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The diversity of the genus Fusarium in soil from the Willem Pretorius Nature Reserve, South Africa

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

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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 [Unpublished]: University of Johannesburg.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

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

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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 was performed on all isolates ...... 56

Figure 4.1.1: Numbers of cultures from large soil particles compared to the small soil particles obtained at the Willem Pretorius nature reserve from the four sites ...... 81

Figure 4.1.2: Germinating spores of a Fusarium species on water agar visualized under a stereo microscope ...... 82

Figure 4.1.3: Colony pigmentation and morphology of Fusarium species on Potato dextrose agar (PDA) after 10 days of growth under dual light (12 hours normal light and 12 hours UV light) conditions. For each pair, the plate on top illustrates the top surface of the plate and the bottom plate illustrates the bottom surface of the plate. The cultures represent isolates identified as; 1. F. fujikuroi (PPRI 19149) 2. F. burgessii (PPRI 19129) 3. F. chlamydosporum (PPRI 19213) 4. F. solani (PPRI 20535) 5. F. acuminatum (PPRI 19235) 6. F. nygamai (PPRI 20740) 7. F. oxysporum (PPRI 20746) 8. F. equiseti (PPRI 21571) 9. F. brachygibbosum (PPRI 21297) 10. F. redolens (PPRI 21574) ...... 83

Figure 4.2: The types and arrangement of the observed morphological characters for the five selected Fusarium species. A-E: Macroconidia of F. solani (A), F. brachygibbosum (B), F. equiseti (C), F. oxysporum (D) and, F. fujikuroi (E). F-I: Microconidia of F. brachygobbosum (F), F. fujikuroi (G), F. oxysporum (H) and, F. solani (I). J, Microconidia borne on polyphialides in situ on CLA (F. fujikuroi). K, Microconidia produced in chains in situ on CLA (F. fujikuroi). L-N; Microconidia borne on monophialides in situ on CLA, F. fujikuroi (L), F. oxysporum (M) and F. solani (N). O-Q Chlamydospores of F. brachygibbosum (O), F. equiseti (P) and, F. oxysporum in situ on CLA (Q). A-Q, scale bar = 20µm ...... 84

Figure 4.3.1: Electrophoretogram of a subset of DNA extracted from the isolated Fusarium species. The genomic DNA bands were visualized on a 2% agarose gel. Lane M: 100 bp DNA ladder. Lane 1: PPRI 19120, lane 2: PPRI 19122, lane 3: PPRI 19123, lane 4: PPRI 19127, lane 5: PPRI 19128, and lane 6: PPRI 19137 ...... 85

Figure 4.3.2: PCR amplification of the EF-1α gene for the same six Fusarium samples as in figure 4.3.1. PCR products were visualized on a 2% agarose gel. Lane M: 100 bp DNA ladder. Lane 1: PPRI 19120, lane 2: PPRI 19122, lane 3: PPRI 19123, lane 4: PPRI19127, lane 5: PPRI 19128, lane 6: PPRI 19137 and lane 7: non-template control (NTC). The fragment size of the amplified gene is ca.700 base pairs...... 85

Figure 4.4.1a&b: Different species complexes and species of the genus Fusarium identified via nBLAST analysis. A-Mycobank database, B-FUSARIUM-ID database ...... 86

Figure 4.4.2a&b: Numbers of Fusarium isolates obtained from the two soil particle sizes. A- Mycobank nBLAST results, B-FUSARIUM-ID nBLAST results ...... 87

Figure 4.4.3a&b: Numbers of Fusarium isolates observed at site 1 and site 2 within the Willem Pretorius nature reserve. A-Mycobank nBLAST results, B-FUSARIUM-ID nBLAST results ...... 88

Figure 4.4.4a&b: Numbers of Fusarium isolates observed at site 3 and site 4 within the Willem Pretorius nature reserve. A-Mycobank nBLAST results, B-FUSARIUM-ID nBLAST results ...... 89

Figure 4.5.1: One of the most parsimonious phylogenetic trees of the Fusarium incarnatum- equiseti species complex (FIESC), including the isolates collected from Willem Pretorius nature reserve, denoted on the tree with PPRI numbers (black font colour) and inferred from the TEF-1α sequence data, as well as isolates from published work indicated by NRRL collection numbers (red font colour). The tree is rooted by F. concolor (NRRL 13459) (purple font colour). Branch support for both MP (purple font colour) and ML (light blue font colour) are indicated on the tree branches with bootstrap support. The FIESC taxa are assembled into clades indicated in the colour blocks. Tree statistics for the FIESC dataset: RI = 0.9028; CI = 0.7301; HI = 0.2699 and, Tree length = 1061 ...... 90

Figure 4.5.2: One of the most parsimonious phylogenetic trees of the Fusarium oxysporum species complex (FOSC), including the isolates collected from Willem Pretorius nature reserve, denoted on the tree with PPRI numbers (black font colour) and inferred from the TEF-1α sequence data as well as isolates from published work denoted on the tree as SARD (orange font colour), AUST (red font colour) and other isolates (dark orange font colour). The tree is rooted by isolate Fusarium sp. (RGB5443). Branch support for both MP (purple font colour) and ML (light blue font colour) are indicated on the tree branches with bootstrap support. The FOSC taxa are assembled into clades indicated in the colour blocks. Tree statistics for the FOSC dataset: RI = 0.9421; CI = 0.8810; HI = 0.1190 and, Tree length = 311 ...... 91

Figure 4.5.3: One of the most parsimonious phylogenetic trees of the genus wide dataset, including the isolates collected from Willem Pretorius nature reserve, denoted on the tree with PPRI numbers (black font colour) and inferred from the TEF-1α sequence data as well as isolates from published work denoted in accession numbers (red font colour). The midpoint rooting method was used to root the genus wide dataset. Branch support for both MP (purple font colour) and ML (light blue font colour) are indicated on the tree branches with bootstrap support. The FOSC taxa are assembled into clades indicated in the colour blocks. Tree statistics for the genus wide dataset: RI = 0.8402; Consistency Index (CI) = 0.4897; HI = 0.5103 and, Tree length = 2079 ...... 92

List of tables

Table 2.1: Table showing a brief summary of the Fusarium systematics history ...... 9

Table 2.2: Mycotoxin-producing Fusarium species in important grasses and their host plants ...... 28

Table 3.1: Fusarium isolates used for the FIESC dataset matrix. The accession numbers of these isolates were obtained from O’Donnell et al. (2009b). The sequences for these isolates were obtained from NCBI Genbank using the NRRL numbers and were combined with the FIESC isolates obtained in the current study to build up the matrix for the FIESC dataset ...... 47

Table 3.2: Fusarium species used for the FOSC dataset matrix. The accession numbers of these isolates were obtained from Laurence et al. (2014). The sequences for these isolates were obtained from NCBI Genbank using the accession numbers and were combined with the FOSC isolates obtained in the current study to build up the matrix for the FOSC dataset ...... 49

Table 3.3: Fusarium isolates used for the genus wide dataset matrix. The accession numbers of these isolates were obtained from Laurence et al. (2016). The sequences for these isolates were obtained from NCBI Genbank using the accession numbers and were combined with the FIESC isolates obtained in the current study to build up the matrix for the genus wide dataset ...... 51

Table 4.1: Fusarium-incarnatum equiseti species complex (FIESC) nucleotide BLAST results from MYCOBANK and FUSARIUM-D databases. The isolates indicated in bold signify contrasting nBLAST results from the two databases ...... 69

Table 4.2: Fusarium oxysporum species complex (FOSC) nucleotide BLAST results from the MYCOBANK and FUSARIUM-ID databases. The isolates indicated in bold signify contrasting nBLAST results from the two databases ...... 72

Table 4.3: Different Fusarium species obtained from nucleotide BLAST analysis from the MYCOBANK and FUSARIUM-ID databases. The isolates indicated in bold signify contrasting nBLAST results from the two databases ...... 74

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Table 4.4: Fusarium brachygibbosum nucleotide BLAST results from the MYCOBANK and FUSARIUM-ID databases. The isolate indicated in bold signify contrasting nBLAST results from the two databases...... 76

Table 4.5: Fusarium solani species complex (FSSC) nucleotide BLAST results from the MYCOBANK and FUSARIUM-ID databases ...... 78

Table 4.6: Fusarium chlamydosporum species complex (FCSC) nucleotide BLAST results from the MYCOBANK and FUSARIUM-ID databases. The isolates indicated in bold signify contrasting nBLAST results from the two databases ...... 80

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List of abbreviations

1F1N one fungus one name acl1 ATP (adenosine triphosphate) citrate lyase AE elution buffer ATP adenosine triphosphate AUST Australia AW wash buffer

C Centigrade CI consistency index CLA carnation leaf agar CBS Centraalbureau voor Schimmelcultures CDC Centres for Disease Control and Prevention EDTA ethylene diamine tetraacetic acid EMBL European Molecular Biology Laboratory DDBJ DNA Data Bank of Japan DAS Diacetoxyscirpenol dH2O distilled water DNA deoxyribonucleic acid dNTP deoxy-nucleotide triphosphate DON Deoxynivalenol F Fusarium FASC Fusarium aywerte species complex FCSC Fusarium chlamydosporum species complex FIESC Fusarium incarnatum-equiseti species complex FFSC Fusarium fujikuroi species complex FGSC Fusarium graminearum species complex FHB Fusarium head blight

FRSC Fusarium redolens species complex FOC Fusarium oxysporum f. sp. cepae FOSC Fusarium oxysporum species complex FSAMSC Fusarium sambucinum species complex FSSC Fusarium solani species complex G Gibberella G–C guanine–cytosine GCPSR Genealogical Concordance Phylogenetic Species Recognition GFSP Gibberella fujikuroi species complex g/l gram per litre GPS geographic positioning system HMG high mobility group HI homoplasy index

ICBN International Code of Botanical Nomenclature IGS intergernic spacer regions ITS internally transcribed spacer regions MAFFT Multiple Alignment using Fast Fourier Transform MAT mating type µM micromolar

MIC minimal inhibitory concentration Mm Millimolar ML maximum likelihood ml Millilitre

MLST Multilocus DNA Sequence Typing MP maximum parsimony mtSSU mitochondrial small subunit rDNA nBLAST nucleotide basic alignment search tool

NCBI National Centre for Biotechnology Information NCF National Collection of Fungi ng Nanogram

NIR nitrate reductase NIV Nivalenol nm Nanometre NRRL Agricultural Research Service Culture Collection NTC no template control ORF open reading frame PAUP Phylogenetic Analysis Using Parsimorny PCNB Pentachloronitrobenzene PCR polymerous chain reaction PDA potato dextrose agar PHO phosphate permase PhyML Phylogeny based on Maximum Likelihood P3 neutralising buffer PPRI Plant Protection Research Institute PS phylogenetic species REMI Restriction Enzyme Mediated Integration rDNA ribosomal deoxyribonucleic acid RI retention index RPB I RNA (Ribonucleic Acid) Polymerase II small subunit

RPB 2 RNA (Ribonucleic Acid) Polymerase II large subunit rpm revolutions per minute s Seconds SFA selective Fusarium agar SIX secreted in xylem

SNA synthetic nutrient agar sp. Species spp. several species T–2 trichothecene mycotoxin TBR tree branching TAE tris base, acetic acid and EDTA TEF 1-α Translation Elongation Factor 1-alpha TFC terminal Fusarium clade tub 2 β-tubulin USA United States of America UV ultra violet V Volts WA water agar WP Willem Pretorius xg times gravity

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Declaration

I, the undersigned, hereby declare that the dissertation submitted herewith for the degree Magister Scientiae at the University of Johannesburg, contains my own independent work and has not been submitted for any degree at any other university.

Mokgaetji Lydia Mojela

December 2017

xiii

Acknowledgements

First and foremost, I would like to thank God the almighty for giving me the strength and perseverance to complete my work as challenging as it was. I could have never gotten this far without him.

The success of this study was made possible by several people and organisations who have assisted, encouraged and, cooperated with me from the beginning to the end of the research project, to you I will forever be grateful:

I would like to say a very big thank you to my supervisor Dr Eduard Venter and co-supervisors Dr. Adrianna Jacobs and, Prof. Brett Summerell, I truly appreciate the support and supervision you have given me from the beginning.

To my parents, my education to you has always been important, you sacrificed so much so that I can get the best education necessary. You always gave me support, have always been my pillar of strength when things got challenging. I will forever be indebted to you. Thank you Mma le Papa, I am who I am because of you.

To my big sister and brothers for always encouraging and having faith in me. You were always there when I needed you most and have always been my number one cheerleaders.

I will forever be thankful to my colleagues and friends at the Department of Botany and Biotechnology for all the assistance they offered none hesitantly, may God bless you. Moreover, I would also like to thank my colleagues at the Agricultural Research Council (ARC) who gave training courses that best aided in the understanding of the study.

I would like to thank the University of Johannesburg, the Department of Botany and Biotechnology and, the ARC for providing me with all the resources and facilities necessary to successfully finish this research.

A big thank you to the National Research Foundation (NRF) for funding my research project, you have truly made dreams come true.

Last but not least to James, I am grateful for all the academic advice you gave me, for always offering to lend a helping hand at all times, for understanding and enduring in this journey with me. Your presence in my life personally and academicaly is truly appreciated.

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CHAPTER 1

INTRODUCTION

Fusarium is a genus initially defined by Link (1809) as Fusisporium and belongs to the phylum Ascomycota. There are many species in the genus and are globally present in soils and plants as saprobes and endophytes (Aoki et al. 2014). The genus Fusarium includes roughly 300 phylogenetically diverse species detected through molecular phylogenetics. However, the majority of these species have not yet been officially named or described (Aoki et al. 2014). Link (1809) defined the Fusarium genus in 1809 and subsequent to that, Wollenweber and Reinking issued a thorough monograph of Fusarium in 1935. They grouped the large number of Fusarium species into 16 divisions containing 65 species, 55 varieties, and 22 forms. However, in 1940, Wollenweber and Reinking’s species were grouped into nine species by Snyder & Hansen (Nalim, 2004). To some extent, the taxonomy of the genus has been controversial since then.

Members of the Fusarium genus are well known for their ability to cause plant diseases and are toxic to both domestic animals and humans (Marasas et al. 1984; Desjardins, 2006). They produce a range of secondary metabolites called mycotoxins that differ greatly in chemical structure (Desjardins, 2006). Fusarium species also produce other secondary compounds of possible importance that are not necessarily restricted to Fusarium strains, such as beauvericin, acuminatum, equisetin, chlamydosporol, butenolide, etc. (Azor et al. 2007; Summerell and Leslie, 2011).

Species concepts have been the main focus of research with all known fungi, with Fusarium being no exception. Currently, the morphological, biological, and phylogenetic species concepts are used within the genus Fusarium (Summerell and Leslie, 2011). The morphological species concept is based on the resemblance of observable morphological characters such as spore shape and size. This concept has dominated for most of the history of Fusarium until the phylogenetic species concept was introduced (Summerell et al. 2010). The biological species concept regards species as groups of populations that effectively or potentially interbreed amongst themselves (Leslie, 1995). The phylogenetic species concept is a much more modern development in mycology. It has become very important in the characterization of Fusarium species. This concept makes use of multiple gene markers (Summerell et al. 2010). According to Taylor et al. (2000), the ecological species concept highlights the adaptation of species to a specific ecological niche and incorporates well into the classification of Fusarium species if studied more intensely.

Fusarium species produce a range of diseases that negatively influence horticulture and agriculture around the world (Windels, 2000). Diseases such as head blight (Windels, 2000) of wheat (Backhouse and Burgess, 2002) and Panama disease that affects bananas have been of great concern (Ploetz, 1990). These diseases and more have had overwhelming sociological and economic impacts towards farmers and communities that depend on such crops as their main source of food (Summerell and Leslie, 2011). Recently, a study by Dean et al. 2012, in the international community of plant pathologists ranked two Fusarium species, F. graminearum Schwabe and F. oxysporum Schltdl. fourth and fifth, on a list of top ten fungal plant pathogens based on scientific/economic importance. Even though Fusarium species can reproduce both sexually and asexually, less than 20% of the species have a recognised sexual cycle and the several different spore types are produced by different species of Fusarium (Ma et al. 2013). Mating in the genus Fusarium has also been identified as a remarkable phenomenon and it occurs between morphologically identical partners. The analysis of mating types using molecular techniques has become an effective tool for investigating the evolutionary life cycles and relationships of species (Christiansen et al. 1998).

Climatic factors are involved in enhancing the growth, dissemination as well as the survival of Fusarium species. These factors could increase the distribution of species and the severity of the diseases they cause. The most crucial climatic factors that affect the production and spread of sexual ascospores and asexual conidia of Fusarium are humidity, light intensity, wind, and temperature (Doohan et al. 2003). Several new species have been discovered in recent studies of the genus Fusarium, e.g. Laurence et al. (2016) and Herron et al. (2015). Most of these species have been isolated from grasses, soil, and pine trees. According to Burgess et al. (2008), some studies have also indicated that several important pathogens, for example, F. thapsinum Klittich, J.F. Leslie, P.E. Nelson & Marasas can be isolated from grasses distant from cropping fields (Burgess et al. 2008). There are four kinds of dispersal patterns that are found in Fusarium species: host associated, anthropogenic, climatic factors, and certainly, cosmopolitan distribution (Backhouse et al. 2001; Summerell et al. 2010).

Phylogeography investigates the processes that affect the geographic dispersal of genetic ancestries, normally based on molecular information and principles suggested by Avise (2000). The fact that most species have a certain degree of population structure amongst temporal and spatial components makes phylogeographic studies possible (Summerell et al. 2010). The biogeography of numerous important Fusarium pathogens can be determined effectively if the biogeography of their closely related non-pathogenic forms can also be correctly identified

(Summerell et al. 2010). Throughout the years, the occurrence of cryptic species has been acknowledged (Klittich et al. 1997; Steenkamp et al. 2000) and although they are either phylogenetically or biologically distinct, cryptic species are normally morphologically indistinguishable (e.g., Britz et al. 1999; Klittich and Leslie, 1992; Nirenberg and O’Donnell, 1998).

During the infection process, the hyphae of Fusarium grows towards the roots of their plant host in reaction to rhizosphere nutrients secreted by plants. They normally multiply on the surfaces of roots prior to penetrating the cortical tissue (Smith, 2007). However, soil-borne pathogens lack the ability to distribute as quickly as those organisms found above ground. Therefore, for successful penetration into the roots within the soil environment, they must have the ability to survive in soil (Smith, 2007). Furthermore, not all Fusarium species will cause plant diseases. The occurrence of Fusarium infections in some cultivated soils is reduced regardless of the presence of a vulnerable host, infectious pathogen and conducive environmental conditions. These types of soils are called Fusarium suppressive soils (Stover, 1962).

In South Africa, peri-urban families that live on severely limited budgets often alternatively grow food at their homes instead of purchasing in supermarkets in order to supplement their supply of food (Iram et al. 2011). Damage by harmful Fusarium species to small-scale crop production has received very limited research attention (van de Walt et al. 2007). In this respect, home cultivates are an essential source of nutrition for resource-poor urban families in Africa and the information on Fusarium species would be more helpful (van de Walt et al. 2007). The information on the incidence of Fusarium species in South African soils has gradually improved during the previous years. The study by Marasas et al. (1988) was the first to report the information on the occurrence of Fusarium species in both undisturbed and cultivated soils found in different climatic areas in South Africa. Furthermore, Klaasen et al. (1991) documented information on the nature and dispersal of Fusarium species in wheat cultivated soils of the Western Cape Province.

Fusarium species identification can be established through the use of DNA sequences originating from the TEF-1α gene that codes for the Translation Elongation Factor 1-α protein. This gene is extremely informative at the species level in Fusarium (Geiser et al. 2004) as there is a high level of phylogenetic variety in the genus Fusarium. Hence, multilocus DNA sequence typing (MLST) that represents a highly robust method for the characterization of the genetic

4 diversity of Fusarium species are currently used (O’Donnell et al. 2009a). The use of MLSTs of mycotic pathogens has distinguished epidemic clones and uncovered cryptic speciation (Short et al. 2014).

Several South African studies such as those by Marasas et al. (1988), Klaasen et al. (1991) and Rheeder and Marasas (1998) have predominantly recovered F. oxysporum, F. incarnatum- equiseti and F. solani, amongst others from both disturbed and undisturbed ecosystems. These three mentioned species are cosmopolitan soil-borne fungi known to occur as both pathogens and saprophytes (Burgess, 1981; Stoner, 1981; McMullen and Stack, 1983). Based on this, it was hypothesized that F. oxysporum, F. incarnatum-equiseti and F. solani will be the most occurring species within the uncultivated ecosystem. The hypotheses was tested in the current study on a collection of isolates from soil samples obtained at the Willem Pretorius Nature Reserve.

The aim of this study was to establish the diversity of Fusarium species in undisturbed soils from the Willem Pretorius nature reserve, located in the Free State Province of South Africa, using the TEF-1α phylogenetic marker. The relationships amongst some of these species were assessed by means of a combined approach using both morphological and phylogenetic analysis. Morphological analysis was performed for five previously described Fusarium species, namely, F. oxysporum, F. equiseti (Corda) Sacc., F. solani (Mart.), F. fujikuroi (Sawada) Wollenw, and F. brachygibbosum Padwick. This was done to provide an overview of some of the morphological characters of Fusarium species obtained in this study. The phylogenetic relationships of strains of the Fusarium oxysporum species complex (FOSC), Fusarium incarnatum-equiseti species (FIESC) and various other Fusarium species were analysed in this study.

The data that this study generated will assist in identifying the possibility of new disease development caused by Fusarium species in future. Therefore, knowing and understanding the type of Fusarium pathogens occurring at certain areas helps us develop strategies to combat future global outbreaks. Some diseases caused by Fusarium species have had overwhelming sociological and economic impacts towards farmers and communities that depend on such crops as their main source of food (Summerell and Leslie, 2011). The study will assist in determining risk evaluation methods for farmers and peri-urban families. The information will educate them on the most prevalent Fusarium species found in certain soil types. However, it

5 is not enough for the grower to only identify the type of Fusarium connected to a disease that may potentially harm their crops, therefore certain control practices will have to be taken. A follow up on this study can also focus on implementing effective control measures for Fusarium species that affect their crops, such as the use of specific fungicides. Therefore, it is important to gather more knowledge on this genus for the purpose of mitigating the damage they cause. Furthermore, the study will also contribute to the information about Fusarium species composition in uncultivated ecosystems of South Africa.

CHAPTER 2

LITERATURE REVIEW

2.1. Introduction

Link (1809) established the genus Fusarium in 1809 and it now encompasses about 70 species (Leslie and Summerell, 2006a) that are both pathogenic and none pathogenic in nature (Tewoldemedhin et al. 2011). Members of this genus can directly cause disease in domesticated animals, humans, and an array of plant species (Leslie and Summerell, 2006a). The Fusarium genus belongs to the phylum Ascomycota, class Sordariomycetes O.E. Erikss. & Winka and the order Hypocreales Lindau (Leslie, 1995). Fusarium species have sexual stages that are generally categorized in the genus Gibberella Sacc. (Nayaka et al. 2011) in addition, for a fewer number of species, were categorized under the genus Albonectria Rossman & Samuels (Rossman et al. 1999). Albonectria was discovered to form a monophyletic clade closely related but distinct to Fusarium (Lombard et al. 2015). Furthermore, recent studies have proposed that Fusarium holds priority over Albonectria sexual stages (Lombard et al. 2015; O’Donnell et al. 2015).

Fusarium contains species that nearly infect every cultivated food crop worldwide. At least 81 out of 101 utmost economically significant plants are hosts to Fusarium species (Nayaka et al. 2011). Plant diseases caused by species in this genus can be initiated at any stage of plant growth. The kinds of diseases caused are relatively diverse and may include seedling blight, seed rot, ear rot, stalk rot, kernel rot, head blight, wilts, leaf diseases, and cankers (Nayaka et al. 2011). It has been a challenge to determine the taxonomic rank of Fusarium species on the basis of phenotypic features alone, including toxigenicity and pathogenicity. Currently, the morphological species concept that has been employed for a long period of time has been implemented together with two other species concepts. Together, the morphological, phylogenetic, and biological species concepts have been used to distinguish Fusarium species (Moretti, 2009; Nayaka et al. 2011; Link, 1809).

2.2. Systematic history

The classification of Fusarium started with the description of the genus by Link in the year 1809 (Table 2.1). During the next century, there were at least a thousand Fusarium species that were named. Many of them were found to have more than one host. In 1935, a detailed monograph was published by Wollenweber and Reinking. In this monograph, the authors took the large number of the identified Fusarium species and arranged them into 16 sections comprising of 65 species, 55 varieties and 22 forms (Wollenweber and Reinking, 1935). This ordered the preceding state of Fusarium species classification and they also documented

synonyms for the species. However, subsequent systems did not support the Wollenweber & Reinking arrangement. Nonetheless, most classifications for Fusarium taxonomy that ensued were based on Wollenweber & Reinking’s work (Nalim, 2004). In the year 1940, Snyder and Hansen took all of Wollenweber & Reinking’s species and grouped them into nine species (Snyder and Hansen, 1940). Moreover, two French researchers, Messiaen and Cassini, created a system moulded around that of Snyder and Hansen in the year 1968. However, they made use of botanical varieties other than cultivars at the sub-specific level (Leslie and Summerell, 2006a). The change by Snyder and Hansen made the diagnosis and classification of Fusarium species much easier. Despite this simplification, the species concepts were polyphyletic and most of the information established on strains classified using these concepts is currently hard to interpret. Fusarium solani and F. oxysporum are two of Snyder and Hansen’s species, that still have a wide usage, but they are also recognised as species complexes (Hillis and Huelsenbeck, 1992; O’Donnell et al. 2000) and require more attention. It was not surprising when several species were described and split from the aforementioned taxa, such as F. virguliforme O'Donnell & T. Aoki and more were likely suspected to follow (Aoki et al. 2003; Summerell et al. 2010).

Table 2.1: Table showing a brief summary of the Fusarium systematic history.

