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Genetische Diversität Des Sternrußtaus (Diplocarpon Rosae) an Rosen

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Genetische Diversität Des Sternrußtaus (Diplocarpon Rosae) an Rosen Genetische Diversität des Sternrußtaus (Diplocarpon rosae) an Rosen Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades einer Doktorin der Naturwissenschaften Dr. rer. nat. genehmigte Dissertation von Dipl.–Ing. agr. Ann­Katrin Lühmann geboren am 30. 12. 1980 in Mölln 2010 Referent: Prof. Dr. Thomas Debener Korreferent: Prof. Dr. Edgar Maiß Tag der Promotion: 17. Dezember 2009 ZUSAMMENFASSUNG Zusammenfassung Diplocarpon rosae, der Sternrußtau an Rosen ist eines der bedeutendsten Pathogene an Freilandrosen. Durch die steigenden Kosten für Fungizide und das gestiegene Umweltbewusstsein der Bevölkerung in den letzten Jahren, erlangt die Resistenzzüchtung neben der in Nutzpflanzen auch in den Zierpflanzen eine immer größere Bedeutung. Um die Gefahr, welche vom Sternrußtau ausgeht, besser einschätzen zu können ist ein genaueres Wissen über dieses Pathogen von großer Wichtigkeit. Die genetische Diversität eines Pathogens spielt eine bedeutende Rolle in der Überwindung von Resistenzen der Wirtspflanze. Je höher die genetische Diversität eines Pathogens ist, desto leichter können Resistenzen überwunden werden und desto wichtiger ist es, dies in der Züchtungsarbeit zu berücksichtigen. Um ein möglichst umfassendes Abbild der genetischen Diversität des Sternrußtaus zu bekommen, wurden in dieser Arbeit molekulare Marker für den Sternrußtau entwickelt, mit denen Einsporisolate, Populationssammlungen sowie weltweite Stichproben analysiert wurden. Zur Analyse eigneten sich hier insbesondere Mikrosatelliten. Es zeigte sich, das die Diversität zwischen den verschiedenen Populationen sehr stark variierte. Die Populationen wiesen Gen‐Diversitäten (nach Nei, 1973) von 0,06 bis 0,44 auf. Dabei zeigte sich, dass die Diversität der Sternrußtaupopulationen mit steigendem Alter der Wirtspflanzenpopulation zunimmt. Des Weiteren zeigte die Diversität der jeweiligen Rosenpopulation sowie der Einsatz von Fungiziden einen Einfluss auf die Diversität der Sternrußtaupopulationen. Anhand von weltweiten Stichprobensammlungen kann darauf geschlossen werden, dass die globale Diversität des Sternrußtaus mit der Diversität einzelner, sehr diverser Sternrußtaupopulationen innerhalb Deutschlands vergleichbar ist. Um die sexuelle Vermehrung des Sternrußtaus und deren Häufigkeit besser charakterisieren zu können, eignet sich die Analyse der Gene für die Kreuzungstypen (Mating‐Types) des Pathogens. Innerhalb dieser Arbeit konnte ein Teil dieses Mating‐Type Locus identifiziert werden. Mit den Ergebnissen aus der Diversitätsanalyse wurden Resistenzuntersuchungen von Rosenpopulationen durchgeführt. Es konnte gezeigt werden, dass das Resistenzniveau gegen den Sternrußtau in Züchtungspopulationen sehr niedrig ist. Als eine Möglichkeit zur effektiveren und schnelleren Selektion auf Sternrußtauresistenz erweisen sich dabei Inokulationen mit einer Konidiensuspension hoher Diversität auf dem Saatbeet. (Schlagwörter: Diplocarpon rosae, Sternrußtau, genetische Diversität, Resistenzzüchtung, pathogenes Potential) I ABSTRACT Abstract Diplocarpon rosae, also called black spot on roses, is one of the most important pathogens infesting field grown roses. Due to raising costs for fungicides and growing environmental consciousness, resistance breeding gets more and more important, beside agricultural crops also in ornamental plants. In order to estimate the pathogenic potential of black spot, detailed knowledge about the pathogen is important. Thereby, the genetic diversity plays an important role. The higher the genetic diversity of a pathogen, the easier to overcome existing resistances. The importance to take this into consideration for resistance breeding raises with higher genetic diversity of the pathogen. To get a comprehensive impression of the genetic diversity of black spot, molecular markers for D. rosae were developed. For the analysis of single spore isolates, population collections as well as global samples, microsatellite markers proved to be very suitable. It turned out, that the diversity between the different analysed populations has a great variation. The populations showed gene diversities (Nei, 1973) from 0.06 to 0.44. The results showed that the diversity of black spot populations increases with the age of rose populations. Furthermore, the diversity of the rose population and the use of fungicides have a great impact on the diversity of the black spot population. Analysis of of global samples of D. rosae showed a similar diversity compared to high diverse german populations. Characterizing the frequency of sexual reproduction of black spot , the analysis of mating‐type genes of the pathogen is useful. Within this thesis