Novel insights into the asexual life-cycle of the wheat-leaf pathogen Zymoseptoria tritici William Tudor Kay Submitted for the degree of Doctor of Philosophy in Biological Sciences, September 2017. This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University. Signature: (William Kay) 1 Thesis Abstract Zymoseptoria tritici, the causal agent of Septoria tritici blotch (STB), is the most economically important pathogen of wheat in temperate climates. EU spending on the control of STB is estimated at more than € 1.3 billion per annum, even when tolerant cultivars are used. This thesis comprises 5 data chapters each looking to ascertain reasons for the success of Z. tritici. Using mainly in vitro methods combined with confocal microscopy, this thesis explores asexual spore submergence in rain, epiphytic development, the potential triggers for pycnidiation, low-nutrient ex planta survival, and soil treatments as a novel method of control. Novel findings include that (i) submergence leads to a form of spore dormancy, (ii) epiphytic development is more extensive than previously thought with hyphal growth and sporulation both occurring readily over a 21 day period, (iii) pycnidiation is a rapid, deterministic developmental process, triggered by contents of lysed plant cells, (iv) asexual spores have the capacity to survive for many weeks in low-nutrient environments, and finally (v) that treating soil-borne environmental spores may be a novel way to reduce field inoculum levels before a new crop is sown. Collectively these results provide many new insights in to the behaviour of Z. tritici during its disease cycle. This thesis proposes an updated version of the life cycle for this devastating fungal pathogen and argues that current methods of control are not adequate long-term. 2 Table of Contents Novel insights into the asexual life-cycle of the wheat-leaf pathogen Zymoseptoria tritici ........ 1 Thesis Abstract ................................................................................................................................... 2 Table of Contents .............................................................................................................................. 3 List of Figures ...................................................................................................................................10 List of Tables ....................................................................................................................................15 Abbreviations ...................................................................................................................................16 Acknowledgments ............................................................................................................................18 General introduction ..................................................................................................................19 1.1. Wheat and the rise of Zymoseptoria tritici ...........................................................................19 1.2. The Z. tritici life cycle ..........................................................................................................20 1.2.1. Spread ..........................................................................................................23 1.2.2. Leaf adhesion .............................................................................................24 1.2.3. Germination ...............................................................................................24 1.2.4. Epiphytic growth .......................................................................................25 1.2.5. Epiphytic survival ......................................................................................26 1.2.6. Epiphytic nutrition ....................................................................................28 1.2.7. Entry into the leaf......................................................................................29 1.2.8. Apoplastic growth .....................................................................................32 1.2.9. Apoplastic survival ....................................................................................33 1.2.10. Apoplastic nutrition ..................................................................................34 1.2.11. Necrotrophic switch .................................................................................35 1.2.12. Plant cell lysis .............................................................................................37 1.2.13. Pycnidiation ................................................................................................38 1.2.14. Environmental survival ............................................................................38 1.3. A final life-cycle ...................................................................................................................40 General materials and methods ................................................................................................41 2.1. Zymoseptoria tritici inoculum .................................................................................................41 2.2. Agar-based media ................................................................................................................41 2.3. Seeds, soil and growth cabinet conditions .......................................................................42 3 2.4. Foliar inoculations and plant incubation periods ...........................................................43 2.5. Experimental repeats and statistical analysis ...................................................................43 2.6. Photography and scanning .................................................................................................44 2.7. Confocal microscopy ..........................................................................................................44 2.8. Assessing growth on agar media .......................................................................................44 Leaving the pycnidium: Conidiospore culturability ...............................................................45 3.1. Introduction .........................................................................................................................45 3.2. Methods ................................................................................................................................45 3.2.1. Assessing the culturability of water-suspended conidiospores ...........45 3.2.2. Assessing the effects of osmotic & nutrient shock during submergence in water .............................................................................................46 3.2.3. Assessing spore viability over time in water cultures ...........................46 3.2.4. Assessing the virulence of conidiospore populations over time ........47 3.2.5. RNA: Extractions ......................................................................................47 3.3. Results ...................................................................................................................................48 3.3.1. Culturability of water-suspended conidiospores is < 30 % ................48 3.3.2. Conidiospore culturability is not affected by suspension concentration ...........................................................................................................49 3.3.3. Reduction in conidiospore culturability is rapid upon suspension ....51 3.3.4. Osmotic and nutrient shock contribute to reduction in culturability 52 3.3.5. Reductions in culturability are not due to conidiospore death ...........54 3.3.6. Virulence of conidiospores reduces over time in water cultures ........55 3.3.7. RNA extractions ........................................................................................57 3.3.8. RNA: NanoDrop™ quantification .........................................................57 3.3.9. RNA: Quantification by agarose gel electrophoresis ...........................58 3.3.10. RNA: Quantification by Agilent 2100 Bioanalyser...............................59 3.3.11. RNA: Quantification by Qubit™ ...........................................................60 3.3.12. RNA: cDNA Library preparation and sample pooling .......................60 3.3.13. RNA: data analysis by MiSeq Nano ........................................................62 3.4. Discussion ............................................................................................................................63 3.4.1. The stress of suspending conidiospores in water .................................64 3.4.2. Conidiospores are viable but not culturable: The VBNC state ..........66 3.4.3. Resuscitation and the maintenance of virulence in VBNC cells ........67 3.4.4. Implications for experimental methodology .........................................68 4 The capacity for epiphytic growth and sporulation ...............................................................70 4.1. Introduction .........................................................................................................................70 4.2. Methods ................................................................................................................................71