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Improvement of Winter Oilseed Rape Resistance to Verticillium Longisporum – Assessment of Field Resistance and Characterization of Ultrastructural Plant Responses

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Improvement of Winter Oilseed Rape Resistance to Verticillium Longisporum – Assessment of Field Resistance and Characterization of Ultrastructural Plant Responses Improvement of Winter Oilseed Rape Resistance to Verticillium longisporum – Assessment of Field Resistance and Characterization of Ultrastructural Plant Responses Dissertation zur Erlangung des Doktorgrades der Fakultät für Agrarwissenschaften der Georg-August-Universität Göttingen vorgelegt von Jessica Knüfer geboren in Wiesbaden Göttingen, Juli 2011 D 7 1. Referent: Prof. Dr. Andreas von Tiedemann 2. Korreferent: Prof. Dr. Petr. Karlovsky Tag der mündlichen Prüfung: 21. Juli 2011 Contents Contents 1. General Introduction ....................................................................................................... 1 1.1 Oilseed rape ................................................................................................................ 1 1.2 Verticillium longisporum ............................................................................................... 1 1.3 Life cycle of V. longisporum ......................................................................................... 2 1.4 Aim of the thesis .......................................................................................................... 4 2. Evaluation of winter oilseed rape resistance to V. longisporum under greenhouse and field conditions ......................................................................................................... 5 2.1 Introduction .................................................................................................................. 5 2.2 Material and Methods .................................................................................................. 6 2.2.1 Plant material for greenhouse trials ....................................................................... 6 2.2.2 Preparation of inoculum and root-dip inoculation of the seedlings ........................ 7 2.2.3 Greenhouse Screening .......................................................................................... 8 2.2.4 Experimental design of field trials and screening of stubbles ................................ 9 2.2.5 Statistical analysis ............................................................................................... 10 2.3 Results ....................................................................................................................... 10 2.3.1 Greenhouse experiment ...................................................................................... 10 2.3.2 Field trials ............................................................................................................ 12 2.3.3 Correlation of greenhouse and field screening data ............................................ 15 2.4 Discussion ................................................................................................................. 22 3. Assessment of field resistance of B. napus to V. longisporum by in planta quantification of fungal DNA with real-time PCR ....................................................... 26 3.1 Introduction ................................................................................................................ 26 3.2 Material and Methods ................................................................................................ 28 3.2.1 Primer design ....................................................................................................... 28 3.2.2 Evaluation of primer specificity and sensitivity ..................................................... 29 3.2.2.1 Fungal isolates ............................................................................................... 29 3.2.2.2 Primer specificity ............................................................................................ 29 3.2.2.3 Primer sensitivity ............................................................................................ 30 i Contents 3.2.3 Amplification of fungal DNA in the presence of non-target DNA ......................... 31 3.2.4 Greenhouse trials ................................................................................................ 32 3.2.4.1 Plant material ................................................................................................. 32 3.2.4.2 Fungal cultures for greenhouse trials ............................................................ 32 3.2.4.3 Cultivation of the plants and root dip inoculation ........................................... 33 3.2.4.4 Evaluation of disease severity in the greenhouse ......................................... 33 3.2.5 Field trial .............................................................................................................. 34 3.2.5.1 Plant material ................................................................................................. 34 3.2.5.2 Experimental design and evaluation of disease severity in the field by screening of stubbles ..................................................................................... 34 3.2.5.3 DNA extraction from field plants and qPCR analysis ..................................... 35 3.2.6 Preparation of DNA standards for qPCR analysis of field samples ..................... 36 3.2.7 Comparison of disease incidence, net AUDPC values and DNA content ........... 37 3.2.8 Statistical analysis ............................................................................................... 37 3.3 Results ....................................................................................................................... 38 3.3.1 Primer sensitivity tests reveal significant differences between primer pairs ........ 38 3.3.2 Primer specificity .................................................................................................. 39 3.3.3 Performance of primer pairs with the iCycler system .......................................... 40 3.3.4 Plant DNA does not affect PCR amplification of fungal DNA .............................. 41 3.3.5 Comparison of performance of ITS and tubulin-based primers ........................... 42 3.3.6 Disease development in the greenhouse ............................................................ 42 3.3.7 Monitoring of disease progress in the field .......................................................... 43 3.3.7.1 Sample preparation for qPCR analysis by combined CTAB/Kit method ....... 43 3.3.7.2 Quantification of fungal DNA in rapeseed stems prior to symptom development .................................................................................................. 44 3.3.7.3 Evaluation of field resistance by visual scoring of stubbles ........................... 46 3.3.8 Relationship between disease incidence, net AUDPC values and DNA content 47 3.4 Discussion ................................................................................................................. 47 ii Contents 4. Histological investigations of resistance factors involved in the interaction of B. napus with V. longisporum ........................................................................................... 52 4.1 Introduction ................................................................................................................ 52 4.2 Material and Methods ................................................................................................ 57 4.2.1 Plant material ....................................................................................................... 57 4.2.2 Fungal isolate ...................................................................................................... 57 4.2.3 Inoculation procedure .......................................................................................... 57 4.2.4 Evaluation of the disease progress ...................................................................... 58 4.2.5 Specimen preparation for TEM ............................................................................ 59 4.2.6 DNA extraction and quantification of V. longisporum in infected plants .............. 60 4.2.7 Statistical analysis ............................................................................................... 61 4.3 Results ....................................................................................................................... 62 4.3.1 Disease development in resistant and susceptible genotypes ............................ 62 4.3.2 Colonization of the hypocotyl by V. longisporum is accelerated in susceptible plants ................................................................................................................... 63 4.3.3 V. longisporum infection induces ultrastructural changes in B. napus xylem-tissue ........................................................................................................ 64 4.4 Discussion ................................................................................................................. 74 5. Assessment of V. longisporum resistance under drought stress conditions......... 80 5.1 Introduction ................................................................................................................ 80 5.2 Material and Methods ................................................................................................ 82 5.2.1 Experimental set-up ............................................................................................. 82 5.2.2 Plant material ....................................................................................................... 82 5.2.3 Fungal isolate and root dip inoculation ...............................................................
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