Date Taxonomic system Number of species 1809 - Link’s description of genus 1809-1935 Unorganised species description More than 1 000 species 1936 - Wollenweber and Reinking 65 species, 16 sections 1940-45 Snyder and Hansen 9 species 1971 Booth 44 species 1982 Gerlach and Nirenberg 78 species 1983 Nelson 30 species (N, T & M, S&M) 1990 - 1997 The use of both molecular phylogenetics and Numerous hundreds of morphology. different species. 1998 Nirenberg and O’Donnell - morphological and molecular Ten species system. Adapted from (Nalim, 2004)

In ‘The genus Fusarium’, Booth (1971) recognized 44 species and seven varieties based on morphological characters. According to his system, the production of microconidia was regarded more as a distinguishing character. Furthermore, Gerlach and Nirenberg documented 73 species and 26 varieties based primarily on the system developed by Wollenweber and Reinking (Gerlach and Nirenberg, 1982). The work done by Booth, Gerlach and Nirenberg, and Nelson invalidated a lot of changes suggested by Snyder and Hansen and preceded towards taxonomic systems greatly derived from Wollenweber and Reinking’s technique. These systems focused more on the careful valuation of morphological characters such as chlamydospores, conidiogenous cells, macroconidia and microconidia on standard media (Summerell et al. 2010), while taking into consideration the difference within species recorded by Snyder and Hansen. A lot has been recorded about the variations between these taxonomic systems, nevertheless, all have made use of most of Wollenweber and Reinking’s sections and species descriptions, and where variations of opinion ensued, they were frequently nomenclatural variations rather than variations in species descriptions (Summerell et al. 2010). These variations were mainly based on the weight given to particular morphological characteristics. In their 1975 publication, Toussoun and Nelson regarded the macroconidia morphology as the primary characteristic for describing most Fusarium species (Nelson, 1992). In 1983, the system by Nelson, Toussoun and Marasas expanded Snyder and Hansen’s nine species to 30 species (Nalim, 2004). In the year 1995, Bilai indicated that the range of variability in certain characters in numerous species was equivalent to that seen in whole sections. Bilai constructed her own amendment of the taxonomy of the genus and suggested merging some of the sections of Wollenweber and Reinking (Leslie and Summerell, 2006a). Classification systems based on morphology had identified 73 species (Gerlach and Nirenberg, 1982) and as little as nine varieties (Snyder and Hansen, 1940). However, molecular phylogenetic studies, introduced in the 1990s discovered a far more complicated picture of species identification in the genus Fusarium (Nalim, 2004). In 2000, Leslie and Summerell (2006b) initiated an annual written laboratory workshop for the purpose of teaching identification techniques on a different basis. The annual laboratory workshops are currently still running. The techniques taught during the workshop are different from those conducted in the 1980s and 1990s in that they are also based on the biological species concept and the phylogenetic species concept rather than assigning more weight to certain morphological characteristics than others.

2.3. Fusarium: one fungus one name Recently, a significant and notable change in the systematics of Fusarium was made. Fungi that reproduce both sexually and asexually (pleomorphic fungi) have been accepted under an exclusive condition of the previous International Code of Botanical Nomenclature (ICBN, Article 59) to possess distinct names that refer to the asexual (anamorph) and the sexual (teleomorph) stages, with reference to the entire fungus (holomorph) (Geiser et al. 2013). The teleomorph name enjoyed preference until introduction of the new amendment. During the 2011 gathering of the Nomenclature Conference of the Botanical Congress hosted in Melbourne, it was concluded that as of the 1st of January 2013, the previous Article 59 will not be applied any further (Hawksworth, 2011). All the names, regardless if they are characterized by a sexual or asexual stages, should be on the same level with regards to priority under the conditions of the ‘one fungus one name’ (1F1N) (Wingfield et al. 2012). Furthermore, the name 1F1N also replaced the ICBN name of Article 59 (Geiser et al. 2013). Therefore, the opportunity remains to preserve anamorph labelled species like those of Fusarium in a manner that the scientific society considers appropriate. This implies that the second-class nomenclatural rank of Fusarium as an asexually (anamorph) reproducing genus has ended, thus allowing the restricted usage of the Fusarium name with the seclusion of other teleomorph names. In recent years, researchers studying the genus Fusarium have been expressing sexual stages with the exclusion of a matching teleomorph name (Covert et al. 2007, Scauflaire et al. 2011). Following the changes made after Article 59, a criterion was formulated for the new circumscription of the genus Fusarium. They encouraged the use of three principles when circumscribing the generic boundaries of Fusarium. The three principles are in terms of monophyly, tradition and, the concept of one Fusarium one name. These three principals have provided an organised system of naming new Fusarium species. Species named after the one fungus one name concept came into effect and have to comply with the basis of the three principles. Species such as those newly described by Laurence et al. (2016), F. coiscis, F. goolgardi, F. mundagurra, F. newnesense, F. tjaetaba and, F. tjaynera have all been described under these principles.

In Geiser et al. (2013), further considerations for the future of the genus Fusarium were also stated. The genus is scattered with names that are in use but characterised vaguely, or entirely lack types. For example, the work previously published by Gräfenhan et al. (2011) would be a good start in the attempt to properly and effectively characterise species of this genus. The construction of a list of current names in the genus, as an attempt to resolve the aforementioned

11 problem, can be made and then be examined one at a time. Moreover, scores of nameless phylogenetic isolates are also being acknowledged, thus a more vigorous scheme for integrating existing taxonomic systems with our fast growing understanding of Fusarium phylogeny is needed (Geiser et al. 2013). More phylogenetic studies, especially those on the basis of much bigger, genome-level information sets, will ultimately be used to recreate a more comprehensive evolutionary past of Fusarium and will make it possible for us to progressively evaluate its monophyly (Gräfenhan et al. 2011; O’Donnell et al. 2013). Finally, and most significantly, our quick and growing understanding of Fusarium diversity needs efficient communication to the relevant society. This motion will need a well-coordinated society effort as well as support mechanisms to aid in the digital classification and sharing of appropriate information and cultures (Geiser et al. 2013).

2.4. Generic placement The genus Fusarium is assumed to be a monophyletic group. However, according to Summerell and Leslie (2011), there is no clear evidence to confirm the argument that the monophyletic description of Fusarium is correct. Furthermore, Wollenweber and Reinking (1935) evidently described Fusarium sensu stricto as polyphyletic (Summerell and Leslie, 2011). However, a more recent study by Lombard et al. (2015) indicated that clades of the genus Albonectria (Rossman et al. 1999) were introduced to accommodate species possessing white to faint yellow ascomata. These species were closely related to Fusarium asexual morphs. The study indicated that this group was defined by a monophyletic clade closely related but distinct from the clades characterising Fusarium, Cyanonectria Samuels & Chaverri and, Geejayessia Schroers, Gräfenhan & Seifert (Lombard et al. 2015). This suggested that the genus Fusarium may indeed be monophyletic. Furthermore, a recent study by O’Donnell et al. (2013) further determined the genetic placement of Fusarium. They made use of both the largest and second largest subunits of the DNA-directed RNA polymerase II (RPB1) and (RPB2) to infer the first thorough and well-substantiated phylogenetic theory of evolutional relationships within the genus Fusarium and 20 of its close relatives. The purpose of this inference was to verify if the terminal Fusarium clade (TFC) is monophyletic. Their analysis showed that the genus Cylindrocarpon produced a basal monophyletic sister group to a TFC. The TFC comprised of 20 strongly substantiated species complexes, and nine monophyletic ancestries that they provisionally documented as Fusarium.

Gräfenhan et al. (2011) made an effort to solve the generic limitations of taxa linked with Fusarium. They have also removed several species into genera such as Fusicolla Bonord.,

Dialonectria (Sacc.) Cooke, Microcera Desm., and numerous others. It may possibly be contended that all other species from the Fusarium genus must be categorized in different genera and be given new names. However, there are counter conflicts that species like those belonging to the F. solani species complex Sacc. should further be allowed under the Fusarium genus (Summerell and Leslie, 2011). They favoured the recommendation of Gräfenhan et al. (2011) with respect to nomenclature. Gräfenhan et al. (2011) recommended that the utilisation of a single nomenclatural scheme based on the name Fusarium can be the best and suitable method for the genus. This would be valid because it is based on the significance of the name and moreover, it expresses utmost information from many points of views. These can either be from pathological, policy, toxicology and, taxonomic backgrounds (Summerell and Leslie, 2011).

2.5. Genetic diversity Genetic diversity is defined as the overall amount of genetic traits in the genetic composition of a certain species. It serves as a method in which populations adapt to constant changing environments. As a result of further variation, it is highly possible that certain individuals will possess a deviation of alleles in a population, which are more suitable for that given environment. Such individuals are more likely to give rise to offspring carrying that allele. This population will endure for longer periods because of the prosperity of these individuals (NBII, 2011). Population, population size and, selection all influence gene diversity. For loci that are neutral, populations that have progressed for longer periods at a single location are more likely to possess more alleles compared to populations that have recently moved to an area. This is due to the ample time obtained for new variants to be introduced and the increase of the occurrences of new alleles to measurable amounts as a result of genetic drift. Therefore, a ‘core of origin’ where a species stems from is expected to possess a greater level of genetic diversity compared to other populations for the reason that it is much older. However, this is not the same for populations that have a low gene diversity because they might have been altered by enormous reductions in the size of the population (McDonald, 1997). Consequently, gene diversity remains low if there is a small number of reproducing individuals, which then results in the occurrence of genetic drift. Numerous alleles within a population at certain frequencies can be maintained through selection but only for genes observed in diploid individuals that display over-dominance (McDonald, 1997). Gene diversity however, is not influenced by the mating system. At distinct loci, asexually reproducing fungi can possess as many alleles as fungi that undergo regular sexual reproduction (McDonald, 1997). Several molecular

13 techniques have been successfully used to differentiate between species and to study genetic diversity within the genus Fusarium (Nayaka et al. 2011). An evaluation at DNA sequence level offers precise grouping of fungal species, courtesy of DNA fingerprinting as one of the techniques successfully used in previous studies to characterise Fusarium. As a result, ecological and evolutionary relationships could be elucidated within the genus Fusarium and several other fungal species (Nayaka et al. 2011; Mule et al. 2005). An increased understanding of the genetic diversity of a crop host is fundamental and serves as a vital management initiative in Fusarium induced diseases (Shahnazi et al. 2014; Koike, 2000).

2.6. Cryptic speciation in Fusarium The taxonomy of the genus Fusarium has been subject to major changes in the past 15 years. During this period, several new Fusarium species have effectively been described (e.g. Britz et al. 2002; Nirenberg and O’Donnell, 1998; Zeller et al. 2003), and in due course, there had been a growing recognition of the occurrence of cryptic species (Klittich et al. 1997; Steenkamp et al. 2000). Although they are either phylogenetically or biologically distinct, cryptic species are normally morphologically indistinguishable (e.g., Britz et al. 1999; Klittich and Leslie, 1992; Nirenberg and O’Donnell, 1998). The identification of cryptic species, also termed sibling species on the basis of either the phylogenetic or biological species concept has modified the taxonomy and nomenclature of several important pathogens occurring globally, species such as F. graminearum (O'Donnell et al. 2004a; Starkey et al. 2007) and F. verticillioides (Sacc.) Nirenberg, that is, the former F. moniliforme J. Sheld. (Leslie, 1991; Seifert et al. 2003). This process is more likely to endure into the future and will generate more accurate species descriptions. However, some species that are currently deemed ‘‘cosmopolitan’’ may end up being further divided into a number of related species. The occurrence of cryptic species thus accentuates the necessity to operate strictly from pure cultures since samples obtained from separate plants tend to be easily populated by numerous Fusarium species (Leslie et al. 1990; Summerell et al. 2011)

Determination of the kinds of speciation procedures that have happened in Fusarium is very challenging. In general, the most frequent type of speciation process is allopatric speciation, in which speciation arises through splits produced by reproductive segregation given the variety of substrates and climate from which the fungi can be retrieved. Another type of the speciation process is sympatric speciation. This is a process whereby two sister groups from the same environment differentiate. However, it is believed to be less common because selection pressures that can occur in this type of differentiation have not yet been identified. Horizontal

14 gene transfer, which is becoming well recognized (Ma et al. 2010), is also significant for these types of fungi, and putative inter-specific crossbreed strains have been discovered under certain field conditions (Leslie et al. 2004a; Leslie et al. 2004b; Summerell et al. 2010).

2.7. Species concepts More difficulties occur in interpreting species concepts than other rudimentary theoretical ideas in the field of biology regardless of the excellent studies of the concept by Mayr (1987; 1988) amongst others. A lot of this misunderstanding seemingly stems from the incorrect belief tolerated by most people that the theoretic species concept remains within the interpretation of systematics, with other authors alleging that evolutionists stole the species concept from systematics. Furthermore, efforts have been made to devise only one species concept relevant to each and every organism (Mishler and Brandon, 1987). However, this is impossible. The species concept forms part of a simple biological hypothesis and must not be deemed similar as the species group as previously stated or suggested by many people (Mishler and Brandon, 1987). The identification of species taxa relies on the description of the species group, apart from the species concept, as indicated by Mayr (1969). Species concepts have remarkably transformed through time and through these transformations, our ideas and beliefs have adapted accordingly regarding species taxa. The foundation of the species theory changed progressively according to the interpretation of the evolutionary theory as improved evolutionary ideas develop (Bock, 2004).

Fusarium studies, like with all fungal genera, are based on the application of species concepts (The hypotheses that demarcate what biologists imply by the phrase species). The genus often serves as a group for the studying and testing of new concepts. Various species concepts produce different answers, for example, on the basis of the biological and morphological species concepts, F. graminearum represents one species but can be several species when applying the phylogenetic species concept (O’Donnell et al. 2004a). The species concepts that are used in the study of the genus Fusarium are based on morphological characteristics (morphological species), crossing experiments (biological species) and, molecular markers (phylogenetic species). Sometimes two or all three of these concepts can be used together (Peay, 2011) because no single technique can guarantee to yield correct identification of all the strains (Leslie and Summerell, 2006a). Although rarely used in Fusarium characterisation, the ecological species concept can also be incorporated into Fusarium studies.

2.7.1. Morphological species concept The classification of Fusarium species is generally based on distinct characters such as the size and shapes of macroconidia and microconidia (Hafizi et al. 2013) (Figure 2.1). Macroconidia are sickle-shaped, multiseptate and long. Microconidia range from pear-shaped to globose and are uniseptate (sometimes biseptate). Macroconidia are normally carried on sporodochia, these are cushion-like stacks of conidiophores. In some instances, macroconidia can be dispersed in the airborne mycelium, in this case, the macroconidia will not be carried on sporodochia. Microconidia are only dispersed by airborne mycelia. Differentiation between some Fusarium species can be done by evaluating the presence or absence of microconidia and macroconidia. However, other species of Fusaria generate both forms of conidia. Either form of conidia, microconidia or macroconidia, amongst the pathogenic species can pose as infectious units (Teetor-Barsch and Roberts, 1983). Chlamydospores (Figure 2.1), defined as resting structures (Smith, 2007), can be produced in both hyphae and conidia. They are either located in the middle or terminally and develop typically when nutrients deplete and when the culture gets old. Chlamydospores only occur in certain Fusarium species and like microconidia and macroconidia are also used to distinguish between Fusarium species. They are frequently found in the soil, possess a thick wall, and may occur individually, in groups, or in chains (Smith, 2007).

Figure 2.1: Fusarium solani. From left to right, macroconidia, microconidia, and chlamydospores. Adapted from Zhang & Sung, (2008).

The morphological species concept is based on the understanding that the morphology of an individual symbolises the variations that exist within an entire species. The description of a species is determined by differences in morphology (Summerell et al. 2010) between species,

16 that is, the species virtually appear different (Mayr, 1969). Fungal taxonomists have used this method for more than 200 years. The Nelson et al. (1983) and Gerlach and Nirenberg, (1982) taxonomic handling of Fusarium are both morphological in nature. They present the base scheme from which the phylogenetic and biological species concepts are being applied (Summerell et al. 2010). The main drawback with this method in mycology stems from the fact that the number of immediate distinguishable morphological traits are far less than the amount of species that need to be differentiated. In spite of these boundaries, the recent widespread use of the morphological criteria by several diagnosticians as well as the possible need to quickly and consistently identify several Fusarium cultures implies that these morphological traits have a potential to remain a vital component of the Fusarium species concept (Summerell et al. 2010).

2.7.2. Biological species concept Mayr described the biological species concept (Mayr, 1940; 1963). This concept regards species as groups of populations that effectively or potentially interbreed amongst themselves. Many practical problems result when using the biological species concept on numerous Fusarium species. This is due to that many Fusarium strains are asexual, they rarely produce sexual stages, and moreover, the same is observed even under research laboratory environments (Summerell et al. (2010). Nonetheless, the biological species concept has been effectively utilized and in most cases has yielded successful results for some groups, particularly in the F. fujikuroi/Gibberella fujikuroi species complex (Leslie, 1995). For these species standards, tester female-fertile strains are accessible and can be utilized in crosses with unknown isolates to verify if the unknown belongs to the same group as the tester strain. According to Summerell et al. (2010) these strains, in conjunction with the development of tests based on the polymerase chain reaction (PCR) for mating types (Kerenyi et al. 2004; Steenkamp et al. 2000), have led to the wider use of the biological species concept for the description of species (Zeller et al. 2003). For example, and more significantly, it is useful as a tool for gathering the data on genetic characteristics as well as the population dynamics exhibited by different fungi (Leslie and Klein, 1996).

2.7.3. Phylogenetic species concept This concept is described by Taylor et al. (2000) as the genealogical concordance species concept which uses a phylogenetic approach to identify species of fungi on the basis of concordance of multiple gene genealogies. It is a modern development in the study of fungi and can simplify several taxonomic difficulties (Summerell et al. 2010). Numerous techniques such as those that make comparisons amongst nucleic acids possible have been utilized to examine the phylogenetic separation and distribution of fungal species. The utilization of molecular methods to analyse phylogenetic relationships has helped in the characterization of morphologically alike but distinct fungi (Geiser et al. 2004). Molecular data is particularly convenient when morphological traits alone are inadequate for the valid and clear identification of taxonomic groups (Nalim, 2004). The phylogenetic species concept makes use of multiple markers, generally variations in DNA sequences of particular genes. This species concept can also produce quantitative measures of genetic similarities (Summerell et al. 2010). When the phylogenetic species concept is used alone, a common setback that taxonomists encounter is where to draw the line amongst species. That is, how dissimilar should two strains be from each other in order to belong to separate taxa? (Summerell et al. 2010). In actual fact, several phylogenetic studies depend on DNA sequences of either one or two loci. These should come from a single or a small amount of representative or isolates that are well characterized. This procedure might result in complications that can be avoided by confirming that sufficient loci and sufficient individuals are examined to attain an exact picture of the difference within the species, at the same time, remaining true to the initial description of phylogenetic species. The initial description depends on populations other than individuals as the unit that is being analysed (Summerell et al. 2010).

2.7.4. Ecological species concept According to Ridley (2003), the concept refers to a theory of species wherein, a species is described as a group of organisms adjusted to a specific array of resources known as a niche, in that particular environment. Ecological studies, especially of species that are closely related and existing in the same region, have clearly proved that the dissimilarities amongst species in behaviour and form are frequently linked to variations in the ecological supplies that the species utilise. A question normally asked by ecological researchers is, why do ecological processes yield distinct species? In the publication by Ridley (2003), it is mentioned that the relationships between parasite and host present an evident example for this question asked. For instance, assume that a certain pathogen, e.g. Fusarium species, exploit two different plant host species.

The plant hosts will vary in certain ways, possibly with regards to where they occur, or based on their form and structure. The pathogen will develop suitable adaptations for successful colonisation of both plant hosts. This supports speciation of the Fusarium pathogen as resources in the environment (plant host in this instance) exist in two discrete forms (Ridley, 2003). The biological species concept and the ecological species concept share a tight relationship. Life, as described by the ecological species concept is presented in the form of distinct species as a result of the adaptations to make use of resources found in nature. Furthermore, interbreeding as described by the biological species concept is similarly influenced by this process. However, the ecological species concept ought to be distinguished from the biological, morphological, and phylogenetic species concepts (Ridley, 2003). According to Taylor et al. (2000), the ecological species concept highlights the adaptation of species to a specific ecological niche, whereas the biological species concept emphasizes the principle of intersterility. The morphological concept however, emphasizes the principles of morphological difference. Finally, the phylogenetic concept highlights nucleotide difference amongst monophyletic lineages (Cai et al. 2011).

2.8. Phylogeography Phylogeography investigates the processes that affect the geographic dispersal of genetic ancestries. It usually makes use of molecular information based on principles suggested by Avise, (2000). According to Summerell et al. (2010), most species have a certain level of population structure that includes both temporal and spatial components, which makes phylogenetic research achievable. The information can then be used to examine biogeographic hypotheses and also to discover processes that lead to evolution within a certain species. The majority of phylogeographic theories suppose that a species is initiated at a specific time and place and that the dispersal of organisms will then be dependent on changes among both time and space (Summerell et al. 2010). The phylogeography of species of Fusarium in natural environments is currently being studied and has shed light on how plant infecting fungi have been distributed as a result of anthropogenic activities within agro-ecosystems, (Summerell et al. 2010).

Molecular data has been widely used within Fusarium to aid in resolving groups that were later defined as distinct species, usually in association with unique morphological characters. For example, it has been determined that mating populations in the F. fujikuroi/G. fujikuroi species complex and groups described using the phylogenetic or biological species concepts are the same (Leslie, 1995; O’Donnell, 2000; O’Donnell et al. 1998b). Depending only on

19 phylogenetic data to reach a verdict on whether separate species are represented by two groups can be difficult. This may lead to assigning one if not more classes of strains as separate individuals, whereas they are portions of identical species. An example of this would be the splitting of F. graminearum into nearly 13 separate phylogenetic species. At first, such groups were given numbers and recognised as a succession of genetic lineages (O’Donnell et al. 2000). Afterwards, names were assigned to the numbered lineages and furthermore, other lineages have also been assigned names other than numbers. About 13 lineages in the FGSC have now been named as phylogenetic species (O'Donnell et al. 2004a; Starkey et al. 2007; Summerell et al. 2010).

2.9. Biogeography The study of distributions together with molecular approaches to phylogeny has recently made substantial advances to our understanding of the biogeography of fungi (Lumbusch and Mueller, 2008). For Fusarium species, the biogeography of several significant pathogens can be determined effectively only if the biogeography of their close relatives (those that are non- pathogenic to important agricultural crops) are correctly assessed. That is, understanding these non-pathogenic species can help determine the biogeography of their related pathogens (infectious species). Therefore, the research of non-infectious fungi in populations comprising of little or limited human influence is required to improve our knowledge with regard to the biogeography of species of Fusarium, together with the relative significance of different plant hosts in Fusarium speciation and dispersal (Summerell et al. 2010). With our current knowledge, it is hard to determine the centre of origin for species of Fusarium. Species of the genus Fusarium have practically been recovered in all sampled environments and are globally dispersed.

The genus Fusarium is likely to be very old and might have emerged early during the evolution of ascomycetes organisms. The diversity of both phylogenetic and morphological traits throughout the genus is coherent with this theory (Summerell et al. 2010). A molecular clock has been calibrated by Taylor and Berbee (2006), specifically for the most important classes of fungi in which Fusarium is included. Given the taxonomic group selected as the point of calibration, the lowest age for species of Fusarium may possibly be around 110, and 250 to 420 millions of years ago. These above mentioned dates all precede the most important breaking up of the present landmasses and as a result, offer help for the early dispersal and the diversity of Fusarium species to all kinds of environments globally. Therefore, host(s) and

20 climate conditions are expected to be more helpful in terms of the origin of many different species of Fusarium, as the geographical locality where they can be retrieved (Summerell et al. 2010).

2.10. Climatic factors Fusarium species obtained from both agricultural and natural ecosystems have different climacteric preferences (Backhouse et al. 2001; Backhouse and Burgess, 2002; Burgess and Summerell, 1992). The variety of species observed can be limited (even if many are present) by climate, as well as local deviations in weather. These conditions can also influence the relative frequency of the species. This means that some species exist that prefer tropical, temperate, or hot arid climates. A fourth class comprises of species that exhibit a cosmopolitan range (Backhouse and Burgess, 2002). The problem with species belonging to the fourth class is that they might be groups of sibling species. The morphology of such species are difficult to distinguish but are genetically distinct, and are still yet to be effectively resolved. For example, maize stalk rot, most probably initiated by F. verticillioides, occurs in maize plants found in moderately warm, dry geographical regions. Contrastingly, the similar disease is found occurring in cooler, moist regions or highland areas and is more likely to be caused by F. subglutinans. Furthermore, crown disease in wheat is caused by F. culmorum (W.G. Sm.) Sacc. and occurs in cooler temperate regions. However, F. pseudograminearum O'Donnell & T. Aoki is more prevalent in arid and warmer subtropical areas for the same disease (Backhouse and Burgess, 2002). Climatic factors encourage the survival, growth, dissemination, and thus the occurrence of Fusarium species, as well as the disease intensity. Temperature, light intensity, wind, and humidity are some of the most serious climatic factors that affect the production as well as the distribution of sexual ascospores and asexual conidia of Fusarium species (Doohan et al. 2003). According to Doohan et al. (2003) climatic circumstances affect distribution frequency of Fusarium species both directly and indirectly. Directly in terms of the effect on the mode of reproduction and indirectly in terms of the effect of vegetation and soil type.

