The diversity of the genus Fusarium in soil from the Willem Pretorius Nature Reserve, South Africa By Mokgaetji Lydia Mojela Dissertation Submitted in fulfilment of the requirements for the degree MAGISTER SCIENTIAE Department of Botany and Plant Biotechnology University of Johannesburg Johannesburg South Africa 2017 Supervisor: Dr. Eduard Venter Co-supervisor(s): Dr. Adrianna Jacobs Prof. Brett A Summerell “And blessed is she who believed that there would be a fulfilment of what was spoken to her from the lord.” Luke 1:45 i COPYRIGHT AND CITATION CONSIDERATIONS FOR THIS THESIS/ DISSERTATION o Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. o NonCommercial — You may not use the material for commercial purposes. o ShareAlike — If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original. How to cite this thesis Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujcontent.uj.ac.za/vital/access/manager/Index?site_name=Research%20Output (Accessed: Date). TABLE OF CONTENTS Page List of figures ..................................................................................................................... iv List of tables ...................................................................................................................... vii List of abbreviations .......................................................................................................... ix Declaration ....................................................................................................................... xiii Acknowledgements .......................................................................................................... xiv Chpter 1 (Introduction) .....................................................................................................1 Chapter 2 (Literature review)...........................................................................................7 2.1. Introduction .......................................................................................................8 2.2. Taxonomic history of the genus ........................................................................8 2.3. Fusarium: one fungus one name .....................................................................11 2.4. Generic placement ..........................................................................................12 2.5. Genetic diversity .............................................................................................13 2.6. Cryptic speciation in Fusarium .......................................................................14 2.7. Species concepts .............................................................................................15 2.7.1. Morphological species concept ............................................16 2.7.2. Biological species concept ...................................................17 2.7.3. Phylogenetic species concept ...............................................18 2.7.4. Ecological species concept ..................................................18 2.8. Phylogeography ..............................................................................................19 2.9. Biogeography ..................................................................................................20 2.10. Climatic factors .............................................................................................21 2.11. Dispersal of Fusarium...................................................................................21 2.12. Sexual and asexual stages .............................................................................22 2.13. Mating types in Fusarium .............................................................................23 2.14. Molecular markers ........................................................................................24 2.15. The Translation Elongation Factor 1-α, the RNA (Ribonucleic Acid) Polymerase II (RPB1 & 2), genes of choice ..............................................................................26 ii 2.16. Mycotoxins and other secondary metabolites ...............................................27 2.16.1. Trichothecenes, Zearalenones, and Fumonisins ..................28 2.16.2. Other secondary metabolites ................................................29 2.17. Infection processes and survival of fusaria in soil ........................................30 2.18. Plant diseases ................................................................................................31 2.19. Human diseases .............................................................................................32 2.20. None-pathogenic Fusarium ..........................................................................33 2.21. Fusarium study in South Africa ....................................................................35 2.22. New technology for Fusarium research ........................................................37 2.23. General climate, vegetation and soil type of the study area (Willem Pretorius nature reserve) ........................................................................................................38 2.24. Conclusion ....................................................................................................39 Chapter 3 (Materials and Methods) ...............................................................................41 3.1. Sampling and sample processing ....................................................................42 3.2. DNA extraction ...............................................................................................44 3.3. Polymerase chain reaction and Cycle Sequencing ..........................................45 3.4. Sequence editing .............................................................................................45 3.5. Phylogenetic analysis ......................................................................................46 3.6. Chapter tables..................................................................................................47 3.7. Chapter figures ................................................................................................54 Chapter 4 (Results) ..........................................................................................................57 4.1. Fungal isolation ...............................................................................................58 4.2. Morphological characterisation ......................................................................58 4.3. DNA extraction and PCR................................................................................59 4.4. Nucleotide BLAST results ..............................................................................59 4.5. Phylogenetic Analysis .....................................................................................63 4.6. Chapter tables..................................................................................................69 4.7. Chapter figures ................................................................................................81 Chapter 5 (Discussion) .....................................................................................................93 Conclusion and future work .........................................................................................107 References .......................................................................................................................108 Summary .........................................................................................................................128 iii List of figures Figure 2.1: Fusarium solani. From left to right, macroconidia, microconidia, and chlamydospores. Adapted from Zhang & Sung, (2008) ....................................................16 Figure 2.2: TEF gene region map of Fusarium with primer locations. Adapted from Geiser et al. (2004) ............................................................................................................................27 Figure 2.3: Diagram showing the diverse structures of some frequently occurring mycotoxins in Fusarium. Taken from Dawson et al. (2006).................................................................29 Figure 2.4: Map showing the five main vegetation types classified by Müller (1986) in the Willem Pretorius Nature Reserve (Adapted from Winterbach, 1999) ...............................39 Figure 3.1: Diagram of the South Africa map showing the location of the Willem Pretorius nature reserve in the Free State Province. The reserve was proclaimed during the building of the Allemanskraal Dam which is bordered by the nature reserve .....................................54 Figure 3.2: The transect method used to obtain soil samples. End samples were pooled and sub-samples were pooled. ..................................................................................................55 Figure 3.3: Flow diagram illustrating the process of isolation and purification using the different media. Morphological characterisation was only performed on selected isolates. DNA extraction and culture preservation