructs a uniform cloning strategy was applied. After transformeation of N. benthamiana plants via Agrobacterium tumefaciens 35 independent transgenic T0 plants were regenerated. The progeny of 23 plants was resistant and 13 CaCV, 6 TSWV, 3 CMV and 1 ToMV resistant line was selected. On selected homozygous T2 lines the stability of virus resistance was proven. Key words: Tospovirus, Begomovirus, RNA-mediated virus resistance, Capsicum chlorosis virus (CaCV), Tomato yellow leaf curl Thailand virus (TYLCTHV), Tobacco leaf curl Thailand virus (TbLCTHV) Zusammenfassung Zusammenfassung Die Produktion von Gemüse unter tropischen und subtropischen Bedingungen stellt eine besondere Herausforderung dar. Eine Ursache ist das Auftreten von Schadinsekten, wobei die direkt an den Pflanzen verursachten Schäden meist nicht der entscheidende Faktor für Ertragsverluste sind. Gravierender ins Gewicht fallen die Möglichkeiten der Virusübertragung und die durch Virusinfektionen ausgelösten quantitativen und qualitativen Ertragseinbußen. Der Anbau von virusresistenten Sorten bietet hier eine vielversprechende Maßnahme, die indirekt auch zu einer Reduktion in der Anwendung von Pflanzenschutzmitteln beitragen könnte. Da nicht für jede Kultur und nicht gegen jede Viruserkrankung ausreichend natürliche Resistenzquellen zur Verfügung stehen, kann der Einsatz einer pathogen-vermittelten transgenen Resistenz eine sinnvolle Alternative darstellen. In Thailand wurden im Rahmen eines Verbundprojektes Tomaten unter geschützten Bedingungen in Foliengewächshäusern kultiviert. Hierbei traten Symptome auf, die unter anderem durch die Infektion mit phytopathogenen Viren ausgelöst wurden. In der hier vorgelegten Arbeit wurden diese Viren näher molekular charakterisiert, sowie die Strategie des RNA-Silencings zur Erzeugung von Virusresistenzen angewandt. Ein Virus wurde zunächst mit Hilfe verschiedener Antiseren als ein Tospovirus mit Verwandtschaft zum Watermelon silver mottle virus und Groundnut bud necrosis virus identifiziert. Für die vollständige molekulare Charakterisierung wurde die komplette Genomsequenz bestimmt. Die phylogenetischen Analysen ergaben die größte Ähnlichkeit zu den in Australien beschriebenen Capsicum chlorosis virus (CaCV) Isolaten, so dass das thailändische Virusisolat, nach seiner Herkunft vom Asian Institute of Technology in Bangkok, als CaCV-(AIT) benannt wurde. Weitere multiple Sequenzvergleiche erlaubte die Einordnung vermeintlich verschiedener in Thailand vorkommender Tospoviren als Isolate des CaCV. Zur Bestimmung des Wirtspflanzenspektrums wurden Infektionsversuche mit CaCV-(AIT) an einem Testpflanzensortiment durchgeführt. Hier zeigten sich Unterschiede des thailändischen Virusisolates zum publizierten Wirtspflanzenkreis der bekannten australischen CaCV Isolate. Ferner konnten zwei Begomoviren als weitere Ursache für die Erkrankung von Tomatenpflanzen identifiziert werden. Aus Blattproben wurden die kompletten Zusammenfassung Genome der beiden unterschiedlicher Begomoviren durch „Rolling circle amplification“ mit Phi29 DNA-Polymerase zunächst angereichert und nachfolgend kloniert. Die Sequenzanalysen zeigten das Vorliegen des erwarteten bipartiten Tomato yellow leaf curl Thailand virus (TYLCTHV), sowie die Infektion der Tomaten mit einem bislang nicht beschriebenen neuen monopartiten rekombinanten Begomovirus, welches vorläufig als Tobacco leaf curl Thailand virus (TbLCTHV) benannt wurde. Die Infektiösität von TYLCTHV und TbLCTHV konnte durch Inokulation von N. benthamiana Pflanzen mit Volllängenklonen gezeigt werden. Hierzu wurden die klonierten DNA Komponenten von TYLCTHV, DNA-A und DNA-B, isoliert, durch Ligation rezirkularisiert und anschließend mit Hilfe der Phi29 DNA-Polymerase vermehrt. Die konkatemeren viralen Kopien wurden mittels Partikelbombardment