2.11. Dispersal of Fusarium The amounts of dispersal processes in the genus Fusarium are practically as many as there are known Fusarium species. One in particular, human transport, makes generalisation concerning dispersal difficult. There are long distance processes that are known at present (Schmale et al. 2006) that possibly could have added to the extensive dispersal of different representatives of the genus throughout the millennia. When combined with anthropogenic dispersal of plant

21 hosts together with their related products, such as soil, there is enough long-range mobility to efficiently mask a large amount of underlying biogeography of Fusarium (Summerell et al. 2010). Long-range mobility of Fusarium fits into three wide categories: dispersal in the air, dispersal in soil, and dispersal within diseased host plants. An example of an air-dispersed species in Fusarium is F. graminearum. Its spores can spread across continental distances via airborne drift (Schmale et al. 2006). It is assumed that numerous other Fusarium species can also be conveyed via long distances through similar mechanisms given the resemblance in spore morphology of various species. It is thus apparent that several Fusarium species are obtained mainly from aerial parts of plants and consequently give rise to dispersal patterns that can be very hard to interpret (Summerell et al. 2010).

According to Backhouse et al. (2001) there are four kinds of dispersal patterns that are found in Fusarium species: host associated, anthropogenic, climatic factors and certainly, a cosmopolitan distribution. Species that colonise plants are reliant on both the presence of the host and a climate appropriate for the host that they colonize. However, most species of Fusarium are believed to have a cosmopolitan distribution. The definition of several species is becoming narrower as species concepts develop and therefore may change the perceived distribution patterns of these species. For instance, both F. solani and F. oxysporum are generally deemed abundant in soils found everywhere across the planet. However, with the continuing evaluation of species concepts particularly for these groups, the presence of species complexes has been apparent. Furthermore, even though a certain member within the species complex may be found in all types of soil, the different members of the complex possess narrower distributions (Summerell et al. 2010). Therefore, biogeographic patterns of these species must be re-examined and modified as a result of these findings. Nonetheless, it might not be unexpected if the dispersal patterns of individuals within the species complexes turn out to be more reflective to those suggested by Backhouse et al. (2001).

2.12. Sexual and asexual stages Several sexual stage genera have been linked with Fusarium species. These associations have preceded the debate of whether Fusarium must be separated based on a one-to-one association between the sexual and asexual genera. A large number of sexually reproducing strains are associated with the order Hypocreales in the Ascomycetes, however, some can also be found in other fungal orders (Summerell et al. 2010). Over the years, several different species such as Plectosporium tabucinum (J.F.H. Beyma) M.E. Palm, W. Gams & Nirenberg and Microdochium nivale (Fr.) Samuels & I.C. Hallett were described from Fusarium. These newly

22 described species were placed into the three sexually reproducing genera being Gibberella, Albonectria and Haematonectria thus creating a link between them as they all have anamorph species that previously resided in Fusarium (Rossman, 1996; Rossman et al. 1999; Samuels et al. 2001). This was a deduction made before the one fungus one name concept.

Associations with the genus Nectria (Fr.) Fr. and other associated genera such as Neocosmospora E.F. Sm. and Cosmospora Rabenh. are less clear (Rossman et al. 1999). However, continued efforts are resolving the relationships between species of Fusarium, together with these sexually reproducing taxa (Schroers et al. 2009). Gibberella is the most frequent of the known sexually reproducing genera, it is associated with most species of Fusarium (Samuels et al. 2001) and compromises most of the significant pathogens such as G. moniliformis (F. verticilloides) and G. zeae (F. graminearum), and all species of the F. fujikuroi/G. fujikuroi species complex (Summerell et al. 2010). Albonectria was previously incorporated in Calonectria De Not. (Rossman et al. 1999), and is linked with a small number of Fusarium species, the most significant being F. decemcellulare Brick, Jahrb. Vereinig. Angew. (Albonectria rigidiuscula (Berk. & Broome) Rossman & Samuels). Haematonectria comprises of H. haematococca (Berk. & Broome) Samuels & Nirenberg, previously identified as Nectria haematococca Berk. & Broome, which is the sexual stage of F. solani (Rossman et al. 1999). However, it was noted that F. solani comprises several known mating populations that almost definitely symbolise different biological species, although only for those species whose Latin binomials have not been given or defined as distinct species (Matuo and Snyder, 1973). Haematonectria is distinguished from Nectria based on both phylogenetic and morphological characters (Summerell et al. 2010). The majority of species named according to their morph composition have fell into disuse following the one fungus one name concept meaning the names previously given no longer apply.

2.13. Mating types in Fusarium Mating in many fungi happens between partners that are morphologically identical. The analysis of mating types using molecular techniques has become an effective tool for investigating the evolution of life cycles and relationships of species (Christiansen, 1998). Mating behaviour is conferred by a mating type locus that consist of idiomorphs occurring in mating couples of most ascomycetous fungi (Pöggeler, 2001). Idiomorphs are defined as different portions of DNA that may comprise one to three open reading frames (ORF’s) in isolates of opposite mating types (Coppin et al., 1997; Pöggeler and Kuck, 2000; Dyer et al.

2001). Mating type in heterothallic species is regulated by one locus that consists of two alleles with idiomorphic characteristics, named MAT-1, and MAT-2 (Turgeon and Yoder, 2000).

Every single MAT idiomorph consists of one gene that codes for a single MAT-specific DNA protein (Kerenyi et al. 2004). These proteins probably perform a vital function in the sexual morphogenesis and cell speciation pathways as transcription factors. MAT alleles consist of a preserved alpha box domain and a high-mobility-group (HMG) box domain, in that order (Yun et al. 2000; Irzykowska and Kosiada, 2011). The sexual form of Gibberella from the genus Fusarium presents a valuable example in which MAT locus evolution can effectively be studied. Heterothallic Fusarium species, like those in the well-recognised F. fujikuroi/G. fujikuroi species complex (GFSP), have a total of three genes in the MAT1 idiomorph (MAT1- 1 to -3) as well as one gene in the idiomorph MAT1-2 (MAT1-2-1) (Arie et al. 1999; Leslie and Summerell, 2006a). All of the four MAT genes share similarity with the ideal species, that is, a model organism for fungi, Neurospora crassa Shear & B.O. Doidge that also harbours four MAT genes (Leslie and Summerell, 2006a). The MAT genes from Fusarium are homologous to those in N. crassa with shared synteny occurring (Glass et al. 1990; Martin et al. 2011; Staben and Yanofsky, 1990).

2.14. Molecular markers Establishing molecular methods for genetic studies has expedited our understanding of fungal genetics and the structure and behaviour of fungal genomes. Fungal taxonomists have greatly benefited from this through fast identification of fungal species and detection of toxic or virulent strains. Furthermore, molecular techniques have been applied to differentiate between species that are closely related, those without or with insufficient morphological variations (Wulff et al. 2010). They have also been used to differentiate strains within a species (Chandra et al. 2010; Lievens and Thomma, 2005). Molecular markers reveal sequence differences between and within species. This also presents the foundation for accurate identification (Moricca et al. 1998). There are generally ten gene sequences frequently utilized to differentiate Fusarium species and not all provide the same level of discrimination for all species (Nayaka et al. 2011); they are fractions of genomic sequences that encode for:

1. The internally transcribed spacer regions (ITS1 and ITS2) found in the ribosomal repeat gene regions; 2. The intergenic spacer regions (IGS); 3. Calmodulin;

4. Nitrate reductase (NIR); 5. Phosphate permase (PHO); 6. β-tubulin (tub2); 7. Mitochondrial small subunit rDNA (mtSSU1); 8. The large subunit of ATP citrate lyase (acl1); 9. Translation Elongation Factor 1-α (TEF 1-α), and 10. The RNA (Ribonucleic Acid) Polymerase II (RPB1 & 2) genes.

The internal transcribed spacer regions (ITS) have been used widely across many genera of fungi including Fusarium, however, the ITS regions were discovered to be ineffective with certain species of the genus. This includes several other Fusarium species such as F. sporotrichioides Sherb., F. langsethiae Torp & Nirenberg, F. arthrosproioides/F. tricinctum (Corda) Sacc., F. avenaceum (Fr.) Sacc., and the ancestors of the F. graminearum species complex (FGSC), and its close relatives (O’Donnell et al. 2000; Yli-Mattila et al. 2002). The gene region is said to change at a frequency greater than that which is seen at the species level, rendering it less effective for use in Fusarium. Furthermore, several Fusarium species within the Gibberela/Fusarium clade have non-orthologous copies of the ITS2 gene region. This can lead to inappropriate phylogenetic inferences (Geiser et al. 2004; O’Donnell and Cigelnik 1997; O’Donnell et al. 1998b). The intergenic spacer regions (IGS) that splits the ribosomal DNA (rDNA) repeat units, is mainly suitable for examining intraspecific relationships (Srinivasan et al. 2010). Recent reports have indicated that the constitution of genetic populations of F. oxysporum can be easily studied with the use of phylogenetic analysis of the IGS gene region (Srinivasan et al. 2010).

In the genus Fusarium, the calmodulin-encoding gene is used to phylogenetically determine discrete species found in the F. fujikuroi species complex (O’Donnell et al. 2000). The NIR and PHO genes are used in Fusarium studies to resolve intra forma specialis within the FOSC (Laurence et al. 2014). β-tubulin amongst other tubulin genes had received growing attention for the purpose of examining evolutional relatedness at all levels. For example, through phylogenetic analyses, it can distinguish at the kingdom level, and in the research of species complexes within fungi, including protists, plants and animals (Einax and Voigt, 2003). However, for the genus Fusarium, it was reported that β-tub2 was not as efficient, especially in the F. solani species complex (FSSC) (Nayaka et al. 2011; Sampietro et al. 2010). The TEF 1-α gene has been the most broadly recognised throughout the genus and provides the most

25 robust level of species discrimination (Cho et al. 1995). The mitochondrial small subunit rDNA is of recent use in the genus Fusarium. This gene region is highly conserved and it effectively resolves deeper nodes within species complexes of the genus Fusarium (Laurence et al. 2014). Another gene of significance in Fusarium studies is the larger subunit of ATP citrate lyase. This gene provides resolution to genera that are closely related to the genus Fusarium (Laurence et al. 2014). The RNA RPB1 & 2 genes are also used within this genus. Laurence et al. (2016) indicated that these loci have been used before to establish deep level phylogeny in Fusarium (Gräfenhan et al. 2011).

2.15. The Translation Elongation Factor 1-α, the RNA (Ribonucleic Acid) Polymerase II (RPB1 & 2), genes of choice. The TEF-1α gene and RNA RPB1 & 2 are three genes that have recently received extensive use in demarcating Fusarium species. The TEF-1α encodes a vital portion of the protein translational machinery. The gene has high phylogenetic importance because of three significant reasons. First, the gene is very informative at species level in the genus Fusarium. Secondly, non-orthologous replicas of the gene have not been discovered in the genus. Finally, universal primers have been created that function throughout the phylogenetic range of the genus. The TEF-1α gene was initially utilized as a phylogenetic marker for inferring generic and species level relationships within Lepidoptera (Cho et al. 1995). The primers were initially synthesized for use in fungi to study ancestries within the F. oxysporum species complex (O’Donnell et al. 1998b). The forward and reverse primers of this gene were constructed on the basis of sites that are shared in exons between Histoplasma capsulatum Darling (Eurotiales/ Eurotiomycetes/ Pezizomycotina/ Ascomycota) and Trichoderma reesei E.G. Simmons (Hypocreales/ Sordariomycetes/ Pezizomycotina/ Ascomycota). These primers can be used on a broader range of filamentous ascomycetes. The primers amplify an approximately 700 base pair region of the TEF-1α gene. The EF1 and EF2 primers flank three introns (Figure 2.2) that equal above half the amplicon’s length in all known Fusaria (Geiser et al. 2004).

Figure 2.2: TEF 1-α gene region map of Fusarium with primer locations. Adapted from Geiser et al. (2004).

The largest subunit of nuclear RNA polymerase II is encoded by RPB1 and comprises eight preserved core regions of similar amino acid composition. This amino acid similarity is associated among all eukaryotic, archaebacterial, and eubacterial genes (Katan, 1999). Some of these gene regions comprise of extremely preserved sequence motifs. PCR primers can be constructed based on these sequence motifs and be used to obtain sequences of the gene encoded by RPB1 from broadly different eukaryotic taxa (Katan, 1999). Due to its large size, the absence of paralogous copies, and moderately constant G-C composition, this gene has shown potential for exploring eukaryotic relationships (Katan, 1999). The second largest subunit of nuclear RNA polymerase II is encoded by RPB2. It is accountable for the transcription of genes that encode proteins (Nelson et al. 1981; Summerell and Rugg, 1992). The RPB2 gene is present in all eukaryotes (Fourie et al. 2009) and its regions are highly conserved in eukaryotes (Katan and Di Primo, 1999; Gordon and Martyn, 1997). Both of these genes are recognised for their informativeness in studies concerning various groups of fungi (Schoch et al. 2009) including Fusarium (Laurence et al. 2014 and 2016; O’Donnell et al. 2010 and O’Donnell et al. 2013).

2.16. Mycotoxins and other secondary metabolites Species within the genus Fusarium produce three of the most significant groups of mycotoxins. These are trichothecenes, zearalenones, and fumonisins (Desjardins, 2006; Marasas et al. 1984). The chemical structures (Figure 2.3) of these compounds were determined over a 30 year period since, the 1960s. Mycotoxins are secondary metabolites that are not very necessary for fungal growth, at least under laboratory conditions. The purpose of secondary metabolites

27 is well recognised. However, some have not been studied in depth and, other than their structures, very little is known about them (Summerell and Leslie, 2011). Fusarium strains have been utilized as the foundation for effective commercial fermentation of the secondary metabolite known as gibberellic acid (used as a growth enhancer). Furthermore, zearalenones have been used as the starting material in the production of the cattle growth promoter hormone. Fumonisins on the other hand are manufactured through the fermentation of Fusarium strains mainly for research purposes (Summerell and Leslie, 2011). Several mycotoxin-producing Fusarium species exist and can be isolated from commercially important grass crops (see table 2.2).

Table 2.2: Mycotoxin-producing Fusarium species in important crops.

Host plant Species responsible for toxin production Maize F. avenaceum, F. graminearum, F. semitectum Berk. & Ravenel, F. sporotrichioides, F. subglutinans (Wollenw. & Reinking) P.E. Nelson, Toussoun & Marasas, F. verticillioides (Sacc.) Nirenberg Barley F. sporotrichioides, F. langsethiae Torp & Nirenberg, F. poae (Peck) Wollenw. Bull. Wheat F. graminearum, F. tricinctum Sorghum F. napiforme Marasas, P.E. Nelson & Rabie, F. proliferatum (Matsush.) Nirenberg Millet F. nygamai L.W. Burgess & Trimboli Rice F. verticillioides, F. proliferatum Adapted from Nayaka et al. (2011).

2.16.1. Trichothecenes, Zearalenones, and Fumonisins

Trichothecenes are the most chemically diverse amongst the three main groups of Fusarium mycotoxins. However, this class of compounds are produced by diverse fungi and some plant species (Desjardins and Proctor, 2006). The trichothecene class of toxins consists of very lethal mycotoxins such as diacetoxyscirpenol (DAS) and T-2 toxins, normally produced by F. sporotrichioides, nivalenol (NIV) produced by F. nivale, as well as the extensively regulated deoxynivalenol that is commonly known as DON or vomitoxin. Vomitoxin is normally produced by F. graminearum and F. roseum (Desjardins, 2006; Summerell and Leslie, 2011 and Gupta, 2007). Fusarium pseudograminearum, F. graminearum, and F. crookwellense L.W. Burgess, P.E. Nelson & Toussoun are species that have been reported to produce the

28 zearalenone toxins. This toxin is also produced by other strains such as those of F. semitectum Berk. & Ravenel and F. equiseti (Merrill et al. 1993). Fumonisins are toxins that comprise of a single amphithallic chemical configuration (Figure 2.3). This chemical structure allows them to integrate with membranes and inhibit sphingolipid metabolism particularly in both humans and animals (Merrill et al. 1993). Symptoms produced by fumonisins are abnormally broad. These include the development of cancer in experimental animals, neural tube deficiencies in new borns, lung edema in swine, and brain lesions in horses (Marasas et al. 1987; O’Donnell and Cigelnik, 1997). Fumonisin B1 is usually synthesized in great quantities and by a large number of strains, thus most studies focus on this toxin (Summerell and Leslie, 2011).

Figure 2.3: Diagram showing the diverse structures of some frequently occurring mycotoxins in Fusarium. Taken from Dawson et al. (2006).

2.16.2. Other secondary metabolites Fusarium species produce several other secondary compounds of possible importance, including: beauvericin, acuminatum, equisetin, chlamydosporol, butenolide, culmorin, fusarins, fusaric acids, fusarochromanones, fusaprolieferins, napthoquinones, wortmannin, moniliformin, sambutoxin, enniatins, and cyclonerodiol. These metabolites do not necessarily have to be produced specifically by Fusarium strains. Beauvericins compounds serve as an example as they were initially found in the fungi Beauveria bassiana (Bals.-Criv.) Vuill. that is known to be entomopathogenic (Azor et al. 2007). Beauvericins represent a group known as the non-ribosomal cyclic depsipeptides, among other groups of compounds made by species of Fusarium including terpenes, sestertepenes, cyclobutenes, polyketides, and a couple of pigments varying in molecular weight from below 100 to 600. When combined, the activities of these metabolites led to them being inadequately understood (Martin et al. 2011) since many

29 scientists only use one or a few of these metabolites at a time. There are several major obstacles that remain in understanding the role that these metabolites perform in both natural and cultivated ecosystems. These include, the identification of several unknown secondary metabolites produced by several Fusarium species, the significant environmental aspects that activate or inhibit their synthesis, and characterisation of the impacts that result from the concurrent exposure to numerous mycotoxins (Summerell and Leslie, 2011).

2.17. Infection processes and survival of fusaria in soil The hyphae of Fusarium species occurring in soil grow towards the roots of their plant host in reaction to rhizosphere nutrients secreted by plants. They normally multiply on the root surfaces prior to penetrating the cortical tissue. Damage to the root or small openings in the cuticle of the root can permit the entry of a fungus (Smith, 2007). For pathogens that cause Fusarium wilt, it is required that the hyphae enter via the cortex to gain entry into the vessels, thereby causing disease. Fungi use specific strategies to colonise their hosts and in the process, plants summon several responses to the attack. The pathogen is able to grow across intercellular spaces within the root cortex. Once inside they can identify cell components of the plant and release enzymes that can attack the cell walls to gain entry. Consequently, the plant’s response to this invasion occurs early whereby they produce callose (a plant polysaccharide) and deposit it around vessel walls and paravascular parenchyma pits where the fungi is likely to attempt entry (Smith, 2007).

Lignification and phytoalexins (antimicrobial substances) appear as secondary metabolites that are harmful to the pathogen. Entry to the xylem vessel is normally not an easy task, however, once the pathogen succeeds, it is met by a series of structural barricades produced by the plant to counteract the fungal invasion (Talboys, 1972). The fungus can successfully enter the vascular tissue only near the root tip where the barricades have not yet developed. Numerous F. oxysporum strains can and do advance within plant vessels. However, development of an invader into a true pathogen is determined by whether it can continuously and completely colonize the host’s vascular system or simply remain restricted near its point of entry. Once the pathogen gains ultimate entry into the xylem vessel, it produces not only hyphae but microconidia and together these move upwards along the transpiration stream. Germination then occurs at a pit where a small germ tube penetrates into an adjacent cell (Beckman et al. 1762; Smith, 2007).

Soil-borne pathogens lack the ability to distribute as quickly as those organisms found above ground. For such pathogens, more time is required for them to initiate disease at a site. As a result, the pathogenic fusaria find means to persevere in soils through intense environmental conditions, together with other rival saprobes and aggressive microflora. They also require some unique advantage so they can reach the plant rhizoplane to initiate infection into the host (Smith, 2007). Therefore, if they achieve and are capable of colonizing the stele of a live plant, they attain the most definite advantage above all other flora residing in that environment. Within this plant tissue, the subsequent generation of inoculum meant for subsequent crops may very well develop. As the plant senesces, the fungal hyphae occupying the vascular system penetrates into the more heavy-walled parenchyma and sclerenchyma tissue where they produce their chlamydospores. When the waste from this plant is restored into the soil, most of the refuse will decay, however the pathogens dormant spores lie secured in the resilient tissue for prolonged periods (Smith, 2007).

Chlamydospores are survival structures of numerous Fusarium species that endure under severe environments (Wollenweber and Reinking, 1935). Further research indicated that chlamydospores only germinate after sensing the secretion of nutrients into the rhizosphere (Smith, 2007). The chlamydospores of certain species of Fusarium germinate and give rise to hyphae in certain soil types more than others. Such soils that permit fungi to germinate and grow are believed to be favourable to the fungi, and should the pathogen be a vascular attacker, the disease is most likely to transpire in this type of soil. On the contrary, soils that do not often permit the chlamydospores to germinate nor grow are thought to be oppressive to the fungi and inhibits disease development. Normally, sandy loam soils that are less-heavy and at low pH levels are more advantageous to the ordinary spores of F. oxysporum formae speciales however, the composition of minerals in the clay (Stotzky et al. 1961), and nutritional factors of the soil are also included. As expected, the common soil-borne saprobes of the species are more inclined to possess chlamydorpores that germinated and grow more rapidly than do those of pathogens (Smith and Snyder, 1972; Smith, 2007).

2.18. Plant diseases Pathogenic species of the genus Fusarium have the ability to attack approximately all grown food crops. At least, 81 out of 101 of the most economically significant plants can be infected by Fusarium pathogens. Plant diseases caused by Fusarium can occur at any phase of the plant’s development resulting in a variety of diseases (Nayaka et al. 2011). These diseases include kernel rot, seedling rot, seed rot, root rot, ear rot, cankers, head blight, leaf diseases,

31 and wilts. Fusarium infections have had numerous and enormous economic impacts worldwide. In the USA alone, several billions of dollars were lost in barley and wheat crops as a result of Fusarium head blight (FHB) caused by F. graminearum sensu stricto (Schmale and Bergstrom, 2003). In the 1960s, a panama wilt outbreak caused devastation in the commercial banana market and was caused by F. oxysporum f. sp. cubense (E.F. Sm.) W.C. Snyder & H.N. Hansen (Ploetz, 1990). The cotton industry in Australia appeared to be under threat as several strains of Fusarium species showed increased pathogenicity to indigenous Gossypium L. species (Wang et al. 2004). In northern Asia and Europe, F. langsethiae, F. poae, and F. sporotrichoides are vital causative agents of scab in both oats and barley. These species were found to be prevalent in outbreaks of Alimentary Toxic Aleukia (ATA), which occurred in the Soviet Union through World War II as they caused high levels of T-2 toxins in various overwintered plants that included millet and wheat (Desjardins, 2006; Nayaka et al. 2011; Yli- Mattila and Gagkaeva, 2010).

Fusarium wilts are mainly caused by species of the F. oxysporum complex. Other species or organisms can also cause wilt but all Fusarium wilt diseases are caused by F. oxysporum. Within the F. oxysporum species complex, specificity occurs due to certain forms in the complex having the ability to cause disease to a particular plant species. For instance, F. oxysporum f. sp. lycopersici (Sacc.) W.C. Snyder & H.N. Hansen produces Fusarium wilt of tomato whereas F. oxysporum f. sp. niveum (E.F. Sm.) W.C. Snyder & H.N. Hansen produces Fusarium wilt of watermelon (Kucharek et al. 2000). This further complicates the situation since the existence of these infectious races within the forma speciales means that one infectious race may trigger disease in particular crop plants but may not in others. Another obstacle presented in the races and forma specialis of F. oxysporum is that they are at most very indistinguishable in their forms. Fusarium solani is also one of the Fusarium species that cause a variety of rots, such as root rot, crown rot, fruit rots, and stem rots. Fusarium subglutinans on the other hand causes disease mainly to sugarcane, sorghum, corn, and several broad leafed plants. For the grower, it is not enough to only identify the type of Fusarium connected to a disease unless certain control practices can be taken (Kucharek et al. 2000).

2.19. Human diseases Human diseases caused by opportunistic pathogenic fungi are turning out to be a growing health concern across the world. The previously reported number of patients vulnerable to infection by intrusive fungal mycoses is approximately 12 million (Parkin et al. 2005, Park et al. 2009, Brown et al. 2012). Around 4.8 million patients are affected by allergic

32 bronchopulmonary aspergillosis, 12 million are affected by allergic fungal sinusitis (To et al. 2012) and six million are affected by fungal eye infections (Lam et al. 2002). Around one billion people worldwide suffer from nail, hair, and skin infections all as a result of invasive fungal mycoses (Vos et al. 2012). In nature, water environments represent reservoirs in which a large variety of microorganisms exist and few of these enter our homes through the tap-water system (Pereira et al. 2010). Consequently, these can potentially present health risks, especially to people who are immune-compromised (Babic et al. 2015). Fusarium species produce a wide range of infections in humans that include superficial (e.g. onychomycosis and keratosis), disseminated or locally invasive infections, with the second exclusively occurring in almost every patient that is immunocompromised (Nucci and Anaissie, 2002). They may also cause the above-mentioned allergic diseases (sinusitis) observed in immunocompetent individuals (Wickern, 1993) and mycotoxicosis in animals and humans after the consumption of food that is poisoned by toxin-producing Fusarium species (Nayaka et al. 2011; Nelson et al. 1994).

Fusarium solani, F. verticilliodes and F. oxysporum are species that most frequently cause fusariosis (Alastruey-Izquierdo, 2008; Guarro and Gene´, 1995; Tortorano et al. 2008). Several other Fusarium species have also been found to trigger human infections. These include F. dimerum Penz. F. incarnatum (Roberge) Sacc. and F. chlamydosporum Wollenw. & Reinking together with species from the F. fujikuroi species complex that include F. nygamai, F. napiforme, F. sacchari (E.J. Butler) W. Gams, and F. proliferatum (Nirenberg and O’Donnell, 1998; Nucci and Anaissie, 2007). Unfortunately, the actual incidence of most of these species is not well understood because they are unknown, and clinical microbiologists and most laboratorians are not particularly aware of their potential occurrence in human infections. This is exacerbated by several Fusarium species that are commonly impervious to all existing antifungal drugs (Azor et al. 2009). Most fusaria display a wide resistance to a variety of antifungal agents. These include amphotericin, echinocandins, azoles, and terbinafine, which normally exhibit high minimal inhibitory concentrations (MIC) activity in vitro (Azor et al. 2007; O’Donnell et al. 2008; Reuben et al. 1989). Regardless of a weak response, liposomal amphotericin B is one of the antifungal drugs that functions relatively well against fusarioses (O’Donnell et al. 1998b; Pujol et al. 1997).

