Pathogen defense mechanisms against Verticillium longisporum in Brassica napus L. – investigation on the role of sulfur-containing compounds Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte Dissertation von Sofia Isabell Rupp, Diplom-Biologin 2020 Referentin: Prof. Dr. rer. nat. Jutta Papenbrock Korreferent: Prof. Dr. rer. hort. Edgar Maiß Tag der Promotion: 04.06.2020 für Hasi Carrell & meine Familie Content Content Summary .......................................................................................................... I Zusammenfassung ......................................................................................... II Abbreviations ................................................................................................. III 1. Introduction.......................................................................................... 1 1.1 Crop plants and their threat from fungal pathogens .................................. 1 1.2 Sulfur in nature and the meaning of sulfur fertilization to strengthen the plant against pathogenic infestation ........................................................... 3 1.2.1 Biogeochemical cycle of sulfur in nature .............................................. 3 1.2.2 Transport and metabolism of sulfur: from root to shoot ........................ 3 1.2.3 Sulfur fertilization – long story short of “Sulfur-Induced Resistance” or “Sulfur-Enhanced Resistance” ............................................................. 6 1.3 The Brassicaceae: a family with many important crop plants ................... 6 1.3.1 The important crop plant Brassica napus ............................................. 7 1.3.2 Evolutionary history and origin of B. napus .......................................... 9 1.4 The plant root as battleground for fungal pathogens ............................... 10 1.4.1 The vascular system of plants ............................................................ 10 1.4.2 The xylem as a niche for vascular wilt pathogens .............................. 11 1.4.3 The genus Verticillium ssp. ................................................................ 12 1.4.4 Verticillium longisporum: the causal agent of Verticillium stem striping ...................................................................................................................... 12 1.4.5 Evolutionary history and origin: hybridization with fatal consequences ..................................................................................................................... .15 1.5 Plant defense mechanisms – when plants make a counterattack ........... 18 1.5.1 Sulfur-containing defense compounds – SDCs .................................. 18 Glucosinolates: the mustard oil bombs .............................................. 20 Glutathione: a versatile protector ....................................................... 22 Hydrogen sulfide: a foul-smelling affair with benefits .......................... 23 1.5.2 The second line of defense – the pathogen-induced mechanisms .... 24 1.6 Through time and defense – does timing matter? .................................... 28 1.6.1 Tick-tock: circadian clocks and diurnal rhythms of plants .................. 28 1.7 Aims of the thesis ....................................................................................... 32 2. Material and methods ........................................................................ 33 2.1 Plant material ............................................................................................... 33 2.1.1 Plant seed surface sterilisation ......................................................... 33 2.1.2 Blake-Kalff medium ............................................................................ 34 2.1.3 Cultivation of Brassica napus in climatic chambers ............................ 35 2.2 Verticillium longisporum strain .................................................................. 35 2.2.1 Verticillium longisporum growth and cultivation .................................. 35 2.2.2 Preparation of Verticillium longisporum spore stock solutions ............ 36 Content 2.3 Various inoculation procedures of Brassica napus with Verticillium longisporum ................................................................................................ 36 2.3.1 Inoculation of 16 d old B. napus plants (previous fertilization with different sulfur supply) with mycelium-spore mixture of V. longisporum…………………………………………………………………36 2.3.2 Inoculation of 16 d old B. napus plants (previous fertilization with different sulfur supply) with different dilutions of mycelium-spore mixture of V. longisporum ............................................................................... 37 2.3.3 Inoculation of 7 d old B. napus seedlings with mycelium-spore mixture of V. longisporum ............................................................................... 37 2.3.4 Inoculation of 7 d old B. napus seedlings with spore solution of V. longisporum ........................................................................................ 37 2.4 General experimental design: the definition of different time-points of inoculation (TPIs)......................................................................................... 38 2.5 Preliminary inoculation experiments of Brassica napus with Verticillium longisporum under diurnal rhythmic and with different sulfur supply .... 39 2.5.1 Preliminary experiment I: inoculation of 7 d old plants with mycelium- spore mixture and subsequent cultivation in sand/soil mixture ........... 39 2.5.2 Pre-experiment II: inoculation of 16 d old plants (previous fertilization with different sulfur supply) with mycelium-spore mixture and subsequent cultivation in sand ........................................................... 41 2.5.3 Pre-experiment IIIa: inoculation of 16 d old plants (previous fertilization with different sulfur supply) with different dilutions of mycelium-spore mixtures and subsequent cultivation in sand ...................................... 43 2.5.4 Pre-experiment IIIb: inoculation of 16 d old plants (previous fertilization with different sulfur supply) with diluted mycelium-spore mixture and subsequent cultivation in sand ........................................................... 44 2.5.5 Pre-experiment IV: inoculation of 7 d old plants with spore solution and subsequent cultivation in sand ........................................................... 45 2.6 Main experiment: inoculation of Brassica napus with Verticillium longisporum under diurnal rhythmic and with different sulfur supply .... 45 2.6.1 Experimental design: different time-points of inoculation of 7 d old plants with mycelium-spore mixture and different sulfur supply .................... 45 2.7 Analytical methods ..................................................................................... 47 2.7.1 DNA-extraction .................................................................................. 47 2.7.2 Real-time quantitative PCR ................................................................ 47 2.7.3 Elemental analysis via ICP-OES ........................................................ 49 2.7.4 Glucosinolate analysis via HPLC ....................................................... 51 2.7.5 Thiol (cysteine and glutathione) analysis via HPLC ............................ 54 2.7.6 Histology: Staining of the plant hypocotyl with toluidine blue .............. 55 2.7.7 Histology: Staining of fungal mycelium with cotton blue ..................... 57 2.8 Statistical analysis ...................................................................................... 58 2.9 Necessary additional notes to the used climatic chambers, biological replicates and amount of plant material .................................................... 59 2.9.1 Climatic chambers from Johnson Controls ......................................... 59 2.9.2 Biological replicates and amount of plant material ............................. 59 Content 3. Results ................................................................................................ 61 3.1 Pre-experiment I: inoculation of 7 d old plants with mycelium-spore mixture and subsequent cultivation in sand/soil mixture ......................... 61 3.1.1 Detection and verification of the infection with V. longisporum in mycelium-spore inoculated B. napus plants potted in sand/soil .......... 61 3.1.2 The occurrence of occlusions in the xylem of mycelium-spore inoculated plants potted in sand/soil.................................................................... 62 3.1.3 Elemental analysis: levels of sulfur, calcium, potassium and iron in mock- and mycelium-spore inoculated plants potted in sand/soil ....... 66 3.1.4 The content of indolic, aliphatic and benzylic glucosinolates in mock- and mycelium-spore inoculated plants potted in sand/soil .................. 70 3.2 Pre-experiment II: inoculation of 16 d old plants (previous fertilization with different sulfur supply) with mycelium-spore mixture and subsequent cultivation in sand ....................................................................................... 79 3.2.1 Detection and verification of the infection with V. longisporum in mycelium-spore inoculated B. napus plants potted in sand ................ 79 3.2.2 Elemental analysis: levels of sulfur, calcium, potassium and iron in mock- and mycelium-spore