zur Inokulation von N. benthamiana Pflanzen eingesetzt und führten genauso erfolgreich zur Infektion der Pflanzen wie die bislang verwendeten multimere Kopien der Begomoviren-Genome, jedoch mit dem Vorteil eines geringeren Arbeitsaufwandes. Zur Erzeugung von Virusresistenzen wurde der Mechanismus des „Post- transkriptionalen Gene Silencings (PTGS)“ genutzt. Dazu wurden invers angeordnete virale Sequenzen des CP- bzw. N-Gens mit 423-548 Nukleotiden die durch das ST- LS1 Intron verbunden wurden, eingesetzt. Ausgewählt wurden vier RNA Viren, die im südostasiatischen Raum Tomatenpflanzen infizieren können: CaCV, Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV) und Tomato mosaic virus (ToMV). Vier nach einer einheitlichen Klonierungsstrategie entwickelte Konstrukte wurden mittels Agrobakterium-vermitteltem Gentransfer in N. benthamina transformiert. Insgesamt wurden 35 unabhängige transgene T0-Pflanzen erhalten. Die Nachkommen von 23 Pflanzen erwiesen sich als resistent, wobei für das CaCV 13 Linien, das TSWV 6 Linien, das CMV 3 Linien sowie für das ToMV 1 Linie selektiert wurden. An ausgewählten homozygoten T2 Linien konnte die Stabilität der Virusresistenz bestätigt werden. Schlagworte: Tospovirus, Begomovirus, RNA-vermittelte Virusresistenz, Capsicum chlorosis virus (CaCV), Tomato yellow leaf curl Thailand virus (TYLCTHV), Tobacco leaf curl Thailand virus (TbLCTHV) Contents Contents 1 General introduction ............................................................................................ 1 2 The complete nucleotide sequence of a capsicum chlorosis virus isolate from Lycopersicum esculentum in Thailand .............................................................. 16 2.1 Abstract .............................................................................................
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  • Management of Viruses and Viral Diseases of Pepper (Capsicum Spp.)

    Management of Viruses and Viral Diseases of Pepper (Capsicum Spp.)

    Chapter Management of Viruses and Viral Diseases of Pepper (Capsicum spp.) in Africa Olawale Arogundade, Titilayo Ajose, Itinu Osijo, Hilary Onyeanusi, Joshua Matthew and Taye H. Aliyu Abstract Increasing outbreaks of virus species infecting pepper (Capsicum spp.) is a major problem for growers in Africa due to a combination of factors, including expansion of pepper cultivation, abundance of insect vectors and climate change. More than 45 viruses have been identified to infect pepper crops causing economic loss in terms of reduced quality and marketable yield, sometimes up to 100%. The Pepper veinal mottle virus (PVMV), Potato virus Y (PVY) and Cucumber mosaic virus (CMV) are endemic in many countries including Uganda, Mali, Cameroon, Morocco and Nigeria. Current management options for virus infection in Capsicum spp. is by the integration of several approaches. More importantly, eradication of infected plants, cultivation of disease resistant varieties, improved cultural prac- tices and judicious use of insecticides especially when plants are young and easily colonized by vectors. In recent years, eco-friendly control measures are needful to reduce occurrence of virus diseases in Capsicum spp. Keywords: climate change, economic loss, outbreaks, management options, virus infection 1. Introduction Peppers (Capsicum spp.) are one of the most important spices and vegetable crops in the economic and social life of people living worldwide [1]. Viruses are among the most important factors threatening Capsicum spp. production in several regions like Australia [2], Europe [3], Asia [4] and Africa [5]. They cause diseases that not only reduce yield and quality of fruits, but also increase the cost of preven- tive measures and cost of producing clean planting materials.