2.20. Non-pathogenic Fusarium The occurrence of Fusarium infections in some cultivated soils is reduced regardless of the presence of a vulnerable host, infectious pathogen, and conducive environmental conditions. These types of soils are called Fusarium suppressive soils (Stover, 1962). Their suppressing

33 capability is normally due to several factors, such as both biotic and abiotic factors (Alabouvette et al. 2004; Domínguez et al. 2001; Louvet et al. 1981). One example is the suppression of the disease Fusarium wilt resulting from the actinomycetous bacteria and other fungi found living in these suppressive soils. Of these, the fluorescent Pseudomonas species and non-pathogenic entities of F. oxysporum are most regularly linked with the suppression of Fusarium wilt (Alabouvette, 1990; Duijff et al. 1999; Scher and Baker, 1982). Initial field trials with non-pathogenic forms of F. solani and F. oxysporum on seedlings of tomato revealed that tomato Fusarium wilt can possibly be suppressed by 50-80% (Chandra et al. 2008), subject to different environmental conditions (Larkin and Fravel, 2002). According to Sneh (1998) non- pathogenic forms of F. oxysporum were used in the field (applied to cuttings of host) to successfully restrain Fusarium wilt in sweet potato known to be produced by F. oxysporum f. sp. batatas (Wollenw.) W.C. Snyder & H.N. Hansen (Belgrove et al. 2011).

Another example is that of Fusarium wilt in banana. Very few studies focused on the influence of chemicals present in soil and physical conditions. Peng et al. (1999) stated that edaphic properties of soil, e.g. calcium (Ca) and iron (Fe) content, water content and temperature influenced the germination of chlamydospores and the severity of Fusarium wilt in banana plants under greenhouse conditions. However, Domínguez et al. (2001) showed that structural strength of soil aggregates varies in soils that favour and suppress Fusarium wilt in banana. Stover et al. (1961) and Stover (1962) indicated that the pH of soil and calcium (Ca) composition of soils in Central America were accountable for Panama disease suppression in the field, and that ammonium (NH4) fertilizer improves disease development (Belgrove et al. 2011).

It is difficult to distinguish non-pathogenic forms of F. oxysporum from their relative pathogenic forms with the use of conventional agar plating methods and contrasting of morphological characters. Furthermore, the formae speciales cannot be identified with the use of molecular markers (Hillis and Huelsenbeck, 1992; Fourie et al. 2009). This is explained by the fact that formae speciales of F. oxysporum do not necessarily form part of a monophyletic group. This is apparent from previously constructed phylogenetic trees whereby isolates showing varying formae speciales and non-pathogenic isolates were clustered together other than with isolates from identical formae speciales. Therefore, host specificity is needed to organise pathogenic isolates into a specific formae speciales, including cultivar specificity to further split the forma specialis simply into races (Armstrong and Armstrong, 1981; Belgrove et al. 2011)

A study by Taylor et al. (2016) was aimed at characterising F. oxysporum strains isolated from onion. They made use of sequencing of housekeeping and pathogenicity-related genes with a specific emphasis on Secreted In Xylem (SIX) genes. With these genes they could distinguish pathogenic from non-pathogenic stains of F. oxysporum species. The presence or absence of these pathogenicity genes was assessed for capacity of the strains to produce disease in both onion seedlings and bulbs. They characterised 31 F. oxysporum strains with the application of pathogenicity tests, sequencing of housekeeping genes, and identification of effectors. Through pathogenicity tests, they discovered that 21 isolates were pathogenic and 10 were non- pathogenic in onion seedling and bulb tests. A concatenated tree generated from housekeeping gene sequences split the F. oxysporum strains into six clades, but could not distinguish pathogenic isolates from non-pathogenic isolates. For the identification of effectors, they discovered ten putative effectors within F. oxysporum f. sp. cepae (FOC) (Hanzawa) W.C. Snyder & H.N. Hansen, together with seven SIX (SIX3, SIX5, SIX7, SIX9, SIX10, SIX12, and SIX14) genes, first reported in F. oxysporum f. sp. lycopersici. Some of which were expressed in pathogenic strains and some not expressed in non-pathogenic strains. They concluded that overall, their results present better insight of pathogenicity in FOC, and possibly might make the analysis and management of Fusarium basal rot of onion in the future much better (Taylor et al. 2016).

2.21. Fusarium research in South Africa In South Africa, research in the genus Fusarium has further improved following the impressive contributions by Marasas et al. (1987 and 1988). Several studies were carried out and have presented a good partial indication of the diversity of the genus in South Africa. A study by Alabouvette et al. (2004) that dealt with species of Fusarium in soils from the Transkei location in the Eastern Cape Province, focused on samples obtained at varying altitudes from cultivated maize soils and uncultivated grassland soils. They discovered that there were two main species of Fusarium in both areas, these being; F. equiseti and F. oxysporum. Fusarium subglutinans and F. moniliforme were among the very few that were retrieved. However, both these species were notably more concentrated in cultivated soils and less so in uncultivated soils (Rheeder and Marasas, 1998). A study by Marasas et al. (1988) evaluated the incidence, dispersal, and ecology of Fusarium species in South African soils from agricultural regions and vegetation types. It was determined from this study that F. oxysporum, F. solani, F. equiseti, F. nygamai, and F. compactum (Wollenw) Raillo were the five most predominant Fusarium species occurring in these regions. Fusarium oxysporum, F. solani, and F. equisetti were frequently

35 isolated more from cultivated than in natural soils. In contrast, F. nygamai was frequently isolated more from natural ecosystems than cultivated ones whereas the occurrence of F. compactum did not vary significantly in both ecosystems. Furthermore, they recorded for the first time in South Africa, two Fusarium species being, F. tricinctum and F. camptoceras Wollenw & Reinking. In another study published by Tewoldemedhin et al. (2011), the pathogenicity of various species of fungi towards apple was examined. These fungi caused an incident identified as apple replant disease. One of these fungal isolates was a Fusarium species. The results for the genus Fusarium indicated that F. oxysporum was broadly distributed; however the strains recovered were not pathogenic. Fusarium avenaceum and F. solani occurred the least with only a few of these isolates having low virulence (Tewoldemedhin et al. 2011).

According to Chen and Swart (2001), strains of F. oxysporum have also been found in association with the root rot (Chen and Swart, 2000) and stem decay (Blodgett et al. 1998) diseases occurring on the plant host Amaranthus hybridus L. Furthermore, the pathogen was the prevalent fungal species carried by Hypolixus haerens L. (pigweed weevil), the main insect that is a pest of A. hybridus in South Africa (Louw et al. 1995; 2002). According to an article by Jacobs and van Heerden (2012), crown rot and root rot of tomato, produced by Fusarium had only been identified in Canada, Japan, USA, and Mexico (Jones et al. 1991). In South Africa, the disease had never been reported before, the only previous reports had been on of Fusarium wilt of tomato (Crous and Baxter, 2000). The authors noticed symptoms such as crown and root rot as well as intense vascular browning on tomato plants that were grown under hydroponic conditions. This led to a study that identified F. oxysporum f. sp. radicis- lycopersici Jarvis & Shoemaker, a causal agent of Fusarium root rot and crown rot from the Eastern Cape part of South Africa (Jacobs and van Heerden, 2012). Panama disease of bananas is prevalent in the Kiepersol and southern KwaZulu-Natal banana production areas in South Africa. Fusarium oxysporum f. sp. cubense Snyder & H.N. Hansen is a causal agent of this disease. The effects of this pathogen on banana plants were observed in the Tzaneen region (Limpopo Province) at a commercial Cavendish plantation in September 2000 (Grimbeek et al. 2001). The outbreak was also observed at another banana plantation site in the Komatipoort region (Mpumalanga Province) in November 2000. It was determined that isolates from both the Tzaneen and Komatipoort regions were F. oxysporum f. sp. cubense. The very same pathogen was discovered to cause Panama disease in Kiepersol and southern KwaZulu-Natal (Grimbeek et al. 2001). Fusarium circinatum Nirenberg & O'Donnell is notorious for causing

36 the pitch canker disease of pines, more specifically Pinus patula Schiede ex Schltdl. & Cham. This pathogen was initially isolated from infected pine seedlings of P. patula in South Africa in 1990. Thereafter, the infectious agent spread to other parts of the country where it infected numerous other species of the genus Pinus (Mitchell et al. 2011).

Agathosma betulina (Berg.) Pillans (buchu) is a South Africa plant used for traditional and medicinal reasons, as an ingredient for beauty products, a source of fragrant oils, and as a colourant for food (Low, 2007). Buchu is susceptible to a fungal pathogen that causes wilts and had the ability to cause great losses in buchu sales. The pathogen was identified as F. oxysporum and this was a newly reported case in South Africa. Jacobs et al. (2010) compared South African Fusarium strains from infected pineapples with those from Brazil following the incidence of a disease similar to that of fusariosis of pineapples produced by F. guttiforme Nirenberg & O'Donnell that was only known to occur in South and Central America. Morphological characters and phylogenetic analyses from this study indicated that the disease observed on pineapple in South Africa was produced by a different Fusarium species newly documented as F. ananatum Jacobs, Marasas & van Wyk (Jacobs et al. 2010). In another South African study by van der Walt et al. (2007), they obtained Fusarium species from maize, air, soil, naturally cultivating morogo vegetables, lerotho, and thepe to examine the occurrence of Fusarium species in household food gardens. They determined that F. proliferatum, F. poae, F. verticillioides, F. oxysporum, F. solani, and F. subglutinans were frequently isolated from these sources. Fusarium semitectum, F. chlamydosporum, and F. equiseti were only recovered where maize was present and all have presented public-health impacts (van der Walt et al. 2007). In South Africa, maize serves as a staple food for over 600K citizens who rely on subsistence farming. However, the maize produced through subsistence farming is often vulnerable to fungal infection. The maize is affected by mycotoxins produced by some Fusarium spp., namely F. verticillioides F. subglutinans, and F. proliferatum. These species were isolated from maize samples collected from subsistence farm areas at four South African Provinces during the growing seasons of 2006 and 2007 (Ncube et al. 2011).

2.22. New technology for Fusarium research Novel technologies specifically for genomic research are improving rapidly. For example, next-generation sequencing present straightforward and robust tools for obtaining nucleic acids from various backgrounds and the generation of sequence data that is an important prerequisite for manufacturing barcodes (Saikia and Kadoo, 2010). Several of these techniques have been implemented for important Fusarium species such as F. sporotrichioides, F. proliferatum, F.

37 verticillioides, F. circinatum, F. oxysporum, and F. graminearum (Saikia and Kadoo, 2010). Gene tagging methods such as transposon tagging and restriction enzyme mediated integration (REMI) are effective tools for uncovering new genes linked with pathogenicity. The techniques are effective because prior knowledge of the gene product is not required. These technologies, along with genome-wide analyses guarantee to improve molecular research in Fusarium (Nayaka et al. 2011).

The establishment of international nucleotide sequence databases (Gene Banks), for example: the USA based National Centre for Biotechnology Information (NCBI), the UK based EMBL Nucleotide Sequence Database, and the DNA Data Bank of Japan (DDBJ) have all become crucial tools for the research of genomes. The databases are internet-based and include phenotypic data such as those for toxin synthesis, and databases for molecular genetics such as fingerprinting patterns have also been suggested for pathogenic fungi (Kang et al. 2002). Annotating strain information together with molecular and biological genetic data, distributed as an internet accessible database, could be a vital tool in the research of genetic diversity for Fusarium species globally (Nayaka et al. 2011).

2.23. General climate, vegetation and soil type of the study area (Willem Pretorius nature reserve)

The Willem Pretorius Nature Reserve is situated in the mixed grasslands of the innermost part of Free State. The main vegetation of the nature reserve can be described as Moist Cool Highveld Grassland (Bredenkamp and Van Rooyen, 1996) and it is one of the only five major conservation regions for this grassland type. The grassland vegetation is the most dominant in the nature reserve and it covers approximately 78% of the terrestrial surface area. An estimated 67% of the grasslands are found on the plains in the south, and between the ridges in the north. The woody vegetation on the reserve is restricted to the ridges and banks of the Sand River, and the dry watercourses (Müller, 1986). Müller (1986) identified five main vegetation types found in the reserve, namely: the tree and shrub communities, the thornveld and riverbank communities, the grassland communities, the hygrophilous communities, and the communities of rocky outcrops (Figure 2.4). The reserve is subjected to temperatures ranging from -6.7°C to 37.8°C and rainfall of c. 600 mm p.a (Müller, 1986; Avenant, 2000). According to Müller (1986), soils found in the reserve are mostly heavy clay from a dolerite origin, but lighter sandy soils from a sandstone origin also occur. The Nature Reserve had been chosen not only because it is described as a Moist Cool Highveld Grassland, which is favourable for Fusarium

38 occurrence, but also because it is one of only five principal conservation areas for this grassland type. Furthermore, the information obtained in this study seeks to provide useful educational and cultural information important in the study of science and local history.

Figure 2.4: Map showing the five main vegetation types classified by Müller (1986) in the Willem Pretorius Nature Reserve (Adapted from Winterbach, 1999).

2.24. Conclusion

Species of the genus Fusarium are found in profuse amounts in grassland soils. Due to their pathogenic nature towards important food crops, enormous losses in crop yield are recorded. For example, in the USA alone, several billion dollars were lost in barley and wheat crops as a result of Fusarium head blight (FHB) caused by F. graminearum sensu stricto (Schmale and Bergstrom, 2003). Peri-urban communities suffer more from these losses as most do not have access to control measures. A good understanding of the composition of populations of Fusarium species is needed to understand the epidemiological features of Fusarium producing diseases occurring on important food crops. Molecular marker technology for quick detection and classification of particular species has received a lot of attention. This technology is essential for sufficient disease management as well as for plant resistance breeding contrary to certain infectious isolates of species in the genus Fusarium. The development of a dependable phylogenetic species concept has allowed more precise species definitions in various groups of fungi that have inadequate distinct morphological or biological characters. Fusarium species contain distinctive mycotoxin profiles, therefore, a quick and correct documentation of the

Fusarium species that occur in plants, at stages of their development, is important in order to estimate the potential toxicological danger to which plants are exposed. This study was aimed at establishing the natural occurrence and distribution of Fusarium species in South African grassland soils. The data that this study generated will assist in identifying the possibility of new disease development caused by Fusarium species in future. Knowing and understanding the type of Fusarium pathogens occurring at certain areas helps us develop strategies to combat future outbreaks globally.

CHAPTER 3

MATERIALS AND METHODS

3.1. Sampling and sample processing

Soil samples were collected from undisturbed soils in the Willem Pretorius (WP) nature reserve, located in the Free State province of South Africa (Figure 3.1). The soil samples (sample 1, 2, 3, and 4) were obtained from four sites within the nature reserve. The GPS coordinates of the four sample sites were: sample 1 (S 28⁰17’07.9”; E 027⁰13’35.5”), sample 2 (S 28⁰19’12.3”; E 027⁰21’10.4”), sample 3 (S 28⁰18’09.8”; E 027⁰17’53.5”), and sample 4 (S 28⁰17’41.2”; E 027⁰13’30.0”). Sample 1 was collected from a Thornveld and riverbank vegetation type, sample 2 and 3 were collected from a grassland vegetation type and sample 4 was collected from a Hygrophilous (plants growing in damp conditions) vegetation type (see figure 2.4). The soil samples were collected in a standardised transect (Laurence et al. 2012) (Figure 3.2) using a core sampler and small spade. That is, the soil was collected in a 15 metres north-south transect bisected by a 15 metres east-west transect. Along these two lines, the samples were taken at one meter intervals and then pooled together. The samples taken at the ends were pooled separately. This was repeated for the other three sites, therefore, resulting in two bags per site.

Three technical replicates of five grams of each obtained soil sample (i.e. each bag) were sieved to separate the large and small particles using a sieve with a 425 µm aperture size. These sieved samples were then plated on selective Fusarium agar (SFA, Leslie and Summerell, 2006a; 200 g/l glucose (dextrose), 5 g/l KHPO4, 20 g/l NaNO3, 5 g/l MgSO4•7H2O, 10 g/l yeast extract, 1 ml of 1% FeSO4•7H2O, 200 g/l agar and, 10 g/l of PCNB) using a direct plating technique. Each fraction was plated on ten separate SFA plates by directly and evenly sprinkling small quantities (5-15 mg) of soil. After three to four days of incubation on the SFA plates, three Fusarium colonies from each of the ten plates were transferred onto three separate ¼ potato dextrose agar (PDA, Merck) plates (Figure 3.3).

Once pure cultures were obtained, each of the Fusarium isolates were subjected to single spore isolation to obtain a single genetic entity that represented the sampled isolate for further studies. This was done by creating a spore suspension through aseptically flooding the colony with 10 ml sterile water. Depending on the spore concentration, a maximum of 1 ml of the spore suspension was evenly spread onto water agar (WA, Merck) and incubated upside down for 24 hours at room temperature. This was done to allow spore germination for both fast to slow germinating spores. A dissecting microscope emitting light from below was used for the spore isolation process. With the use of a fine needle, a single germinating spore was removed and

42 each placed on a separate full strength PDA plate in replicates of four per culture. The result of this process were pure single spored cultures.

The purified cultures were then allowed to grow for seven to 10 days using a cycle of 12 hours cool white light alternating with 12 hours darkness (near UV light, 320-420 nm). These cultures were incubated at controlled temperatures between 21-24 oC. For cultures whose morphological characters (those selected to illustrate the basic morphological characters of Fusarium species) had to be further studied, mycelium from the full strength PDA plates were transferred onto synthetic nutrient agar (SNA, Leslie and Summerell, 2006a; 10 g/l KHPO4, 10 g/l KNO3, 5 g/l MgSO4•7H2O, 5 g/l KCl, 02 g/l, glucose, 2 g/l, sucrose, 200 g/l agar). This was done to induce the production of chlamydospores on carnation leaf agar for the production of both chlamydospores and macroconidia and again on full strength PDA for determining culture pigmentation and colony morphology (Leslie and Summerell, 2006a). The incubation duration was for a period of ten days under similar incubation as mentioned above. These cultures were then studied for their morphological characters the following day using a Zeiss Axio Imager.A2 microscope.

The obtained cultures were preserved using three preservation methods, namely, oil slants, freeze drying, and ultralow freezing. For preservation of the cultures in oil, twice autoclaved paraffin oil was used. The pure cultures were inoculated onto half strength PDA slants in McCartney bottles and allowed to grow until the medium was completely covered. Then enough oil was poured aseptically onto these slants to fill the bottle. For cultures producing aerial hyphae, the hyphae were gently pushed down before pouring the oil. The bottles were then labelled appropriately and stored at temperatures of 15-17oC. To freeze dry the cultures, two petri-dishes were prepared for each pure and sporulating culture. To remove the spores, 5 ml of a 10% skimmed milk and 5% myo-inositol mixture was poured onto each plate. The spores were washed off using a sterile swab and 1.5 ml of the re-suspended spores were aseptically transferred into separate vials (four to six replicates of vials per culture) and frozen overnight in a freezer. The next day, the vials were dried using the Edwards Mudulyo freeze drier (Thermo ScientificTM, USA) at -45oC and left for 24 hours. Each vial was sealed with a metal crimp seal after breaking the vacuum of the freeze drier. These were then stored for long term storage. Storage at ultralow temperatures was another method used for culture preservation. For this method, 4-6 ml of 15% sterile glycerol was placed onto a mite free culture plate. The mycelium was scraped off from the plate using sterile tips. The suspension was divided equally between two cryovials and the tubes then marked appropriately. The tubes were

43 placed into Mr FrostiesTM (Thermo Scientific, USA) and then placed in an ultralow freezer for 4 hours. The tubes were removed after the 4 hours and were placed in relevant waxed boxes in the ultralow freezer. These cultures preserved in oil, via freeze drying, and ultralow freezing all formed part of the South African National Collection of Fungi (NCF) under the live culture collection (PPRI).

3.2. DNA extraction The DNA plant mini kit (Qiagen, Hilden, Germany) was used to isolate DNA from the obtained single spored fungal strains using the manufacturer’s protocol for fungal tissue. The protocol for extraction is as follows: The samples were disrupted using a mortar and pestle in liquid nitrogen and transferred to a 1.5 ml tube. Buffer AP1 (400 µl) and RNase A (4 µl) were added inside the tube and incubated for ten minutes at 65oC. The tube was inverted two to three times during incubation. Buffer P3 (130 µl) was then added to the tube and the contents were mixed by vortexing and incubated on ice for five minutes. Following incubation, the lysate was centrifuged for five minutes at 20 000 xg (14 000 rpm). The lysate was then pipetted into a QIAshredder spin column placed in a 2 ml collection tube and was centrifuged for two minutes at 20 000 xg. The flow-through was then transferred into a new 1.5 ml tube without disturbing the pellet. Buffer AW1 (750 µl) was added to the tube and mixed by pipetting. Then, 650 µl of the mixture was transferred into a DNeasy Mini spin column placed in a 2 ml collection tube and centrifuged for one minute at ≥ 6 000 xg (≥ 8 000 rpm). The flow-through was discarded and the process was repeated with the remaining sample. The spin column was then placed into a new 2 ml collection tube and 500 µl buffer AW2 was added and centrifuged for one minute at ≥ 6 000 xg. The flow-through was discarded. Another 500 µl buffer AW2 was added and centrifuged at 20 000 xg for two minutes. The spin column was then transferred to a new 1.5 ml tube and 100 µl buffer AE was added and incubated at room temperature (15-25oC) for five minutes. Following incubation, the tube, along with the spin column were centrifuged at ≥ 6 000 xg for one minute. The step was repeated by adding 50 µl buffer AE to complete the elution step. The DNA was eluted in 150 µl of AE buffer and stored at -20 ºC until further use. The DNA concentration was determined spectrophotometrically by diluting 5 µl of the extracted DNA into a volume of 45 µl of dH2O. The absorbance of DNA was determined at 260 nm and 280 nm on a SmartSpecTM Plus spectrophotometer (BIO-RAD). The quality of the DNA was also observed using 2% agarose (Lonza) gel electrophoresis whereby the DNA HyperLadderTM 100 bp (Bioline) was used to determine the size of the fragments. All gels were made with and

44 run in 1X TAE (40Mm Tris, 20mM acetic acid, and 1 Mm EDTA) at 3-5 V cm-1. Ethidium bromide was used to visualise the DNA under UV light using a UV Light transilluminator.

3.3. Polymerase chain reaction and cycle sequencing Polymerase chain reaction (PCR) was carried out in a 20 µl reaction volume containing 1x PCR buffer, 1 U Exsel Taq polymerase (JMR Holdings), 2.5 mM of each dNTPs (Bioline), 10 µM of EF1 (forward primer; 5’-ATGGGTAAGGARGACAAGAC-3), 10 µM of EF2 reverse primer; 5’-GGARGTACCAGTSATCATGTT-3’ (O’Donnell et al. 1998a), and 200 ng of fungal DNA template. Amplifications were performed in a T100TM thermocycler (BIO-RAD) using an initial denaturation for 2 minutes at 94°C and this was followed by 30 cycles of 30 s at 94°C, and 30 s at 72°C. A final elongation was performed for 5 minutes at 72°C and the reaction held at 20°C. All PCR reactions were evaluated using agarose gel electrophoresis as described above. The TEF-1α PCR amplicons were sequenced from both ends using the forward and reverse primers, respectively. Cycle sequencing was carried out in a 10 µl reaction volume containing 0.3 µl BIG DYE terminator sequence mix (Life Technologies), 1x sequencing buffer, 10 µM of the respective primers and 1 µl of the cleaned PCR product as template DNA. The cycle sequencing protocol used were 96oC for 10 s, 50oC for 5 s, and 60oC for 4 minutes over 26 cycles. After cycle sequencing, the samples were submitted to a sequencing facility for sequencing electrophoresis.

3.4. Sequence editing The obtained sequence data were edited on BioEdit version 7.0.9.0 software to verify base calling and for removal of poor sequences. The edited sequences were then aligned on the BioEdit software for each isolate to minimize the occurrence of ambiguous nucleotides. For each of the aligned sequences, a consensus sequence was attained with minimum manual manipulation. Once the sequences were edited, the TEF-1α sequence from an isolate of interest was analysed at MYCOBANK (http://www.mycobank.org/) Robert et al. (2013), and FUSARIUM-ID (fusariumdb.org/) Geiser et al. (2004) linked to the BLAST server. The TEF- 1α sequences were then used as a query for comparison to the database. Nucleotide BLAST (nBLAST) retrieved the closest correspondences to the TEF-1α query sequence. The closest corresponding query sequence chosen was the one with a high percentage similarity and a high sequence overlap.

3.5. Phylogenetic analysis Alignment of DNA sequences was performed using MAFFT (Katoh and Kuma, 2000) through the insertion of gaps. Gaps were regarded as missing data in the subsequent analysis. Phylogenetic analysis for all datasets, FOSC (O’Donnell et al. 2009b), FIESC (O’Donnell et al. 2009b), and the genus wide dataset (Laurence et al. 2016) was performed based on Maximum Parsimony (MP) and Maximum Likelihood (ML). Maximum Parsimony was performed using PAUP 4.0* (Swofford, 2002) and Maximum Likelihood (ML) was performed with the use of a web server (PhyML - ATGC: Montpellier Bioinformatics platform) (Guindon et al. 2010). The best models determined by PhyML for the FOSC, FIESC, and the genus wide datasets were GTR+G+F, TN93+G+F and TN93+G+F, respectively. Heuristic searches were completed with the random addition of sequences (100 replicates), and tree bisection- reconnection (TBR) branch swapping was employed to infer maximum parsimony. The Retention Index (RI) and Consistency Index (CI) were determined to show the amount of homoplasy in the respective datasets. Trees were rooted using the outgroup method for the FOSC and FIESC datasets, while midpoint rooting was used for the genus wide analyses. The outgroup for the FOSC and FIESC phylogenetic trees were isolates Fusarium sp. (J397074) and F. concolor (NRRL 13459), respectively. Bootstrap analyses were executed to establish branching point confidence intervals (1 000 replicates) for the most parsimonious trees produced for the TEF-1α data sets. The visualisation of trees was performed using FigTree v1.4 (Rambaut, 2013).