a part of the sequence of the mating type locus of D. rosae could be identified. With the results from the analysis of diversity of black spot, resistance screenings of rose populations were carried out. It turned out, that the level of resistance against black spot in the breeding populations is very low. Inoculations of seedling beds with conidia suspension with a high diversity proved to be a good possibility for a faster selection for black spot resistance. (keywords: Diplocarpon rosae, black spot, genetic diversity, resistance breeding, pathogenic potential) II INHALTSVERZEICHNIS Inhaltsverzeichnis 1 Einleitung...................................................................................................................................................1 1.1 Rosen..................................................................................................................................................................1 1.1.1 Charakterisierung von Resistenzen in Rosen ..............................................................................2 1.2 Sternrußtau an Rosen .................................................................................................................................3 1.2.1 Arbeiten zur Diversität des Sternrußtaus .....................................................................................6 1.3 Bedeutung der genetischen Diversität von pilzlichen Phytopathogenen..............................8 1.3.1 Entstehung der genetischen Diversität ..........................................................................................9 1.3.2 Gen‐Diversität und genotypische Diversität .............................................................................12 1.3.3 Abschätzung des pathogenen Potentials von Pflanzenpathogenen für die Resistenzzüchtung ...............................................................................................................................13 1.4 Ziele der Arbeit ...........................................................................................................................................14 2 Material und Methoden...................................................................................................................... 15 2.1 Verwendete Einsporisolate....................................................................................................................15 2.2 Pflanzenmaterial ........................................................................................................................................15 2.3 Verbrauchsmaterialien............................................................................................................................15 2.3.1 Bakterienstamm, Plasmid und Enzyme.......................................................................................16 2.3.2 Primer........................................................................................................................................................16 2.4 Kultur des Pilzes Diplocarpon rosae in­vivo ....................................................................................16 2.5 Kultur von Mycel und Konidien in­vitro ...........................................................................................16 2.6 Sammlung der Freilandproben ............................................................................................................17 2.7 Aufbereitung der Proben........................................................................................................................17 2.8 Aufbereitung getrockneter Proben ....................................................................................................17 2.9 Resistenzuntersuchungen von Rosenpopulationen....................................................................18 2.10 DNA‐Extraktion aus Konidien...............................................................................................................19 2.11 DNA‐Extraktion aus Mycel.....................................................................................................................19 2.12 Plasmid‐Extraktion ...................................................................................................................................20 2.13 Ligation in pGEM‐tEasy ...........................................................................................................................20 2.14 Herstellung elektrokompetenter E. coli ‐ Zellen (DH10B)........................................................20 2.15 Transformation von elektrokompetenten E. coli ‐ Zellen (DH10B)......................................20 2.16 Kolonie Blot..................................................................................................................................................20 2.17 Hybridisierung
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