Fusarium isolates used to construct matrices for each dataset were obtained from previously published work (O’Donnell et al. 2009b: Laurence et al. 2012, Laurence et al. 2016). These are given in table 3.1, 3.2 and 3.3.

3.6. Chapter tables

NRRL NO. FIESC MLST ISOLATE SOURCE 3020 10-a Unknown 3214 10-a Unknown 5537 8-a Fescue hay 6548 12-a Wheat 13335 21-a Alfalfa 13379 23-b Rice 13402 9-b (F. scirpi) Pine soil 20423 4-a (F. lacertarum) Lizard skin 20697 14-b (F. equiseti) Beet 20722 27-a Chrysanthemum sp. 22244 25-a Rice 25795 5-c Disphyma crassifolium seed 26417 26-a Leaf litter 26419 14-a (F. equiseti) Soil 26921 12-a Wheat 26922 9-c (F. scirpi) Soil 28029 3-b Human eye 28577 28-a Grave stone 28714 26-b Acacia sp. branch 29134 9-a (F. scirpi) Pasture soil 31011 12-a Thuja sp. 31160 15-c Human lung 31167 18-a Human sputum 32175 15-a Human sputum 32181 15-c Human blood 32182 15-b Human blood 32522 18-b Human diabetic cellulitis 32864 17-a Human

Table 3.1 continued 32865 21-b Human endocarditis 32866 23-a Human cancer patient 32867 23-a Human 32868 25-c Human blood 32869 15-c Human cancer patient 32871 5-a Human abscess 32993 25-b Human nasal tissue 32994 15-c Human ethmoid sinus 32995 15-c Human sinus 32996 15-c Human leg wound 32997 7-a Human toenail 34001 15-e Human foot wound 34002 22-a Human ethmoid sinus 34003 20-a Human sputum 34004 16-a Human BAL 34005 24-a Human intravitreal fluid 34006 15-a Human eye 34007 15-a Human sputum 34008 15-d Human lung 34010 15-c Human maxillary sinus 34011 15-a Human sputum 34032 5-a Human abscess 34034 1-c Human leg 34035 5-d Human sinus 34037 5-b Human abscess 34039 1-b Human 34056 16-b Human bronchial wash 34059 16-c Human blood 34070 17-c Tortoise 36123 4-b Unknown 36136 14-a (F. equiseti) Unknown 36269 12-b Pinus nigra seedling 36318 3-a Unknown 36321 14-a (F. equiseti) Soil 13459 (F. concolor) Outgroup Plant debris Adapted from O’Donnell et al. (2009b). NRRL numbers downloaded from NCBI GenBank.

Table 3.2: Fusarium species used for the FOSC dataset matrix. The accession numbers of these isolates were obtained from Laurence et al. (2012). The sequences for these isolates were obtained from NCBI Genbank using the accession numbers and were combined with the FOSC isolates obtained in the current study to build up the matrix for the FOSC dataset.

AUST Voucher no. SARD Voucher no. OTHER Voucher no. OUTGROUP 5 44930 221 42 7 O 0017 conglutinans 256 Fusarium sp. RBG5443 84 44913 191 12 4 F6152 conglutinans 252 103 44917 209 17 6 F6325 raphani 254 2 119 44935 90 41 8 120 44926 260 1 1 122 44914 259 2 1 129 44911 258 2 1 142 46475 208 5 3 165 46477 261 1 1 172 46478 94 13 2 214 46617 158 10 3 217 46622 263 1 1 226 46681 19 5 2 242 46619 262 1 1 293 46679 285 1 1 351 46664 265 1 1 359 46645 22 3 1 378 46647 264 4 1 387 52659 201 10 1 421 52650 268 1 1 459 52647 267 4 3 508 52816 274 6 1

Table 3.2 continued 545 52817 275 1 1 556 52807 273 1 1 557 52627 266 1 1 564 52820 276 1 1 571 52646 6 1 1 572 52654 269 1 1 582 52669 271 2 1 584 52663 270 4 2 589 52673 272 1 1 603 53113 282 1 1 605 53077 277 1 1 610 53079 278 1 1 629 53088 281 1 1 638 53081 279 1 1 641 53084 280 1 1 642 683 689 693 695 696 697 699 706 713 AUST numbers adapted from Laurence et al. (2012).

Accession no. (GENBANK) Fusarium Species Isolate TEF-1α F. acutatum NRRL13308 AF160276 F. anthophilum NRRL13602 AF160292 F. aywerte RBG5743 KP083250 F. aywerte RBG5736 KP083248 F. aywerte RBG5741 KP083249 F. bactridioides NRRL20476 AF160290 F. begoniae NRRL25300 AF160293 F. brevicatenulatum NRRL25446 AF160265 F. bulbicola NRRL13371 KF466415 F. cerealis TUR057 JN541063 F. coicis sp. nov. RBG5368/NRRL66233 KP083251 F. coicis sp. nov. RBG5369/NRRL66234 KP083252 F. commune NRRL22903 AF008513 F. concentricum NRRL25181 AF160282 F. culmorum VI01002 AJ543541 F. denticulatum NRRL25302 AF160569 F. dlaminii NRRL13164 AF160277 F. foetens NRRL31852 AY320087 F. foetens NRRL52749 JF740825 F. globosum NRRL26131 AF160285 F. goolgardi sp. nov. RBG5412/NRRL66248 KP083253 F. goolgardi sp. nov. RBG5418/NRRL66249 KP083254 F. goolgardi sp. nov. RBG5411/NRRL66250 KP101123 F. hostae NRRL29889 AF331817 F. kyushuense VI01325 AJ427274 F. langsethiae VI01280 AJ427272 F. mundagurra sp. nov. RBG5717/NRRL66235 KP083256 F. mundagurra sp. nov. RBG5599/NRRL66236 KP083255

Table 3.3 continued F. napiforme NRRL13604 AF160266 F. nelsonii NRRL13338 GQ505402 F. newnesense sp. nov. RBG5443/NRRL66237 KJ397074 F. newnesense sp. nov. RBG5444/NRRL66238 KP083257 F. newnesense sp. nov. RBG5445/NRRL66239 KP083258 F. newnesense sp. nov. RBG5446/NRRL66240 KP083259 F. newnesense sp. nov. RBG610/NRRL66241 KP083261 F. newnesense sp. nov. RBG6847 KP083262 F. newnesense sp. nov. RBG609/NRRL66242 KP083260 F. nygamai NRRL13488 AF160273 F. oxysporum NRRL22902 AF160312 F. palustre NRRL54054 GQ856949 F. phyllophilum NRRL13617 AF160274 F. poae VI01265 AJ420839 F. pseudocircinatum NRRL22946 AF160271 F. pseudograminearum CS5791 JN541056 F. pseudograminearum RGB3580 HQ667168 F. pseudonygamai NRRL13592 AF160263 F. ramigenum NRRL25208 KF466423 F. redolens FRCO-681 AF331816 F. sibiricum NRRL53429 HM744683 F. sporotrichioides VI1313 AJ420818 F. subglutinans NRRL22016 AF160289 F. succisae NRRL13613 AF160291 F. tjaetaba sp. nov. RBG5361/NRRL66243 KP083263 F. tjaetaba sp. nov. RBG5363/NRRL66244 KP083264 F. tjaetaba sp. nov. RBG5364/NRRL66245 KP083265 F. tjaynera sp. nov. RBG5367/NRRL66246 EF107152 F. tjaynera sp. nov. FRL19318 EF107150 F. tjaynera sp. nov. RBG5366/NRRL66247 KP083266 F. tjaynera sp. nov. FRL19315 EF107151 F. tjaynera sp. nov. FRL11240 EF107155 F. udum NRRL22949 AF160275 F. werrikimbe FRL19361 EF107132 F. sibiricum NRRL53430 HM744684

Table 3.3 continued F. lactis NRRL25200 AF160272 Fusarium sp. NRRL25184 AF008514 Fusarium sp. NRRL36351 GQ915500 Fusarium sp. NRRL46670 GU250579 Fusarium sp. CML908 EU574681 Fusarium sp. NRRL25204 AF160299 Fusarium sp. NRRL5537 GQ505588 Fusarium sp. NRRL29123 AF160310 Fusarium sp. CML914 EU574682 Fusarium sp. NRRL29123 AF160310 Fusarium sp. NRRL29124 AF160311 Fusarium sp. NRRL25195 AF160298 Fusarium sp. NRRL25623 AF160300 Fusarium sp. NRRL25807 AF160305 Fusarium sp. NRRL26756 AF160307 Fusarium sp. NRRL26757 AF160308 Fusarium sp. NRRL25346 AF160296 Fusarium sp. NRRL25622 AF160301 Fusarium sp. NRRL25615 AF160304 Fusarium sp. NRRL26793 AF160309 Fusarium sp. NRRL25303 AF160283 Fusarium sp. NRRL25309 AF160284 Fusarium sp. NRRL26427 AF160286 Fusarium sp. NRRL26061 AF160303 Fusarium sp. NRRL26152 AF160306 Fusarium sp. NRRL26794 AF160287 Fusarium sp. NRRL28852 AF160288 Fusarium sp. NRRL25221 AF160268 Fusarium sp. NRRL26064 AF160302 F. commune HM804942.1 F. inflexum AF331814.1 F. polyphialidicum DQ295144.1 Adapted from Laurence et al. (2016), Accession numbers downloaded from NCBI GenBank.

3.7. Chapter figures

Sampling and sample processing

Figure 3.1: Map of South Africa 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 that is bordered by the nature reserve.

Figure 3.2: The transect method used to obtain soil samples. End samples were pooled and sub- samples were pooled.

5g soil sample (3X) Separated into two fractions

Large soil particles Small soil particles (3X) (3X)

Plate on SFA (3X10)

Transfer to ¼ PDA (30X3)

Single spore suspension on WA

Morphological Transfer spore to PDA (4 characterisation reps/culture) Plate onto SNA, CLA, and PDA

DNA extraction and culture preservation

CHAPTER 4

RESULTS

4.1 Fungal isolation

The soil samples that were collected from the Willem Pretorius nature reserve were separated into small fine soil particles and large soil particles containing relatively larger plant debris such as roots, dried leaves, and twigs. From this, a total of 452 isolates were gathered. Of these 452 isolates, 273 were obtained from large soil particles (94 from site 1, 83 from site 2, 63 from site 3, and 33 from site 4) and 179 from small soil particles (76 from site 1, 78 from site 2, 20 from site 3, and 5 from site 4) (Figure 4.1.1). To obtain single genetic entities, each Fusarium culture was subjected to single spore isolation on water agar. The time for germination to occur depended on the different cultures and ranged from 10-12 hours. Figure 4.1.2 illustrates spore germination observed during the 10-12 hours. Following this, the colony pigmentation of the single spore isolates were observed on PDA after 10 days of growth under dual light conditions (12 hours normal light and 12 hours UV light). The colours observed ranged from brown, violet, orange, yellow, grey, and white (Figure 4.1.3).

4.2. Morphological characterisation

The morphological characterisation of four well-known and previously well described species of Fusarium were evaluated to provide an overview of some of the morphological characters of Fusarium species obtained in this study. These were F. solani, F. equiseti, F. oxysporum, and F. fujikuroi. The fifth species F. brachygibbosum, represented a less well described species with limited morphological characterisation (Figure 4.2). The observed morphological characters of four of the previously described species were in line with the descriptions of Leslie and Summerell (2006). All the species produced macroconidia and microconidia except F. equiseti, which only produced macroconidia. All these species, except F. fujikuroi, are known to produce chlamydospores predominantly after two – four weeks of plating on CLA, Leslie and Summerell (2006). These structures were observed in F. brachygibbosum, F. equiseti, and F. oxysporum within the ten days of plating in this study.

Morphological features of the five studied Fusarium species are also shown in figure 4.2. The macroconidia produced by F. oxysporum were slightly curved with a medium length (65.3x3.11 µm). They had a tapered and curved apical cell, the basal cell was foot shaped and were three septated. The microconidia were non-septated and oval shaped (20.4x3.24 µm). Monophialides were short and conidia were produced in false heads. The chlamydospores were observed singly at the terminal ends of hyphae. The F. solani macroconidia were relatively wide, stout, and robust (85.6x6.16 µm). They had a blunt and rounded apical cell, a distinct

58 foot cell, and were five septated. The microconidia observed were non-septated or contained one septum, and had an oval shape (47.3x10.18 µm). The species had long monophialides and the microconidia accumulated in false heads. No chlamydospores were observed for this species during the study of the morphological characters and can be ascribed to the growth stage the cultures were in. The F. fujikuroi macroconidia were of medium length (66.0x7.62 µm). They had a tapered apical and a poorly developed foot cell and were three septated. The microconidia were non-septate, oval, and club shaped with a flattened base (24.9x4.09 µm). They had both monophialides and polyphialides with aerial mycelium producing false heads and chains of medium length. The shape of F. equiseti macroconidia were relatively long and slender (89.5x7.56 µm). They had a tapered and slightly elongate apical cell and the basal cell was foot shaped and had five septa. The chlamydospores were observed in pairs and occurred in the agar. The observed morphological characters of the F. brachygibbosum isolate were defined by five septate, medium length macroconidia, wide and thick-walled at the centre (80.0x5.88 µm). The macroconidia were characterized by a foot shaped and barely notched basal cell and a hooked apical cell. The microconidia were relatively large, oval shaped, and had three septa (46.0x5.45µm). Chlamydospores were produced in chains in hyphae located in the agar.

4.3. DNA extraction and PCR

Total DNA extracted from 357 of the 452 Fusarium cultures yielded high quality DNA (Figure 4.3.1). The average A260/A280 ratio for all isolates was between 1.5 and 1.9. The TEF-1α gene was successfully amplified using PCR with no spurious amplification occurring in the no template control sample (NTC) (Figure 4.3.2). An amplicon of approximately 700 base pairs was observed as expected.

4.4 Nucleotide BLAST results

The nucleotide BLAST under Mycobank results of partial TEF-1α sequences of the 357 Fusarium isolates produced five species complexes, ten species, and an unknown group of isolates designated as Fusarium spp. (Figure 4.4.1a). The five complexes were the F. fujikuroi species complex (FFSC), F. incarnatum-equiseti species complex (FIESC), F. oxysporum species complex (FOSC), and F. solani species complex (FSSC) and the F. chlamydosporum species complex (FCSC). The different species types were represented by F. acuminatum Ellis & Everh., F. brachygibbosum Padwick., F. boothii O’Donnell, T. Aoki, Kistler & Geiser, F. burgessii M.H. Laurence, Summerell & E.C.Y Liew, F. caucasicum Letov., F. inflexum R.

Schneid., F. nygamai L.W. Burgessii & Trimboli, F. polyphialidicum Marasas, P.E Nelson, Toussoun & van Wyk, F. redolens Wollenw., and F. sporotrichioides Sherb. The FIESC accounted for 27% (96 isolates) and were the majority of Fusarium species discovered at the undisturbed soils of the Willem Pretorius nature reserve. It was followed by F. brachygibbosum, the FOSC and the FSSC that accounted for 20% (71 isolates), 18% (65 isolates), and 11% (40 isolates) respectively. Fusarium nygamai, the FCSC, the FFSC, and F. burgessii were obtained in moderate amounts and accounted for 5% (18 isolates), 5% (17 isolates), 3% (12 isolates), and 3% (10 isolates) respectively. The unknown Fusarium spp. accounted for 2% (eight isolates) whereas F. polyphialidicum (five isolates), F. acuminatum (three isolates), F. caucasicum (three isolates), F. inflexum (two isolates), and F. sporotrichioides (two isolates) each accounted for 1% of the species discovered. Fusarium boothii and F. redolens, with one isolate each, made up less than 1% of the total number of species. These two species, including those making up the remaining 1% of the total amount, were the least obtained species in the grassland soils.

Nucleotide BLAST under the FUSARIUM-ID database produced the same five species complexes, a group of unknown Fusarium species also designated as Fusarium spp. (Figure 4.4.1b), however, only nine Fusarium species were identified, unlike the ten identified from the Mycobank database. The amount of isolates representing the species complexes based on the FUSARIUM-ID database vary from that of the Mycobank database. The FIESC, FOSC, FSSC, FCSC, and FFSC accounted for 26% (93 isolates), 21% (74 isolates), 13% (46 isolates), 5% (18 isolates), and 5% (17 isolates), respectively. Fusarium brachygibbosum, Fusarium spp., F. acuminatum, and F. redolens were also identified from the FUSARIUM-ID database and accounted for 20% (seven isolates), 7% (26 isolates), 1% (three isolates) and less than 1% (one isolate) respectively. Six of the nine species discovered were only unique to the FUSARIUM-ID database. These are F. venenatum Nirenberg, (three isolates), F. beomiforme P.E. Nelson, Toussoun & L.W Burgess, (two isolates), both accounted for 1% each, and F. armeniacum (G.A. Forbes, Windels & L.W. Burgess) L.W Burgess & Summerell, (one isolate), F. hostae Geiser & Juba, (one isolate), F. commune K, Skorg., O’Donnell & Nirenberg, (one isolate), and F. concolor Reinking, (one isolate) that accounted for less than 1% each.

For results obtained from the Mycobank database, the majority of the isolates obtained from small soil particles belong to the FIESC, followed by the FOSC, F. brachygibbosum and the FSSC, as seen in figure 4.4.2a. The rest of the isolates, excluding F. acuminatum and F.

60 redolens, were found in moderate to low numbers from small soil particles. There was no representative of F. acuminatum and F. redolens in this soil fraction. Both the FIESC and F. brachygibbosum species accounted for the majority of species recovered from large soil particles. The third and fourth highest species recovered in this soil fraction were members of the FOSC and FSSC, respectively. The rest of the isolates, besides F. boothii (not recovered in the large soil particles), were accounted for by moderate to low numbers from large soil particles. From results obtained under the FUSARIUM-ID database (Figure 4.4.2b), the FIESC, FOSC, F. brachygibbosum, the FSSC, and Fusarium spp. contributed to the majority of frequently recovered species from the small soil particles. The FFSC, FCSC, F. venenatum, F. beomiforme, and F. concolor were found in moderate to low numbers from small soil particles. Fusarium acuminatum, F. commune, F. armeniacum, F. hostae, and F. redolens were not recovered from small soil particles. The FIESC, FOSC, F. brachygibbosum, and the FSSC were recovered in high amounts and Fusarium spp., the FFSC, the FCSC, F. acuminatum, F. beomiforme, F. armenicum, F. commune, F. hostae, and F. redolens were found in moderate to low numbers from large soil particles. Fusarium venenatum and F. concolor were not recovered from large soil particles. The numbers of isolates for each species complex and each species recovered separately from the two soil types in figure 4.4.2a and 4.4.2b are indicated by column height.

The Mycobank database nBLAST results (Figure 4.4.3a) showed that isolates of the FOSC, FIESC, FSSC, and F. brachygibossum occurred frequently at site one and two. Fusarium nygamai, F. burgessii, Fusarium spp., F. polyphialidicum, and F. caucasicum were represented in moderate to low amounts at both site one and two. Fusarium inflexum, F. sporotrichioides, F. boothii, and F. redolens also occurred in moderate to low amounts and were only observed at site one whereas isolates of the FFSC were only observed at site two. Nucleotide BLAST results from the FUSARIUM-ID database (Figure 4.4.3b) also indicated that FOSC, FIESC, FSSC, and F. brachygibossum were the four most frequently occurring species at site one and two. Fusarium spp. isolates, the FFSC, and F. venenatum occurred in moderate amounts at both sites. Isolates of the FCSC, F. hostae, and F. redolens were only observed at site one and occurred in low numbers. Fusarium beomiforme and F. concolor were only observed at site two and were also represented in low amounts. Fusarium acuminatum (from both the Mycobank and FUSARIUM-ID database), F. armeniacum, and F. commune were neither represented at site one or two but were observed at site four (see figure 4.4.4a&b). Fusarium nygamai, F. burgessii, F. caucasicum, F. inflexum, F. sporotrichioides, F. boothii, and F.

61 polyphialidicum were uniquely identified via nBLAST from the Mycobank database, whereas F. armeniacum, F. beomiforme, F. commune, F. concolor, and F. venenatum were uniquely identified via nBLAST from the FUSARIUM – ID database.

The Mycobank database nBLAST results (Figure 4.4.3a) for site three and four revealed that the FIESC and FCSC were the most frequently occurring at both these sites. Fusarium brachygibbosum was observed to occur frequently only at site three. The FOSC and F. nygamai isolates occurred in moderate numbers at both sites. The FSSC, FFSC, and F. burgessii only occurred at site three in low numbers. Fusarium acuminatum, the Fusarium species, and F. sporotrichioides were only observed at site four and also occurred in low numbers. Fusarium polyphialidicum, F. caucasicum, F. inflexum, F. boothii, and F. redolens were neither observed at site three or four but were represented at site one and two (see figure 4.4.3a). FUSARIUM- ID database nBLAST results, similar to Mycobank database results, showed that the FIESC and FCSC were the most represented isolates at both site three and four. Furthermore, F. brachygibbosum was only observed at site three and frequently occurred at this site. Similarly, the FSSC and F. beomiforme occurred only at site three but in moderate and low numbers, respectively. The FOSC was observed at both sites and occurred more at site four than at site three. The FFSC and Fusarium species occurred at equally low numbers at both sites. Fusarium acuminatum, F. armeniacum, and F. commune were observed only at site four in low numbers. Fusarium venenatum, F. concolor, F. hostae, and F. redolens were only represented at site one and two (see figure 4.4.3b).

All 354 isolates from this study obtained through nucleotide BLAST analysis under the Mycobank and FUSARIUM-ID databases are tabulated in table 4.1, 4.2, 4.3, 4.4, 4.5, and 4.6. Most of the hits for each isolate correlated on both databases whereas a few gave contrasting IDs with nearly the same percentage similarities (indicated in bold). In some instances however, as indicated in table 4.3, only the Mycobank nBLAST results resolved identification of isolates to species level whereas the FUSARIUM-ID database resolved the identification of isolates to genus level. Each isolate was allocated a unique PPRI voucher number. Isolates with high percentage similarity ranging between 98-100% were accepted to represent species that have been previously described. Those with a percentage similarity of 90-97% were considered new species or new species in previously described species complexes. In this study, all the species complexes fall in the 98-100% similarity range, with only a few isolates falling in the 90-97% range. The unnamed Fusarium spp., F. caucasicum, F. inflexum, F. redolens, and F. nygamai also fall within the 98-100% similarity. Most species such as F. burgessii, F.

62 polyhialidicum, F. sporotrichioides, F. boothii, F. beomiforme, F. concolor, F. venenatum, F. hostae, and F. brachygibbosum fell within the 90-97% similarity range.

4.5 PHYLOGENETIC ANALYSIS

Fusarium incarnatum-equiseti species complex (FIESC, figure 4.5.1): The phylogenetic analysis of the TEF-1α gene for the FIESC dataset resolved the dataset into eight distinct clades encompassing the various MLSTs within the FIESC. The dataset comprised 152 isolates that included 96 PPRI isolates from the current study and 56 isolates from the O’Donnell et al. (2009b) publication. Clade one, represented isolates from the current study that were phylogenetically unresolved. Clade two, and seven only presented isolates from O’Donnell et al. (2009b). Clade two, encompassed various MLST FIESC species defined as MLST 15-18, 20-28 whereas clade seven encompassed MLST 14 species. Clade three to eight comprised of isolates from both this study and that by O’Donnell et al. (2009b). These clades encompassed MLST 10, MLST 12, MLST five, MLST one, three and four, MLST 14, and MLST nine species respectively. The MP analysis provided strong bootstrap support for the resolution of these clades, whereas the ML analysis provided less to no support for the clades. Bootstrap values of ≥70% were utilised as a basis at which topological incongruences’ were identified (O’Donnell et al. 2009b) for both MP and ML. The MP bootstrap values were obtained using heuristic search methods and the starting tree(s) were obtained via stepwise addition. Parsimony analysis of the dataset produced a CI of 0.7301, RI of 0.9028 and a HI of 0.2699. The ML tree was based on the best model TN93+G+F determined by PhyML (Guindon et al. 2010). Fusarium concolor (NRRL 13459) was used as an outgroup to the dataset.

The first clade encompassed three isolates identified through nBLAST analysis as Fusarium incarnatum-equiseti species complex MLST 10-a, 5-d, and 9-b. These isolates were phylogenetically related but also appear to have grouped out of their respective clades that were observed across the phylogenetic tree. The clade resolved with a bootstrap support of 100% from the MP analysis and none from the ML analysis. Clade two comprised of isolates from the O’Donnell et al. (2009b) study that grouped together. These isolates had been isolated from various human sources such as blood, leg, eye, and crops such as rice from different countries and were supported by an overall MP bootstrap support of 78%. Clade three was supported by a MP bootstrap of 81% and it encompassed six PPRI isolates from the current study with all identified via nBLAST analysis as MLST 10-a. These species were closely related to two isolates from O’Donnell et al. (2009b) described as F. equiseti (NRRL3020, MLST 10-a; host

63 and origin unknown) and NRRL3214, MLST 10-a; host and origin unknown). Clade four consisted of one isolate (PPRI 20790) from the current study identified as MLST 12-c and was closely related to a F. equiseti (NRRL 36269, MLST 12-b) isolate from a Pinus nigra J.F. Arnold tree in Croatia. The clade was supported by a strong MP bootstrap of 100%. Clade five, with MP bootstrap support of 88%, consisted of 59 isolates from the current study, 42 of these isolates were identified as MLST 5-f, 14 as MLST 5-d, and three as MLST 5-e. The 59 species grouped together with four isolates from O’Donnell et al. (2009b) defined as F. equiseti and thus share similarity. These four isolates were isolated from human abscesses (NRRL 32871, 34032 and 34037), human sinus (NRRL 34035), and from Disphyma crassifolium seed (NRRL 25795). The sixth clade encompassed one isolate (PPRI 20758) from the current study identified as MLST 1-a. It was closely related to six isolates from O’Donnell et al. (2009b) that grouped together with a MP bootstrap support of 87%. Of these six isolates, PPRI 20758 is more closely related to NRRL20423 (F. lacertarum MLST 4-a, host: lizard skin; origin: India) and NRRL36123 (MLST 4-b, host and origin unknown). It is then distantly related to NRRL34034 (MLST 1-c, host: human leg; origin: Arizona), NRRL34039 (MLST 1-b, host: human; origin: Connecticut), NRRL28029 (MLST 3-b, host: human eye; origin: California), and NRRL36318 (MLST 3-a, host and origin unknown) within the same clade. Clade seven, similar to clade two, only consisted of isolates from the O’Donnell et al. (2009b) study. All four isolates appeared to be the same MLST and were supported by a MP bootstrap value of 100% and a ML bootstrap value of 98%. Clade eight consisted of 26 isolates from the current study that grouped together with three isolates from O’Donnell et al. (2009b) described as F. scirpi that were isolated from soil. The PPRI isolates in this clade were all identified as MLST 9-b excluding PPRI 19187, which was identified as MLST 5-d via nBLAST analysis but phylogenetically grouped with MLST 9-b isolates. The clade was supported by a MP bootstrap value of 80%. Significant differences were observed between the MP and ML phylogenies and as a result, several ML bootstrap values could not be assigned concordantly with MP bootstrap values for clade support.

Fusarium oxysporum species complex (FOSC, figure 4.5.2): Phylogenetic analysis of the TEF- 1α for the FOSC dataset resolved the dataset into two phylogenetic species as previously described by Laurence et al. (2014). The dataset comprised of 156 isolates that included 70 PPRI isolates from the current study, 46 AUST isolates obtained from Laurence et al. (2012) and 37 SARD isolates obtained. Also included in this dataset were isolate O0017 F. oxysporum f. sp. conglutinans 256, F6152 F. oxysporum f. sp. conglutinans 252, and F6325 F. oxysporum

64 f. sp. raphani 254 isolates. The clades encompassed various formae speciales of the FOSC that grouped accordingly into phylogenetic species 1 and 2 (PS1 and PS2). Two separate clades comprising PS2 isolates were observed in this study and were designated PS2a and PS2b. PPRI isolates from the current study were grouped within PS1 and PS2a. Both MP and ML analysis provided limited bootstrap support for the FOSC dataset and as a result, the two phylogenies were partially incongruent. Bootstrap values of ≥70% were utilised as a basis at which topological incongruences were identified (O’Donnell et al. 2009a) for both MP and ML. The MP bootstrap values were obtained using heuristic search methods and the starting tree(s) were obtained through stepwise addition. Parsimony analysis of the dataset produced a CI of 0.8810, RI of 0.9421 and a HI of 0.1190. The ML tree was based on the best model GTR+G+F determined by PhyML (Guindon et al. 2010). Fusarium sp. (J397074) was used as an outgroup for the dataset.

The phylogenetic species 1, consisted of three PPRI isolates 20715, 22778, and 20540. The former was identified through nBLAST as F. oxysporum f. sp. perniciosum Toole. and the two latter isolates were identified as F. oxysporum f. sp. cubense W.C. Snyder & H.N. Hansen. These isolates were grouped with 15 AUST isolates, indicating close relatedness. The clade was supported by a MP bootstrap value of 92% and a ML bootstrap value of 72%. Phylogenetic species 2a (PS2a) lacked bootstrap support from both the MP and ML analyses. It comprised of 66 PPRI isolates from the current study. This clade consisted mainly of four different formae speciales of F. oxysporum, i.e., F. oxysporum f. sp. cubense W.C. Snyder & H.N. Hansen, F. oxysporum f. sp. raphani W.C. Snyder & H.N. Hansen, F. oxysporum f. sp. lini W.C. Snyder & H.N. Hansen, and F. oxysporum f. sp. melonis W.C. Snyder & H.N. Hansen identified via nBLAST analysis. These species appeared to share similarity to most of the SARD isolates compared to AUST isolates that all represented species of the FOSC. PPRI 20537 was phylogenetically related to F6325 F. oxysporum f. sp. raphani 254. Isolates identified as F. oxysporum f. sp. cubense via nBLAST were observed in both phylogenetic species (PS1 and PS2a). These were the two previously mentioned PPRI 22778 and PPRI 20540 that grouped within PS1 and PPRI 20526 and PPRI 20476 that grouped within PS2a. There were inconsistencies observed in clade two in that isolates of this clade did not cluster within their designated clades but instead were scattered across the phylogenetic tree. Phylogenetic species 2b cluster comprised AUST and SARD isolates and none of the FOSC isolates from the present study grouped within this cluster. Similar with the FIESC phylogenetic analysis, significant

65 differences were also observed between MP and ML phylogenies and therefore were incongruent.

GENUS WIDE (Figure 4.5.3): Phylogenetic analysis of the TEF-1α gene for the genus wide dataset resolved the dataset into 16 clades. The dataset comprised of 150 isolates that included 66 PPRI isolates from the current study and 84 isolates from the Laurence et al. (2016) publication. Clade one, 11, 15, and 16 represented groups of isolates from the current study that were phylogenetically unresolved. Clade five, nine, 13, and 14 represented isolates in which only isolates from Laurence et al. (2016) grouped. These clades comprised of the FFSC, newly described F. newesense sp. nov isolates, the F. aywerte species complex (FASC), and FCSC respectively. Clade two, three, four and six encompassed isolates of the FFSC, clade seven encompassed isolates of the FOSC, whereas clade ten and twelve encompassed isolates of the F. redolens species complex (FRSC) and F. sambucinum species complex (FSAMSC), respectively. Clade eight encompassed F. commune isolates. The MP analysis provided strong bootstrap support for these clades whereas the ML analysis provided significantly less support for the clades. Bootstrap values of ≥70% were utilised as a basis at which topological incongruences were identified (O’Donnell et al. 2009a) for both MP and ML. The MP bootstrap values were obtained using heuristic search methods and the starting tree(s) were obtained via stepwise addition. Parsimony analysis of the dataset produced a CI of 0.4897, RI of 0.8402 and a HI of 0.5103. The ML tree was based on the best model TN93+G+F determined by PhyML (Guindon et al. 2010). The midpoint rooting method was used for the genus wide analyses.

Clade one with a MP bootstrap support of 82%, encompassed six PPRI isolates identified as F. burgessii, from the Mycobank database. Four of these isolates (PPRI 19129; PPRI 21062; PPRI 21072; PPRI 21097) were only identified as Fusarium sp. from the FUSARIUM-ID database. The other two isolates, PPRI 21079 and PPRI 21048, were identified as F. beomiforme and F. concolor respectively from the FUSARIUM-ID database. Four other isolates (PPRI 22744; PPRI 21070; PPRI 21263; PPRI 22787) also identified as F. burgessii from the Mycobank database, did not group within this clade and were observed as outliers. Ten PPRI isolates were grouped under clade two and these were identified as species of the F. fujikuroi species complex through nBLAST analysis. PPRI 22756 and PPRI 19149 were closely related to an undescribed isolate AF160268 Fusarium sp. (origin: Zimbabwe), and three other PPRI isolates (PPRI 21033; PPRI 21055; PPRI 21272) also appeared to share close similarity with two other described F. mundagurra sp. nov species bearing accession numbers KP083255 (origin:

Australia) and KP083256 (origin: Australia). These PPRI isolates may also potentially be new undescribed species.

Clade three comprised of 15 PPRI isolates identified as F. nygamai and was only supported by a MP bootstrap value of 85%. These isolates grouped with a described F. nygamai isolate (AF160273, origin: Australia). Clade four was supported by a MP bootstrap value of 77% and comprised of three PPRI isolates (PPRI 20741; PPRI 22795; PPRI 20533) bearing contrasting identities as determined through nBLAST analysis. These isolates were identified as F. nygamai and FFSC from the Mycobank and FUSARIUM-ID databases, respectively. The three isolates were phylogenetically closely related to an undescribed Fusarium species with accession number AF160309 (host: Striga hemanthica (Dellie) Benth., origin: Sudan). Clade five lacked bootstrap support and comprised of the FFSC isolates obtained from NCBI Genbank, also used in the publication by Laurence et al. (2016). Isolate PPRI 19224 identified as a species of the FFSC was grouped in clade six and it was closely related to F. dlamini (AF1602227, host and origin unknown), two undescribed isolates Fusarium spp. (AF160303, origin: Madagascar and AF160306 origin: Niger) that were determined to be members of the FFSC through nBLAST. A strong MP bootstrap value of 100% supported the similarity. Clade seven comprised of three distinct species, i.e., species of the FOSC, F. inflexum, both from the current study and F. foetens. These were supported by a MP bootstrap value of 94%. Six of the current study’s PPRI isolates were identified as Fusarium sp. and two were identified as F. inflexum from the Mycobank database but contrastingly, all eight PPRI isolates were identified as species of the FOSC from the FUSARIUM-ID database. The isolates appeared related to both described species F. oxysporum (AF160312) and F. inflexum (AF331814).

Clade eight with a MP bootstrap of 100% comprised of a single isolate from the current study, PPRI 19124. The isolate was identified as a restricted item under the Mycobank database and as F. commune under the FUSARIUM-ID database. The isolate was confirmed to be F. commune (origin: unknown) as it shared close similarity with a described species F. commune (HM804942) obtained from NCBI Genbank. Clade nine consisted of four isolates, three of these isolates were newly described as F. newnesense sp. nov. The other Fusarium sp. isolate (AF008514, host: Grape; origin: Europe) was undescribed and shared similarity with the three newly described isolates by Laurence et al. (2016). Clade 10 with a MP bootstrap of 100% comprised of a single isolate from the current study, PPRI 21574. The isolate was identified as F. redolens via nBLAST analysis. The isolate was confirmed to be F. redolens as it shared close similarity with a described species F. redolens (AF331816, origin: unknown) obtained

67 from NCBI Genbank. Clade eleven comprised of four PPRI isolates identified as F. polyphialidicum under the Mycobank database and as Fusarium sp. under the FUSARIUM-ID database and were collectively supported by a MP bootstrap support of 98%. These isolates were distantly related to F. polyphialidicum (DQ295144) that appeared to be basal to this clade. This suggests that the four F. polyphialidicum isolates may be distinct unknown species as indicated by the FUSARIUM-ID nBLAST results.

Clade twelve comprised of three isolates from the current study. The PPRI 19183 isolate was confirmed to be related to F. palustre (GQ856949, origin: USA). Both PPRI 20541 and PPRI 20719 were confirmed to be related to F. kyushuense (AJ427274, origin: Japan). These isolates were grouped with described isolates of the FSAMSC. Clade 13 and 14 consisted of isolates also used in a study by Laurence et al. (2016) that represented the FASC and an isolate of the FCSC, F. nelsoni Marasas & Logrieco respectively. These isolates were not closely related to any of the isolates from the current study. Clade 15 comprised of three isolates from the current study, PPRI 21378; PPRI 20721, and PPRI 21092, identified as F. caucasicum under the Mycobank database and as species of the FSSC under the FUSARIUM-ID database. These isolates obtained a strong bootstrap support of 100%. Clade 16 comprised of five PPRI isolates, three (PPRI 22745, PPRI 19235, PPRI 19153) that were identified as F. acuminatum and were supported by a MP bootstrap of 100%. The other two isolates (PPRI 21053, PPRI 21303) also received a MP bootstrap support value of 100% but obtained contrasting identities from both nBLAST databases.

4.6. Chapter tables

Table 4.1: Fusarium-incarnatum equiseti species complex (FIESC) nucleotide BLAST results from MYCOBANK and FUSARIUM-ID databases. The isolates indicated in bold signify contrasting nBLAST results from the two databases.

PPRI MYCOBANK DATABASE FUSARIUM-ID DATABASE MYCOBANK % FUSARIUM-ID NO. SIMILARITY % SIMILARITY 19120 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_FIESC 9-b NRRL13402 99.791 99.79 19122 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.844 99.84 19123 FIESC (NRRL 43623; MLST type: 5-e) GQ505661, N1 FD_01684_GFSC NRRL43623 99.688 99.68 19127 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 96.141 96.14 19128 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 96.091 96.09 19137 FIESC (NRRL 34035; MLST type: 5-d) GQ505637, N1 FD_01660_ FIESC 5-d NRRL34035 99.406 99.4 19141 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 96.289 96.28 19142 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 19151 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.793 99.79 19152 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 19158 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 99.586 99.58 19163 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 19164 FIESC (NRRL 34035; MLST type: 5-d) GQ505637, N1 FD_01660_ FIESC 5-d NRRL34035 99.804 99.8 19165 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.802 99.8 19171 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 99.799 99.79 19174 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_ 01695_FIESC 5-f NRRL45997 100 100 19178 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.838 99.83 19180 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 96.97 96.96 19181 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 19182 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.587 99.58 19187 FIESC (NRRL 34035; MLST type: 5-d) GQ505637, N1 FD_01660_ FIESC 5-d NRRL34035 96.488 96.48 19189 FIESC (NRRL 43623; MLST type: 5-e) GQ505661, N1 FD_01684_GFSC NRRL43623 99.595 99.59 19190 FIESC (NRRL 34035; MLST type: 5-d) GQ505637, N1 FD_01660_ FIESC 5-d NRRL34035 99.792 99.79 19193 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 19195 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 99.84 19197 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.844 99.84 19198 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.375 99.37 19199 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 19208 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 19210 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 99.84 99.84 19214 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 19215 FIESC (NRRL 34035; MLST type: 5-d) GQ505637, N1 FD_01660_ FIESC 5-d NRRL34035 99.433 99.43 19225 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100

Table 4.1. Continued

19226 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 19230 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 99.806 98.8 19242 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 19243 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.803 99.8 20524 F. scirpi CBS 731.87 GQ505600, N8 FD_01618_ FIESC 9-b NRRL13402 96.27 96.8 20528 F. equiseti CBS 394.93 GQ505597, N8 FD_01695_ FIESC 5-f NRRL45997 99.644 100 20542 F. equiseti CBS 394.93 GQ505597, N8 FD_01695_ FIESC 5-f NRRL45997 99.612 100 20546 F. scirpi CBS 731.87 GQ505600, N8 FD_01618_ FIESC 9-b NRRL13402 96.257 96.79 20547 F. equiseti CBS 394.93 GQ505597, N8 FD_01660_ FIESC 5-d NRRL34035 98.879 99.81 20703 F. equiseti CBS 394.93 GQ505597, N8 FD_01695_ FIESC 5-f NRRL45997 99.647 100 20706 F. equiseti CBS 394.93 GQ505597, N8 FD_01695_ FIESC 5-f NRRL45997 99.643 100 20707 F. equiseti CBS 394.93 GQ505597, N8 FD_01684_GFSC NRRL43623 96.127 99.64 20709 F. scirpi CBS 448.84 GQ505605, N8 FD_01618_ FIESC 9-b NRRL13402 97.996 99.81 20713 F. equiseti CBS 394.93 GQ505597, N8 FD_01695_ FIESC 5-f NRRL45997 99.643 100 20714 FIESC (NRRL 3020; MLST type: 10-a) GQ505586, N1 FD_01613_ FIESC 10-a NRRL3214 98.81 98.9 20731 FIESC (NRRL 36401; MLST type: 2-a) GQ505651 , N1 FD_01613_ FIESC 10-a NRRL3214 90.566 96.02 20736 FIESC (NRRL 45997; MLST type: 5-f) GQ505672 , N1 FD_01695_ FIESC 5-f NRRL45997 100 100 20747 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 96.197 96.4 20749 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.321 99.32 20756 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 96.141 96.14 20758 FIESC (NRRL 45996; MLST type: 1-a) GQ505671, N1 FD_01694_ FIESC 1-a NRRL45996 95.37 95.37 20761 FIESC (NRRL 3214; MLST type: 10-a) GQ505587, N1 FD_01613_ FIESC 10-a NRRL3214 98.986 99.05 20762 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.841 99.84 20783 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 98.72 98.72 20786 FIESC (NRRL 3020; MLST type: 10-a) GQ505586 , N1 FD_01613_ FIESC 10-a NRRL3214 92.822 93.08 20788 FIESC (NRRL 34035; MLST type: 5-d) GQ505637, N1 FD_01660_ FIESC 5-d NRRL34035 99.669 99.51 20789 FIESC (NRRL 34035; MLST type: 5-d) GQ505637, N1 FD_01660_ FIESC 5-d NRRL34035 99.532 99.53 20790 F. scirpi CBS 731.87 GQ505600, N8 FD_01673_ FIESC 12-c NRRL36392 98.539 98.27 20791 FIESC (NRRL 34035; MLST type: 5-d) GQ505637 , N1 FD_01660_ FIESC 5-d NRRL34035 99.377 99.37 21034 FIESC (NRRL 34035; MLST type: 5-d) GQ505637 , N1 FD_01660_ FIESC 5-d NRRL34035 99.031 99.03 21035 FIESC (NRRL 3020; MLST type: 10-a) GQ505586 , N1 FD_01613_ FIESC 10-a NRRL3214 94.009 94 21036 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 99.355 99.35 21045 FIESC (NRRL 3214; MLST type: 10-a) GQ505587 , N1 FD_01613_ FIESC 10-a NRRL3214 88.684 88.68 21060 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 97.119 97.26 21077 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 99.844 99.84 21080 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 96 95.91 21089 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 99.846 99.84 21091 FIESC (NRRL 34035; MLST type: 5-d) GQ505637, N1 FD_01660_ FIESC 5-d NRRL34035 99.685 99.68 21099 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 99.37 99.37 21258 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 96.271 95.94 21271 FIESC (NRRL 34035; MLST type: 5-d) GQ505637, N1 FD_01660_ FIESC 5-d NRRL34035 99.515 99.51 21274 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 98.259 99.67 21367 FIESC (NRRL 34035; MLST type: 5-d) GQ505637 , N1 FD_01660_ FIESC 5-d NRRL34035 98.701 98.32

Table 4.1. Continued

21571 F. equiseti CBS 394.93 GQ505597, N8 FD_01660_ FIESC 5-d NRRL34035 98.746 99.63 21572 FIESC (NRRL 3214; MLST type: 10-a) GQ505587, N1 FD_01613_ FIESC 10-a NRRL3214 98.095 98.38 21575 FIESC (NRRL 34035; MLST type: 5-d) GQ505637, N1 FD_01660_ FIESC 5-d NRRL34035 99.831 99.83 22748 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.844 99.84 22751 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.839 99.83 22752 FIESC (NRRL 34035; MLST type: 5-d) GQ505637, N1 FD_01660_ FIESC 5-d NRRL34035 99.682 99.68 22757 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 99.842 99.84 22758 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 98.268 98.13 22762 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.689 99.68 22763 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 22764 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.685 99.84 22765 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.842 100 22768 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 99.37 99.37 22772 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 22777 F. equiseti CBS 394.93 GQ505597, N8 FD_01695_ FIESC 5-f NRRL45997 99.638 100 22789 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 99.665 99.66 22792 F. equiseti CBS 394.93 GQ505597, N8 FD_01695_ FIESC 5-f NRRL45997 99.455 99.81 22796 FIESC (NRRL 13402; MLST type: 9-b) GQ505592 F. scirpi, N1 FD_01618_ FIESC 9-b NRRL13402 93.939 95.02 22797 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 99.83 22802 FIESC (NRRL 45997; MLST type: 5-f) GQ505672, N1 FD_01695_ FIESC 5-f NRRL45997 100 100 PPRI = Living fungal collection of the South Africa National Collection of Fungi, Plant Protection Research Institute, ARC, Pretoria, South Africa; MLST = Multiloci sequence type; BLAST = Basic Local Alignment Search Tool; NRRL = Agricultural Research Service culture collection, United States Department of Agriculture, Illinois, United States of America; CBS = Centraalbureau voor Schimmelcultures, Filamentous fungi and Yeast Collection, Netherlands.

PPRI NO. MYCOBANK DATABASE FUSARIUM-ID DATABASE MYCOBANK % FUSARIUM-ID % SIMILARITY SIMILARITY 19136 F. oxysporum f. sp. melonis CBS 420.90 EF056790, N8 FD_00801_FOSC 231 NRRL38885 99.497 99.49 19207 F. oxysporum f. sp. melonis CBS 420.90 EF056790, N8 FD_00801_ FOSC 231 NRRL38885 99.677 99.68 19216 FOSC (NRRL 38591; MLST type: 191) FJ985379 FD_01194_ FOSC 4 NRRL26178 100 100 19217 FOSC (NRRL 36286; MLST type: 154) FJ985344 FD_01375_ F. oxysporum 99.01 99.8 19218 FOSC (NRRL 38591; MLST type: 191) FJ985379 FD_01194_ FOSC 4 NRRL26178 99.807 99.8 19221 FOSC (NRRL 38545; MLST type: 222) FJ985409 FD_01375_ F. oxysporum 100 100 19239 FOSC (NRRL 38545; MLST type: 222) FJ985409 FD_01375_ F. oxysporum 100 100 19240 FOSC (NRRL 26408; MLST type: 65) FJ985289 FD_00142_ FOSC 65 NRRL26408 100 100 19242 FOSC (NRRL 26408; MLST type: 65) FJ985289 FD_01695_FIESC 5-f NRRL45997 100 100 21570 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 20526 F. oxysporum f. sp. cubense CBS 102031 FJ985331, N8 FD_00801_ FOSC 231 NRRL38885 99.182 99.18 20529 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 20530 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 99.66 20531 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 20532 F. oxysporum f. sp. lini CBS 176.33 FJ985344, N8 FD_01375_ F. oxysporum 99.674 99.51 20534 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 20537 F. oxysporum f. sp. raphani CBS 488.76 FJ985273, N8 FD_01216_ FOSC 2 NRRL20433 98.366 98.22 20538 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 20539 F. oxysporum f. sp. raphani CBS 488.76 FJ985273, N8 FD_01216_ FOSC 2 NRRL20433 100 100 20540 F. oxysporum f. sp. cubense CBS 102018 FJ985326, N8 FD_01191_ FOSC 17 NRRL22550 99.642 99.64 20545 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 99.678 99.52 20704 F. oxysporum f. sp. lini CBS 216.49 FJ985349 , N8 FD_01618_ FOSC 9-b NRRL13402 100 100 20705 F. oxysporum f. sp. dianthi CBS 416.90 FJ985284, N8 FD_01249_ FOSC 46 NRRL28401 99.651 99.6 20711 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 20715 F. oxysporum f. sp. perniciosum CBS 794.70 AF008506, N8 FD_00619_ FOSC 17 NRRL38344 99.338 99.35 20716 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 20724 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 20725 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 20730 FOSC (NRRL 36357; MLST type: 159) FJ985349, N1 FD_01194_ FOSC 4 NRRL26178 100 100 20732 FOSC (NRRL 38591; MLST type: 191) FJ985379, N1 FD_01194_ FOSC 4 NRRL26178 100 100 20733 FOSC (NRRL 36357; MLST type: 159) FJ985349, N1 FD_01194_ FOSC 4 NRRL26178 100 100 20737 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 99.833 99.83 20743 FOSC (NRRL 22519; MLST type: 4) FJ985266 , N1 FD_01194_ FOSC 4 NRRL26178 98.618 97.9 20744 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 99.836 99.67 20746 F. oxysporum f. sp. cubense CBS 102031 FJ985331, N8 FD_00801_FOSC 231 NRRL38885 99.466 99.3 20751 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 99.83

Table 4.2. Continued

20752 F. oxysporum f. sp. lini CBS 216.49 FJ985349 N8 FD_01194_ FOSC 4 NRRL26178 100 100 20757 F. oxysporum f. sp. melonis CBS 420.90 FJ985357, N8 FD_01375_F. oxysporum 100 100 20775 FOSC (NRRL 38591; MLST type: 191) FJ985379, N1 FD_01194_ FOSC 4 NRRL26178 100 100 20776 FOSC (NRRL 36286; MLST type: 154) FJ985344, N1 FD_01375_ F. oxysporum 99.677 99.52 21037 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 21059 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 99.663 99.66 21064 F. oxysporum f. sp. melonis CBS 420.90 FJ985357, N8 FD_01375_ F. oxysporum 97.9 97.93 21076 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 98.347 98.15 21095 FOSC (NRRL 36357; MLST type: 159) FJ985349, N1 FD_00785_ FOSC 191 NRRL38591 100 100 21254 F. oxysporum f. sp. melonis CBS 420.90 EF056790, N8 FD_00801_ FOSC 231 NRRL38885 99.52 99.53 21291 FOSC (NRRL 36357; MLST type: 159) FJ985349, N1 FD_00785_ FOSC 191 NRRL38591 100 100 21304 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_00785_ FOSC 191 NRRL38591 100 99.51 21305 FOSC (NRRL 36471; MLST type: 166) FJ985356, N1 FD_01194_ FOSC 4 NRRL26178 99.306 98.85 21311 FOSC (NRRL 38591; MLST type: 191) FJ985379, N1 FD_01194_ FOSC 4 NRRL26178 99.841 99.69 21313 FOSC (NRRL 38308; MLST type: 189) FJ985377, N1 FD_01269_ FOSC 87 NRRL26960 99.831 99.83 21368 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 99.837 99.22 21372 F. oxysporum CBS 132481 OPC055EF1, N8 FD_00226_ F. oxysporum 100 99.5 21373 FOSC (NRRL 38326; MLST type: 196) FJ985384, N1 FD_00785_ FOSC 191 NRRL38591 99.673 99.68 21374 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 97.107 97.1 21375 FOSC (NRRL 38326; MLST type: 196) FJ985384, N1 FD_00785_ FOSC 191 NRRL38591 99.668 99.67 19244 F. oxysporum f. sp. raphani CBS 488.76 FJ985273, N8 FD_01216_ FOSC 2 NRRL20433 99.806 99.8 22778 F. oxysporum f. sp. cubense CBS 102018 FJ985326, N8 FD_01191_ FOSC 17 NRRL22550 99.633 100 22779 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC NRRL26178 100 100 22780 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 22781 F. oxysporum f. sp. raphani CBS 488.76 FJ985273, N8 FD_01216_ FOSC 2 NRRL20433 100 100 22782 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 22788 F. oxysporum f. sp. melonis CBS 420.90 FJ985357, N8 FD_01375_ F. oxysporum 100 100 22794 F. oxysporum f. sp. melonis CBS 420.90 FJ985357, N8 FD_01375_ F. oxysporum 99.832 99.67 22798 F. oxysporum f. sp. melonis CBS 420.90 FJ985357, N8 FD_01375_ F. oxysporum 100 100 22801 F. oxysporum f. sp. lini CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 99.834 99.83 PPRI = Living fungal collection of the South Africa National Collection of Fungi, Plant Protection Research Institute, ARC, Pretoria, South Africa; MLST = Multiloci sequence type; BLAST = Basic Local Alignment Search Tool; NRRL = Agricultural Research Service culture collection, United States Department of Agriculture, Illinois, United States of America; CBS = Centraalbureau voor Schimmelcultures, Filamentous fungi and Yeast Collection, Netherlands.

PPRI NO. MYCOBANK DATABASE FUSARIUM- ID DATABASE MYCOBANK % FUSARIUM – ID SIMILARITY % SIMILARITY 19149 FFSC (NRRL 20476; MLST type: none), N1 FD_01179_FFSC NRRL25221 100 100 19224 FFSC (NRRL 26756; MLST type: none), N1 FD_01151_ FFSC NRRL26061 99.58 99.57 19231 FFSC (NRRL 20476; MLST type: none), N1 FD_01179_ FFSC NRRL25221 98.459 98.64 21033 FFSC (NRRL 20476; MLST type: none), N1 FD_01179_FFSC NRRL25221 98.4 98.4 21053 FFSC (NRRL 20476; MLST type: none), N1 FD_01179_FFSC NRRL25221 98.689 98.68 21055 FFSC (NRRL 20476; MLST type: none), N1 FD_01179_FFSC NRRL25221 97.642 98.24 21071 FFSC (NRRL 20476; MLST type: none), N1 FD_01179_FFSC NRRL25221 97.756 97.9 21253 FFSC (NRRL 20476; MLST type: none), N1 FD_01179_FFSC NRRL25221 97.709 97.7 21272 FFSC (NRRL 20476; MLST type: none), N1 FD_01179_FFSC NRRL25221 98.243 98.55 21300 FFSC (NRRL 20476; MLST type: none), N1 FD_01179_FFSC NRRL25221 98.24 98.24 21306 FFSC (NRRL 20476; MLST type: none), N1 FD_01179_FFSC NRRL25221 98.265 97.55 22756 FFSC (NRRL 20476; MLST type: none), N1 FD_01179_FFSC NRRL25221 98.428 98.31 19184 F. nygamai CBS 140.95 HM347121, N8 FD_01855_Fusarium sp. 99.793 99.79 19196 F. nygamai CBS 140.95 HM347121, N8 FD_01855_Fusarium sp. 99.406 99.4 19219 F. nygamai CBS 140.95 HM347121, N8 FD_01855_Fusarium sp. 99.803 99.8 19229 F. nygamai CBS 140.95 HM347121, N8 FD_01855_Fusarium sp. 99.802 99.8 20533 F. nygamai CBS 454.97 AF160309, N8 FD_01145_FFSC NRRL26793 98.51 98.5 20740 F. nygamai CBS 140.95 HM347121, N8 FD_01855_Fusarium sp. 98.901 98.9 20741 F. nygamai CBS 454.97 AF160309, N8 FD_01145_FFSC NRRL26793 98.801 98.8 21051 F. nygamai CBS 140.95 HM347121, N8 FD_01855_ Fusarium sp. 99.836 99.67 21056 F. nygamai CBS 140.95 HM347121, N8 FD_01855_ Fusarium sp. 98.313 99.07 21098 F. nygamai CBS 140.95 HM347121, N8 FD_01855_ Fusarium sp. 99.205 99.2 21255 F. nygamai CBS 140.95 HM347121, N8 FD_01855_ Fusarium sp. 99.838 99.83 21265 F. nygamai CBS 140.95 HM347121, N8 FD_01855_ Fusarium sp. 98.313 99.07 21294 F. nygamai CBS 140.95 HM347121, N8 FD_01855_ Fusarium sp. 99.67 99.2 21308 F. nygamai CBS 140.95 HM347121, N8 FD_01855_ Fusarium sp. 98.901 99.22 21573 F. nygamai CBS 140.95 HM347121, N8 FD_01855_ Fusarium sp. 99.678 99.83 22759 F. nygamai N8 CBS 140.95 HM347121, N8 FD_01855_ Fusarium sp. 99.537 99.53 22785 F. nygamai N8 CBS 140.95 HM347121, N8 FD_01855_ Fusarium sp. 100 100 22795 F. nygamai N8 CBS 454.97 AF160309, N8 FD_01145_FFSC NRRL26793 98.556 98.55 22744 F. burgessii CBS 125537 CBS 125537, N8 FD_01329_F. beomiforme 93.825 88.12 22787 F. burgessii CBS 125537 CBS 125537, N8 FD_00942_F. hostae 92.883 90.23 21048 F. burgessii CBS 125537 CBS 125537, N8 FD_01854_ Fusarium sp. 93.073 92.12 21062 F. burgessii CBS 125537 CBS 125537, N8 FD_01854_ Fusarium sp. 93.606 91.45 21070 F. burgessii CBS 125537 CBS 125537, N8 FD_01854_ Fusarium sp. 93.388 89.35 21072 F. burgessii CBS 125537 CBS 125537, N8 FD_01329_ F. beomiforme 90.508 95.97

Table 4.3. Continued

21079 F. burgessii CBS 125537 CBS 125537, N8 FD_01847_ F. concolor 93.517 89.52 21097 F. burgessii CBS 125537 CBS 125537, N8 FD_01854_ Fusarium sp. 93.606 91.45 21263 F. burgessii CBS 125537 CBS 125537, N8 FD_01854_ Fusarium sp. 92.773 90.57 20525 F. polyphialidicum CBS 463.91 HM347119, N8 FD_01854_ Fusarium sp. 94.464 94.87 20543 F. polyphialidicum CBS 463.91 HM347119, N8 FD_01854_ Fusarium sp. 91.528 95.82 20779 F. polyphialidicum CBS 463.91 HM347119, N8 FD_01854_ Fusarium sp. 94.479 94.47 21257 F. polyphialidicum CBS 463.91 HM347119, N8 FD_01854_ Fusarium sp. 99.676 99.2 22800 F. polyphialidicum CBS 463.91 HM347119, N8 FD_01854_ Fusarium sp. 96.205 96.2 20721 F. caucasicum CBS 179.35 DQ247543, N8 FD_01443_FOSC 3+4-aa NRRL32343 98.786 99.08 21092 F. caucasicum CBS 179.35 DQ247543, N8 FD_01443_FOSC 3+4-aa NRRL32343 100 99.69 21378 F. caucasicum CBS 179.35 DQ247543, N8 FD_01443_FOSC 3+4-aa NRRL32343 99.445 99.44 20759 F. inflexum CBS 716.74 AF008479, N8 FD_01216_FOSC 2 NRRL20433 99.842 99.84 20784 F. inflexum CBS 716.74 AF008479, N8 FD_01216_FOSC 2 NRRL20433 99.681 99.68 20719 F. sporotrichioides CBS 178.64 EU128186, N8 FD_01306_ F. venenatum 90.928 92.12 19183 F. sporotrichioides CBS 178.64 EU128186, N8 FD_01305_ F. armeniacum 93.097 99.01 21574 F. redolens CNRMA12.291, N5 FD_01081_ F. redolens 99.843 99.84 20541 F. boothii CBS 110250 AF212443, N8 FD_01306_ F. venenatum 88.83 91.77 19124 RESTRICTED ITEM FD_01014_ F. commune 100 100 19191 CBS 214.49 FJ985348, N8 FD_01375_ F. oxysporum 100 100 20760 CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 20763 CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 100 21078 CBS 216.49 FJ985349, N8 FD_01194_ FOSC 4 NRRL26178 100 99.83 21303 CBS 159.57 FJ985339, N8 FD_00781_ FOSC 225 NRRL38587 99.485 99.21 21312 CBS 216.49 FJ985349, N8 FD_00785_ FOSC 191 NRRL38591 99.841 99.69 21376 CBS 216.49 FJ985349, N8 FD_00785_ FOSC 191 NRRL38591 99.659 99.67 PPRI = Living fungal collection of the South Africa National Collection of Fungi, Plant Protection Research Institute, ARC, Pretoria, South Africa; MLST = Multiloci sequence type; BLAST = Basic Local Alignment Search Tool; NRRL = Agricultural Research Service culture collection, United States Department of Agriculture, Illinois, United States of America ; CBS = Centraalbureau voor Schimmelcultures, Filamentous fungi and Yeast Collection, Netherlands.

Table 4.4: Fusarium brachygibbosum nucleotide BLAST results from the MYCOBANK and FUSARIUM-ID databases. The isolate indicated in bold signify contrasting nBLAST results from the two databases.

PPRI NO. MYCOBANK DATABASE FUSARIUM-ID DATABASE MYCOBANK % FUSARIUM-ID % SIMILARITY SIMILARITY 19093 F. brachygibbosum (NRRL 34033; MLST type: none), N1 FD_01841_ F. brachygibbosum 91.756 92.95 19116 F. brachygibbosum (NRRL34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.348 94.71 19118 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.461 93.46 19125 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.635 94.63 19133 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.695 94.54 19134 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.435 94.36 19135 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.613 93.61 19138 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 95.032 95.03 19139 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 95.011 95.01 19143 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.775 93.92 19145 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.291 94.29 19148 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.389 94.54 19150 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.147 94.07 19154 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.467 94.46 19161 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.318 94.31 19162 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.035 94.03 19166 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.572 94.57 19167 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.291 94.6 19168 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.617 94.61 19169 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.572 94.57 19170 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 92.812 93.62 19175 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.454 94.38 19188 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.583 94.58 19232 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.422 94.74 19233 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.303 94.61 19236 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.628 94.79 20527 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.729 93.8 20720 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.366 93.85 20729 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 92.8 93.61 20734 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.949 93.94 20745 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 92.916 93.31 20755 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.087 93.49 21041 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.058 93.31 21042 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.613 93.61 21049 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.974 93.91

Table 4.4. Continued

21050 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01306_ F. venenatum 85.757 89.8 21052 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.631 95.01 21058 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.939 93.93 21061 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.32 93.61 21063 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.949 94.46 21081 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.991 93.99 21082 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.581 94.5 21084 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.754 94.67 21086 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.584 93.58 21087 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.92 94.23 21088 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.333 93.07 21093 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.951 95.23 21094 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 95.04 95.59 21100 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.62 94.74 21101 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.904 95.43 21102 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.175 94.1 21275 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.312 93.31 21276 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.569 95.1 21277 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.003 94.12 21290 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.762 94.68 21292 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.462 95.25 21296 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 100 94.76 21297 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.703 94.62 21301 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.991 93.99 21307 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.518 94.44 22744 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.435 93.66 22746 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.563 94.48 22747 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.798 93.79 22760 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.74 93.74 22771 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.701 93.65 22773 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.698 93.5 22774 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 93.779 93.77 22775 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.108 94.03 22776 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 92.912 93.69 22786 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.391 94.39 22743 F. brachygibbosum (NRRL 34033; MLST type: none) , N1 FD_01841_ F. brachygibbosum 94.018 94.01 PPRI = Living fungal collection of the South Africa National Collection of Fungi, Plant Protection Research Institute, ARC, Pretoria, South Africa; MLST = Multiloci sequence type; BLAST = Basic Local Alignment Search Tool; NRRL= Agricultural Research Service culture collection, United States Department of Agriculture, Illinois, United States of America.

Table 4.5: Fusarium solani species complex (FSSC) nucleotide BLAST results from the MYCOBANK and FUSARIUM-ID databases.

PPRI NO. MYCOBANK DATABASE FUSARIUM-ID DATABASE MYCOBANK % FUSARIUM- SIMILARITY ID % SIMILARITY 19147 FSSC (NRRL 32737; MLST type: 5-k) DQ247057, N1 FD_01529_ FSSC 5-k NRRL32737 99.804 99.8 19200 NOT FOUND ON DATABASE FD_01567_ FSSC 12-e NRRL32821 NA 96.03 19202 FSSC (NRRL 32810; MLST type: 5-m) DQ247118, N1 FD_01457_ FSSC 3+4-ee NRRL32505 99.416 99.54 19206 NOT FOUND ON DATABASE FD_01567_ FSSC 12-e NRRL32821 NA 94.03 19238 NOT FOUND ON DATABASE FD_01567_ FSSC 12-e NRRL32821 NA 94.28 19246 FSSC (NRRL 28559; MLST type: 3+4-o) DQ246900 F. falciforme, N1 FD_01372_ F. solani 100 99.8 20523 FSSC (NRRL 22783; MLST type: 5-f) DQ246851, N1 FD_01489_FSSC 3+4-pp NRRL32720 99.83 100 20535 FSSC CNRMA14.336, N5 FD_01524_ FSSC 5-g NRRL25388 99.662 99.66 20544 FSSC (NRRL 32810; MLST type: 5-m) DQ247118, N1 FD_01531_ FSSC 5-m NRRL32810 99.698 99.84 20549 FSSC (NRRL 22779; MLST type: 5-e) DQ246848, N1 FD_01371_ F. solani 99.382 99.39 20710 FSSC (NRRL 32484; MLST type: 5-j) DQ246982, N1 FD_01524_ FSSC 5-g NRRL25388 99.371 99.37 20712 FSSC (NRRL 22783; MLST type: 5-f) DQ246851 FD_01519_ FSSC 5-d NRRL43681 99.832 99.83 20717 FSSC CNRMA14.216, N5 FD_01528_ FSSC 5-j NRRL32484 99.668 99.66 20718 FSSC (NRRL 32492; MLST type: 5-c) DQ246990 , N1 FD_01519_ FSSC 5-d NRRL43681 99.837 99.83 20722 FSSC (NRRL 22779; MLST type: 5-e) DQ246848, N1 FD_01489_ FSSC 3+4-pp NRRL32720 99.845 100 20742 FSSC CNRMA15.557, N5 FD_01519_ FSSC 5-d NRRL43681 99.391 99.39 20753 FSSC (NRRL 22783; MLST type: 5-f) DQ246851, N1 FD_01519_ FSSC 5-d NRRL43681 99.691 99.69 20777 FSSC CNRMA15.557, N5 FD_01527_ FSSC 5-i NRRL31168 99.848 99.84 20778 FSSC (NRRL 32810; MLST type: 5-m) DQ247118, N1 FD_01531_ FSSC 5-m NRRL32810 99.849 99.84 20781 FSSC CNRMA15.557, N5 FD_01527_ FSSC 5-i NRRL31168 100 100 20785 FSSC CNRMA15.557, N5 FD_01371_ F. solani 98.778 99.12 21044 FSSC CNRMA15.557, N5 FD_01372_ F.solani 95.387 96.91 21074 FSSC (NRRL 28559; MLST type: 3+4-o) DQ246900 F. falciforme, N1 FD_01489_ FSSC 3+4-pp NRRL32720 95.11 99.8 21090 FSSC CNRMA14.336, N5 FD_01524_ FSSC 5-g NRRL25388 99.558 99.55 21096 FSSC CNRMA15.557 N5 FD_01372_ F. solani 99.398 99.85 21256 FSSC (NRRL 32810; MLST type: 5-m) DQ247118, N1 FD_01372_F. solani 96.148 96.82 21260 FSSC CNRMA14.216, N5 FD_01373_ F. solani 99.698 99.54 21262 FSSC CNRMA15.557, N5 FD_01372_ F. solani 99.256 99.85 21268 FSSC (NRRL 28559; MLST type: 3+4-o) DQ246900 F. falciforme, N1 FD_01489_ FSSC 3+4-pp NRRL32720 99.364 99.22 21295 FSSC CNRMA14.684, N5 FD_01519_ FSSC 5-d NRRL43681 99.843 99.68 21298 FSSC (NRRL 28559; MLST type: 3+4-o) DQ246900, N1 FD_01489_ FSSC 3+4-pp NRRL32720 98.183 99.64 21299 FSSC (NRRL 32738; MLST type: 3+4-tt) DQ247058 F. falciforme , N1 FD_01497_ FSSC 3+4-tt NRRL32738 99 99.32 21310 FSSC CNRMA14.684, N5 FD_01372_ F. solani 99.02 99.52 21369 FSSC CNRMA14.684, N5 FD_01371_ F. solani 99.095 99.09 21576 FSSC (NRRL 22783; MLST type: 5-f) DQ246851, N1 FD_01519_ FSSC 5-d NRRL43681 99.839 99.84

Table 4.5. Continued

22755 FSSC (NRRL 32505; MLST type: 3+4-ee) DQ247002 F. falciforme, N1 FD_01457_ FSSC 3+4-ee NRRL32505 99.846 99.54 22783 FSSC (NRRL 32778; MLST type: 3+4-yy) DQ247088 F. falciforme, N1 FD_01492 _FSSC 3+4-r NRRL28565 99.838 99.83 23239 FSSC (NRRL 28565; MLST type: 3+4-r) DQ246906 F. falciforme, N1 FD_01492 _FSSC 3+4-r NRRL28565 99.841 99.84 22793 FSSC (NRRL 32484; MLST type: 5-j) DQ246982, N1 FD_01524_ FSSC 5-g NRRL25388 99.679 99.67 22799 FSSC (NRRL 28565; MLST type: 3+4-r) DQ246906 F. falciforme, N1 FD_01492 _FSSC 3+4-r NRRL28565 99.842 99.54 22942 FSSC (NRRL 32810; MLST type: 5-m) DQ247118, N1 FD_01531_ FSSC 5-m NRRL32810 99.679 99.53 PPRI = Living fungal collection of the South Africa National Collection of Fungi, Plant Protection Research Institute, ARC, Pretoria, South Africa; MLST = Multiloci sequence type; BLAST = Basic Local Alignment Search Tool; NRRL = Agricultural Research Service culture collection, United States Department of Agriculture, Illinois, United States of America; NA = Percentage similarity not available due to there being no match of the query sequence on the database.

PPRI NO. MYCOBANK DATABASE FUSARIUM-ID DATABASE MYCOBANK % FUSARIUM-D % SIMILARITY SIMILARITY 19117 FCSC (NRRL 43630; MLST type: 2-a) GQ505426, N1 FD_01717_ FCSC 2-a NRRL43630 98.207 98.2 19119 FCSC (NRRL 13338; MLST type: 4-a) GQ505402 F. nelsonii, N1 FD_01724_ FCSC 4-a NRRL13338 99.697 99.69 19132 FCSC (NRRL 43630; MLST type: 2-a) GQ505426, N1 FD_01724_ FCSC 4-a NRRL13338 99.605 99.53 19146 FCSC (NRRL 43630; MLST type: 2-a) GQ505426, N1 FD_01717_ FCSC 2-a NRRL43630 98.232 99.6 19156 FCSC (NRRL 43630; MLST type: 2-a) GQ505426, N1 FD_01717_ FCSC 2-a NRRL43630 99.385 98.23 19157 FCSC (NRRL 36539; MLST type: 1-k) GQ505422, N1 FD_01717_ FCSC 2-a NRRL43630 97.753 99.38 19173 FCSC (NRRL 13338; MLST type: 4-a) GQ505402 F. nelsonii, N1 FD_01844_ FCSC 1-i NRRL45992 99.696 97.74 19177 FCSC (NRRL 13338; MLST type: 4-a) GQ505402, N1 FD_01724_ FCSC 4-a NRRL13338 99.63 99.69 19179 FCSC (NRRL 13338; MLST type: 4-a) GQ505402, N1 FD_01724_ FCSC 4-a NRRL13338 99.414 99.63 19192 FCSC (NRRL 43630; MLST type: 2-a) GQ505426, N1 FD_01724_ FCSC 4-a NRRL13338 98.246 99.41 19213 FCSC (NRRL 43630; MLST type: 2-a) GQ505426, N1 FD_01717_ FCSC 2-a NRRL43630 100 98.24 19228 FCSC (NRRL 43630; MLST type: 2-a) GQ505426, N1 FD_01717_ FCSC 2-a NRRL43630 98.821 100 19228 FCSC (NRRL 43630; MLST type: 2-a) GQ505426, N1 FD_01717_ FCSC 2-a NRRL43630 98.821 100 19237 FCSC (NRRL 13338; MLST type: 4-a) GQ505402, N1 FD_01717_ FCSC 2-a NRRL43630 99.594 98.82 19245 FCSC (NRRL 13338; MLST type: 4-a) GQ505402, N1 FD_01724_ FCSC 4-a NRRL13338 99.609 99.59 20754 FCSC (NRRL 13444; MLST type: 2-a) GQ505403, N1 FD_01724_ FCSC 4-a NRRL13338 99.522 99.6 20782 FCSC (NRRL 13444; MLST type: 2-a) GQ505403, N1 FD_01717_ FCSC 2-a NRRL43630 99.347 99.52 22938 FCSC (NRRL 13338; MLST type: 4-a) GQ505402 F. nelsonii, N1 FD_01717_ FCSC 2-a NRRL43630 99.531 99.34 PPRI = Plant Protection Research Institute; MLST = Multiloci sequence type; BLAST = Basic Local Alignment Search Tool; NRRL= Agricultural Research Service culture collection, United States Department of Agriculture, Illinois, United States of America.

4.7. Chapter figures Fungal isolation

large soil particles small soil particles 100 90 80 70 60 50 40 30 20 10 0 Site 1 Site 2 Site 3 Site 4

Figure 4.1.1: Numbers of cultures from large soil particles compared to the small soil particles obtained at the Willem Pretorius nature reserve from the four sites.

Figure 4.1.2: Germinating spores of a Fusarium species on water agar visualized under a stereo microscope.

1 2 3 4 5

6 7 8 9 10

Figure 4.1.3: Colony pigmentation and morphology of Fusarium species on Potato dextrose agar (PDA) after 10 days of growth under dual light (12 hours’ normal light and 12 hours UV light) conditions. For each pair, the plate on top illustrates the top surface of the plate and the bottom plate illustrates the bottom surface of the plate. The cultures represent isolates identified as; 1. F. fujikuroi (PPRI 19149) 2. F. burgessii (PPRI 19129) 3.

F. chlamydosporum (PPRI 19213) 4. F. solani (PPRI 20535) 5. F. acuminatum (PPRI 19235) 6. F. nygamai (PPRI 20740) 7. F. oxysporum (PPRI

20746) 8. F. equiseti (PPRI 21571) 9. F. brachygibbosum (PPRI 21297) 10. F. redolens (PPRI 21574).

F H B D

A C E G I

K O

J L M N P Q

Figure 4.2: The types and arrangement of the observed morphological characters for the five selected Fusarium species. A – E:

Macroconidia of F. solani (A), F. brachygibbosum (B), F. equiseti (C), F. oxysporum (D) and, F. fujikuroi (E). F – I: Microconidia of

F. brachygobbosum (F), F. fujikuroi (G), F. oxysporum (H) and, F. solani (I). J, Microconidia borne on polyphialides in situ on CLA (F. fujikuroi). K, Microconidia produced in chains in situ on CLA (F. fujikuroi). L – N; Microconidia borne on monophialides in situ on CLA, F. fujikuroi (L), F. oxysporum (M) and F. solani (N). O – Q Chlamydospores of F. brachygibbosum (O), F. equiseti (P) and, F. oxysporum in situ on CLA (Q). A – Q, scale bar = 20µm.

DNA extraction and PCR

Figure 4.3.1: Electrophoretogram of a subset of DNA extracted from the isolated Fusarium species. The genomic DNA bands were visualized on a 2 % agarose gel. Lane M: 100 bp DNA ladder. Lane 1: PPRI 19120, lane 2: PPRI 19122, lane 3: PPRI 19123, lane 4: PPRI 19127, lane 5: PPRI 19128, and lane 6: PPRI 19137.

800bp

700bp

Figure 4.3.2: PCR amplification of the TEF-1α gene for the same six Fusarium samples as in figure 4.3.1. PCR products were visualized on a 2 % agarose gel. Lane M: 100 bp DNA ladder. Lane 1: PPRI 19120, lane 2: PPRI 19122, lane 3: PPRI 19123, lane 4: PPRI19127, lane 5: PPRI 19128, lane 6: PPRI 19137 and lane 7: no-template control (NTC). The fragment size of the amplified gene is ca.700 base pairs.

A B

Figure 4.4.1a&b: Different species complexes and species of the genus Fusarium identified via nBLAST analysis. A – Mycobank database, B –

FUSARIUM-ID database.

Small soil particles Large soil particles Small soil particles Large soil particles

60 60

50 50

40 40

30 30

20 20

10 10

0 0

A B

Figure 4.4.2a&b: Numbers of Fusarium isolates obtained from the two soil particle sizes. A-Mycobank nBLAST results, B-FUSARIUM-ID nBLAST results.

site 1 site 2 site 1 site 2

50 60 45 50 40 35 40 30 25 30 20 20 15 10 10 5 0 0

A B

Figure 4.4.3a&b: Numbers of Fusarium isolates observed at site 1 and site 2 within the Willem Pretorius nature reserve. A-Mycobank nBLAST results, B-FUSARIUM-ID nBLAST results.

Site 3 Site 4 site 3 site 4

25 25

20 20

15 15

10 10

5 5

0 0

A B

Figure 4.4.4a&b: Numbers of Fusarium isolates observed at site 3 and site 4 within the Willem Pretorius nature reserve. A-Mycobank nBLAST results, B-FUSARIUM-ID nBLAST results

Phylogenetic Analysis

NRRL13459 F. concolor 100 PPRI 21045 PPRI 19215 Clade 1 PPRI 21060 87 NRRL 34002 NRRL 13335 NRRL 32865 100 NRRL 3400 NRRL 1337 NRRL 32866 NRRL 32867 NRRL 34003 NRRL 31160 NRRL 32175 NRRL 32181 NRRL 32869 NRRL 32994 NRRL 32995 80 NRRL 32996 94 NRRL 34001 78 NRRL 34006 NRRL 34007 Clade 2 (MLST 15-18; MLST 20-28) NRRL 34008 NRRL 34010 NRRL 34011 NRRL 32182 NRRL 34004 NRRL 34056 78 NRRL 34059 NRRL 31167 NRRL 32522 100 NRRL 32864 NRRL 34070 95 NRRL 20722 NRRL 28577 100/ 99 NRRL 22244 100 NRRL 32868 NRRL 32993 NRRL 26417 100/ 95 NRRL 28714 PPRI 20731 PPRI 20714 PPRI 20761 81 PPRI 21572 Clade 3 (MLST 10) 73 NRRL 3020 NRRL 3214 PPRI 20786 PPRI 21035 NRRL 5537 NRRL 32997 100 PPRI 20790 NRRL 36269 NRRL 6548 Clade 4 (MLST 12) 80/ 98 NRRL 26921 NRRL 31011 PPRI 19122 PPRI 19142 PPRI 19163 PPRI 19174 PPRI 19181 PPRI 19193 PPRI 19199 PPRI 19214 PPRI 19226 PPRI 22772 PPRI 22777 PPRI 22802 PPRI 20542 PPRI 20703 PPRI 20706 PPRI 22765 PPRI 19152 PPRI 19195 PPRI 19242 PPRI 19208 PPRI 19225 PPRI 22763 PPRI 20528 PPRI 20762 PPRI 22789 PPRI 20736 PPRI 20713 PPRI 22797 PPRI 19243 PPRI 19165 88 PPRI 22764 PPRI 22751 PPRI 19182 Clade 5 (MLST 5) PPRI 19151 PPRI 22748 PPRI 20749 PPRI 19198 NRRL 25795 PPRI 19178 PPRI 22762 PPRI 22792 PPRI 19197 PPRI 19137 PPRI 19164 PPRI 19190 PPRI 22752 PPRI 21571 PPRI 20547 PPRI 21091 PPRI 20788 PPRI 20789 PPRI 20791 NRRL 34035 PPRI 21271 PPRI 21034 PPRI 21575 PPRI 21367 PPRI 22758 95/93 NRRL 34037 NRRL 32871 NRRL 34032 100 PPRI 19123 PPRI 19189 PPRI 20707 87 PPRI 20758 100 NRRL 20423 75 NRRL 36123 NRRL 34034 Clade 6 (MLST 1, 3 & 4) NRRL 34039 NRRL 28029 NRRL 36318 100/98 NRRL 20697 NRRL 26419 Clade 7 (MLST 14) NRRL 36136 NRRL 36321 PPRI 19127 PPRI 19180 PPRI 19128 94 PPRI 20756 PPRI 21080 100 PPRI 21258 PPRI 19187 PPRI 22796 PPRI 19141 100 PPRI 20524 PPRI 20546 NRRL 26922 100 PPRI 20783 NRRL 29134 PPRI 19120 Clade 8 (MLST 9) PPRI 19171 PPRI 22757 PPRI 19230 PPRI 20709 PPRI 21089 PPRI 19210 NRRL 13402 80 PPRI 21077 PPRI 21274 PPRI 21099 PPRI 21036 PPRI 20747 83 PPRI 22768 PPRI 19158 Figure 4.5.1: One of the most parsimonious phylogenetic trees of the FIESC, including the isolates collected from Willem Pretorius nature reserve, denoted on the tree with PPRI numbers (black font colour) and inferred from the TEF-1α sequence data, as well as isolates from published work indicated by NRRL collection numbers (red font colour). The tree is rooted by F. concolor (NRRL 13459) (purple font colour). Branch support for both MP (purple font colour) and ML (light blue font colour) are indicated on the tree branches with bootstrap support. The FIESC taxa are assembled90 into clades indicated in the colour blocks. Tree statistics for the FIESC dataset: RI = 0.9028; CI = 0.7301; HI = 0.2699 and, Tree length = 1061.

RBG5445 Fusarium sp. SARD 52659 201 10 1 AUST 556 100/ 99 80 AUST 242 PS2b 86 AUST 695 AUST 629 71 AUST 683 AUST 508 AUST 572 AUST 582 AUST 387 AUST 421 AUST 571 PPRI 20715 92/ 78 PPRI 22778 PPRI 20540 PS1 AUST 122 AUST 589 AUST 642 74 AUST 564 AUST 638 AUST 584 AUST 689 AUST 557 71 AUST 605 PPRI 20705 AUST 214 AUST 172 SARD 44911 258 2 1 AUST 459 AUST 706 AUST 699 SARD 46478 94 13 2 SARD 53079 278 1 1 95 AUST 603 AUST 641 SARD 52654 269 1 1 SARD 46475 208 5 3 SARD 46477 261 1 1 SARD 52646 6 1 1 AUST 142 AUST 378 SARD 46645 22 3 1 97 SARD 46647 264 4 1 AUST 545 AUST 119 70 AUST 697 AUST 293 70 AUST 103 F. sp. conglutinans 256 AUST 693 87 SARD 44926 260 1 1 AUST 217 84 SARD 44914 259 2 1 SARD 52669 271 2 1 SARD 52663 270 4 2 SARD 53088 281 1 1 SARD 52672 272 1 1 AUST 351 AUST 359 PPRI 20526 AUST 84 AUST 713 SARD 52817 275 1 1 PPRI 19240 PPRI 20539 PPRI 19244 SARD 52816 274 6 1 SARD 53113 282 1 1 SARD 46681 19 5 2 PPRI 22781 PPRI 20545 PPRI 19216 PPRI 20744 SARD 52627 266 1 1 PPRI 20730 SARD 52820 276 1 1 PPRI 20531 PPRI 20534 PPRI 19218 AUST 165 PPRI 20775 SARD 44917 209 17 6 PPRI 20732 SARD 44913 191 12 4 SARD 44935 90 41 8 PPRI 20743 PPRI 20724 PPRI 21078 PPRI 20763 PPRI 20760 PPRI 20529 PS2a PPRI 20711 PPRI 20538 PPRI 21570 SARD 52807 273 1 1 SARD 46619 262 1 1 PPRI 21291 PPRI 21368 SARD 53077 277 1 1 SARD 46664 265 1 1 SARD 46679 285 1 1 PPRI 20530 PPRI 22801 PPRI 21037 PPRI 21095 PPRI 22779 PPRI 22780 PPRI 22782 PPRI 20725 PPRI 20716 PPRI 20704 PPRI 21076 PPRI 21304 PPRI 20737 PPRI 20751 PPRI 21305 PPRI 20733 PPRI 20752 PPRI 21311 PPRI 21312 PPRI 21373 PPRI 21375 PPRI 21376 PPRI 21374 F. sp. raphani 254 2 SARD 52647 267 4 3 PPRI 20537 SARD 53081 279 1 1 SARD 53084 280 1 1 AUST 129 PPRI 21254 PPRI 19207 AUST 120 PPRI 19136 SARD 46617 158 10 3 PPRI 20757 AUST 226 SARD 46622 263 1 1 PPRI 22798 PPRI 19221 PPRI 22788 PPRI 22794 PPRI 19191 PPRI 19239 SARD 44930 221 42 7 86 PPRI 21059 PPRI 19217 PPRI 20532 PPRI 20776 PPRI 21313 AUST 696 AUST 610 F. sp. conglutinans 252 SARD 52650 268 1 1 PPRI 20746 87 PPRI 21372 AUST 5

Figure 4.5.2 One of the most parsimonious phylogenetic trees of the FOSC, including the isolates collected from Willem Pretorius nature reserve, denoted on the tree with PPRI numbers (black font colour) and inferred from the TEF- 1α sequence data as well as isolates from published work denoted on the tree as SARD (orange font colour), AUST (red font colour) and other isolates (dark orange font colour). The tree is rooted by isolate Fusarium sp. RGB5443. Branch support for both MP (purple font colour) and ML (light blue font colour) are indicated on the tree branches with bootstrap support. The FOSC taxa are assembled into clades indicated in the colour blocks. Tree statistics for the FOSC dataset: RI = 0.9421; CI = 0.8810; HI = 0.1190 and, Tree length = 311. 91

PPRI 22744 PPRI 19129 82 PPRI 21072 PPRI 21079 Clade 1 PPRI 21048 PPRI 21062 PPRI 21097 PPRI 21070 PPRI 21263 PPRI 22787 AF160271 F. pseudocircinatum PPRI 19231 99 PPRI 22756 PPRI 19149 80 AF160268 Fusarium sp. PPRI 21300 86 PPRI 21306 Clade 2 (FFSC) KP083256 F. mundagurra sp. nov. KP083255 F. mundagurra sp. nov. PPRI 21033 99 PPRI 21055 78 PPRI 21272 PPRI 21071 PPRI 21253 PPRI 19196 PPRI 21051 PPRI 21294 PPRI 22759 85 PPRI 19184 PPRI 21573 PPRI 21255 PPRI 20740 Clade 3 (FFSC) PPRI 21308 PPRI 19229 PPRI 19219 89/90 PPRI 21098 PPRI 21056 PPRI 21265 PPRI 22785 AF160273 F. nygamai AF160272 F. lactis AF160309 Fusarium sp. 77 73 PPRI 20741 PPRI 22795 86 PPRI 20533 82 AF160266 F. napiforme KF466423 F. ramigenum AF160263 F. pseudonygamai Clade 4 (FFSC) AF160265 F. brevicatenulatum KP083263 Fusarium sp. KP083264 Fusarium sp. KP083265 Fusarium sp. 100 AF160304 Fusarium sp. KP083251 Fusarium sp. 97/71 KP083252 Fusarium sp. AF160276 F. acutatum AF160302 Fusarium sp. AF160274 F. phyllophilum 99 AF160275 F. udum AF160301 Fusarium sp. AF160289 F. subglutinans AF160296 Fusarium sp. AF160307 Fusarium sp. AF160308 Fusarium sp. 100 AF160305 Fusarium sp. AF160298 Fusarium sp. AF160300 Fusarium sp. Clade 5 (FFSC) AF160292 F anthophilum 72 AF160291 F. succisae AF160290 F. bactridioides KF466415 F bulbicola EF107132 Fusarium sp. 88/82 AF160293 F. begoniae EU574681 Fusarium sp. EU574682 Fusarium sp. AF160299 Fusarium sp. 100/96 AF160310 Fusarium sp. AF160311 Fusarium 10 AF160277 F. dlaminii sp.PPRI 19224 AF160303 Fusarium sp. AF160306 Fusarium sp. 91 AF160286 Fusarium sp. AF160282 F. concentricum Clade 6 (FFSC) 77 AF160283 Fusarium sp. AF160284 Fusarium sp. AF160285 F. globosum AF160287 Fusarium sp. AF160288 Fusarium sp. AF160312 F. oxysporum 100 PPRI 19191 PPRI 20759 PPRI 20784 86 AF331814 F. inflexum 91 PPRI 21078 PPRI 20760 Clade 7 (FOSC) PPRI 21376 PPRI 20763 PPRI 21312 97 AY320087 F. foetens JF740825 F. foetens 100 AF008513 Fusarium sp. PPRI 19124 Clade 8 (F. commune) HM804942 F commune 10 AF008514 Fusarium sp. KP083260 F. newnesense sp. nov. Clade 9 (F. newnesense sp.) 82 /99 KP083261 F. newnesense sp. nov KP083262 F. newnesense sp. nov nov. 100 AF331817 F hostae PPRI 21574 Clade 10 (FRSC) 100 AF331816 F redolens PPRI 22800 98 PPRI 20525 PPRI 20779 Clade 11 PPRI 21257 PPRI 20543 DQ295144 F. polyphialidicum GQ505588 Fusarium sp. PPRI 19183 GQ856949 F palustre GQ915500 Fusarium sp. 94 KP083253 F. goolgardi sp. nov. KP083254 F. goolgardi sp. nov. 100 AJ420818 F. sporotrichioides 70 HM744683 F. sibiricum HM744684 F. sibiricum Clade 12 (FSAMSC) AJ427272 F. langsethiae 92 JN541056 F. pseudograminearum 8 AJ543541 F. culmorum 6 JN541063 F. cerealis 93 AJ420839 F. poae AJ427274 F. kyushuense PPRI 20541 100 PPRI 20719 89 KP083248 F. aywerte EF107155 F. tjaynera sp. nov. EF107151 F. tjaynera sp. nov. EF107152 F. tjaynera sp. nov. Clade 13 (FASC) EF107150 F. tjaynera sp. nov. KP083266 F. tjaynera sp. nov. 100 GQ505402 F. nelsonii GU250579 Fusarium sp. Clade 14 (FCSC) 100 PPRI 21378 PPRI 20721 Clade 15 PPRI 21092 100 PPRI 22745 PPRI 19235 PPRI 19153 Clade 16 100 PPRI 21053 PPRI 21303

Figure 4.5.3: One of the most parsimonious phylogenetic trees of the genus wide dataset, including the isolates collected from Willem Pretorius nature reserve, denoted on the tree with PPRI numbers (black font colour) and inferred from the TEF-1α sequence data as well as isolates from published work denoted in accession numbers (red font colour). The midpoint rooting method was used to root the genus wide dataset. Branch support for both MP (purple font colour) and ML (light blue font colour) are indicated on the tree branches with bootstrap support. The genus wide taxa are assembled into clades indicated in the colour blocks. Tree statistics for the genus wide dataset: RI = 0.8402; Consistency Index (CI) = 0.4897; Tree length = 2079 and, Homoplasy Index (HI) = 0.5103.

CHAPTER 5

DISCUSSION

Members of the genus Fusarium are well known for their ability to cause plant diseases and the production of toxins detrimental to animal and human health (Marasas et al. 1984; Desjardins, 2006). The kinds of diseases caused are relatively diverse and may include seedling blight, seed rot, ear rot, stalk rot, kernel rot, head blight, wilts, leaf diseases, and cankers (Nayaka et al. 2011). These diseases have had overwhelming sociological and economic impacts towards farmers and communities that depend on such crops as their main source of food (Summerell and Leslie, 2011). Therefore, it is important to gather more knowledge on this genus for the purpose of mitigating the damage they cause. The information on the incidence of Fusarium species in South African soils is limited. This information has been recorded in only two systematic surveys involving both undisturbed and cultivated soils found in different climatic areas (Marasas et al. 1988; Klaasen et al. 1991). These surveys have made most use of the morphological species concept and are yet to introduce the phylogenetic species concepts as a tool to efficiently characterise Fusarium species. Therefore, with the use of these two species concepts, our study aimed at characterising species in the genus Fusarium obtained from undisturbed soils in the South African grassland biome. This was done to further contribute to the information about Fusarium species composition in uncultivated ecosystems of South Africa. Furthermore, it will assist in determining risk evaluation methods for farmers and peri-urban families.

Accurate isolation procedures play a major role on recovering species of the genus Fusarium from soil, plant debris, and diseased plants (Leslie and Summerell, 2006a). According to Summerell et al. (2003) the isolation method used should increase chances of recovering the fungal genus of interest and repress the growth of non-Fusarium species. The method used in this study to isolate Fusarium species from grassland soils proved conducive in only isolating Fusarium species. These were then purified into pure cultures for further characterisation. The taxonomy, physiology and several other applications in fungal studies were all performed on pure cultures (Zhange et al. 2013). All cultures used in the current study were successfully derived from a single germinating spore.

Microbes have the ability to synthesize pigments such as anthraquinones, carotenoids, flavonoids, quinines, etc. It is the anthraquinone compounds from which the different colours of fungi are derived (Sharma et al. 2012). According to Leslie and Summerell (2006a), the variety of pigments that Fusarium species synthesize on PDA media determine their physical colony morphology. Furthermore, Leslie and Summerell (2006a) indicated that the type of media and growth conditions under which the cultures are grown are crucial for the synthesis

94 of pigments to be used for comparisons. Growth conditions could be those related to light exposure as some cultures may be more sensitive to light than others and media conditions such as pH may also influence pigmentation of cultures. In the present study, we observed a variety of colour pigmentation produced by the different species of Fusarium on PDA plates. The colours observed were coherent with most findings from published literature.

Morphological characters are the keystone criteria for classifying fungal species (Begerow et. al. 2010). According to Wingfield et al. (2001) and McNeil et al. (2004), the correct identification of species, especially those that are pathogenic to plants, is fundamental for all aspects concerning plant diseases from diverse ecosystems. In the current study, morphological characters of four well described species of the genus Fusarium and one poorly studied species were examined for the purpose of evaluating the basic morphological characters of Fusarium species. Morphological characters of four of these species, F. equiseti, F. fujikuroi, F. oxysporum, and F. solani were consistent with published information on these species. The morphological characters of F. brachygibbosum strains observed in this study were not documented in previous studies suggesting that it may be a new species closely related to F. brachygibbosum. A study by Mirhosseini et al. (2014) reported the first occurrence of F. brachygibbosum on oleander in Iran. They indicated that the morphological characters of this species were similar to the previously described F. brachygibbosum isolate by Padwick. The observed macroconidia were infrequent and dispersed and included three to four septa (25.92x4.43 μm). The microconidia were vaguely curved, fusiform, ovoid, and include zero to two septa (11.05x3.97 μm). These features differ with those observed for isolate PPRI 19213 that was identified via nBLAST as an isolate that shares similarity to F. brachygibbosum. The difference is supported by the fact that the macroconidia of this PPRI isolate were five septate, medium in length (80.0x5.88 µm), wide, and thick-walled at the centre. Although less abundant, the microconidia were relatively large (46x5.48 µm), oval shaped, and had three septa. The observed chlamydospores were in chains but were more oval than spherical in shape. Macroconidia are produced by all species from the genus Fusarium and are the most essential morphological characters employed for identifying Fusarium species, and that in many instances, a culture can be identified to species level solely from the morphology of this spore (Leslie and Summerell 2006a). Our results indicated that this character was observed in all the five species examined, whereas microconidia, which are not produced by all Fusarium species, were produced by all the species whose morphological characters were examined, except for F. equiseti. According to literature, F. equiseti does not produce microconidia (Nirenberg and

O’Donnell, 1998) and their absence alone was therefore an important character (Leslie and Summerell, 2006a). After ten days of incubation, chlamydospores were only observed for F. brachygibbosum, F. equiseti, and F. oxysporum isolates that may have received enough UV light. Most but certainly not all Fusarium species that produce chlamydospores actively and abundantly do so at two to four weeks of incubation when nutrients are depleted or when the culture becomes older (Leslie and Summerell 2006a; Smith, 2007). This may be the reason chlamydospores were not observed in the F. solani isolate, or that the culture may have not been subjected to enough UV light to offset favourable conditions and thereby triggering the production of chlamydospores. This isolate will be re-plated in a future study and will be subjected to precise incubation standards and will be processed at a precise timeframe. The approach of demarcating Fusarium species using morphological characters has presented significant drawbacks. The biggest problem is that the number of distinct morphological characters available is far less than the amount of Fusarium species that need to be characterised (Summerell et al. 2010). Regardless of these shortcomings, the use of morphological characters to describe species is more likely to remain an essential aspect of species concepts for the genus Fusarium because of two important reasons. The first reason is that the morphological criteria is currently still widely used by diagnosticians worldwide; and secondly, the convenience and practical need to quickly and routinely classify several Fusarium cultures has proven to be effective (Summerell et al. 2010).

Our study recovered five Fusarium species complexes and nine or ten species types based on the FUSARIUM-ID and Mycobank databases, respectively. This included several unknown Fusarium spp. In the case of the unknown Fusarium spp. O’Donnell et al. (2010) clarifies that most of the sequences added in these databases are recorded as Fusarium spp. simply because they symbolise unnamed species based on numerous genealogical concordance phylogenetic species recognition (GCPSR) based reports. However, annotations are often incorporated in the accession records in order to aid in identifying phylogenetic species and/or multilocus haplotypes (O’Donnell et al. 2015). The majority of the nucleotide BLAST results from both databases corresponded with the other. However, some isolates gave two contrasting top hits for the same sequence and this could be due to a number of reasons. O’Donnell et al. (2015) advise that taxon names should be scrutinised carefully as homogenous or nearly homogenous sequences added under multiple identities may signify sequence misidentification. Furthermore, these contrasting hits may also be due to dual nomenclature that was observed before the one fungus one name change, for example, Gibberella zeae (Schwein.) Petch and F.

96 graminearum representing the same species. Ultimately, phylogenetic analysis performed in the current study indicated that most of the contrasting identities are closely related species and that some are classified within the same species complex. For example, isolates identified as F. caucasicum in the Mycobank database and as FSSC in the FUSARIUM-ID database are closely related. Fusarium caucasicum and F. solani were previously documented to share similar morphological characters (Capek and Hanc, 1960). Furthermore, two isolates identified as F. sporotrichioides in the Mycobank database were separately identified as F. venenatum and F. armeniacum in the FUSARIUM-ID database. These three species are closely related in that they are grouped within the same species complex, that is, the FSAMSC (Laurence et al. 2016). Another contrasting identification that was resolved includes isolates identified as F. inflexum and the FOSC in the Mycobank and FUSARIUM-ID database, respectively. Fusarium inflexum and F. oxysporum are closely related and reside in the FOSC (O’Donnell et al. 1998b; O’Donnell et al. 2000). The identity of an isolate identified in the current study was restricted in the Mycobank database and identified as F. commune in the FUSARIUM-ID database. This isolate was phylogenetically matched with a described F. commune (HM804942) isolate. Moreover, F. nygamai isolates were identified to species level in the Mycobank database but only to genus level in the FUSARIUM-ID database. These isolates phylogenetically clustered within the FFSC.

Fusarium consists of species that are widely distributed in soils across the world (Burgess, 1981). These species are typically associated with plant debris and organic matter (Rheeder and Marasas, 1998). We aimed to characterise Fusarium species from soil samples obtained from the undisturbed grassland soils of the Willem Pretorius nature reserve. From the various Fusarium species, including species of the FIESC, FOSC, FSSC, FCSC, and F. brachygibbosum, were obtained. These were the five most dominant morphological species recovered from the grassland soil fractions. Other species in the present study occurred in moderate to low frequencies (Figure 4.4.3a&b). Therefore, the diversity of Fusarium species recovered in the present study substantiated the fact that soils from non-cultivated ecosystems represent conservation areas of a primary biodiversity whereby microbial populations have not been impacted by human activities (Edel-Hermann et al 2015). The four most predominant Fusarium species recovered by Marasas et al. (1988) in a previous South African study were F. oxysporum, F. solani, F. equiseti, and F. nygamai. The first three species are also among our most frequently isolated species, with F. nygamai being the sixth most observed species occurring in moderate numbers in the grassland soils. In the study by Marasas et al. (1988),

97 these three species (F. oxysporum, F. solani, and F. equiseti) occurred more frequently in cultivated soils compared to undisturbed soils. The opposite was true for F. nygamai that was more prevalent in undisturbed soils. Fusarium nygamai was also fairly represented in undisturbed soils from the present study. This suggests that F. nygamai may be indigenous to South Africa and may be linked to certain native plants. Furthermore, it is not likely to compete well with strains such as F. equiseti and F. oxysporum that are prevalent in disturbed soils (Marasas et al. 1988). Fusarium nygamai was initially described in Australia and associated with root rot and basal stalk rot of sorghum along with other Fusarium species and groups of fungi (Summerell et al. 2011). Fusarium nygamai has also been isolated from sorghum field soils or plant tissue in both Australia and South Africa. This statement is supported by the fact that in the present study, 15 of our isolates identified as F. nygamai via nBLAST were closely related to a described isolate of F. nygamai (AF160273) isolated from sorghum in Australia (Glenn, 2007). However, the species phylogenetically clusters within the African clade of the fujikuroi species complex. Another South African study by Rheeder and Marasas (1998) substantiated the aforementioned frequently occurring species as it compared the diversity of Fusarium species in both uncultivated grassland soils and cultivated maize. They discovered that members of F. oxysporum and F. equiseti were both frequently isolated from plant debris in the two ecosystems. Both of these species are cosmopolitan soil-borne fungi known to occur as both pathogens and saprophytes (Burgess, 1981; Stoner, 1981; McMullen and Stack, 1983). Moreover, F. solani was their fourth highest frequently isolated species and occurred in moderately high frequencies (Rheeder and Marasas, 1998). In a study by Burgess and Summerell (1992), F. chlamydosporum was the most isolated from drier sampling regions of grassland soils. Though not obtained in high numbers in the grassland soils of the present study, F. chlamydosporum received a moderate representation as it ranked at number five (as per Mycobank nBLAST results). In the same study, F. chlamydosporum was followed by F. equiseti, F. nygamai, and F. solani that were amongst their five most frequently recovered species. They ranked respectively at position two, four, and five. Isolates identified as F. brachygibbosum via nBLAST were in the top three of the most occurring species in the present study. However, there are very few reports of F. brachygibbosum from South Africa. The first report was in a conference proceeding by Van Coller et al. (2013) and was isolated from wheat kernels and not from soil. Furthermore, a recent review article by Beukes et al. (2017) documented a broad overview of mycotoxin producing Fusarium associated with grain plants in South Africa, particularly maize, sorghum and wheat. Fusarium brachygibbosum was one of the Fusarium species found in association with crown rot, root rot, and head blight of wheat.

There is also not enough information available about the distribution and occurrence of F. brachygibbosum and therefore warrants further study. The findings from the three studies are thus concurrent with the outcome of the current study in relation to frequently isolated species of the FIESC, F. brachygibbosum, the FOSC, FSSC, FCSC, and F. nygamai in natural ecosystems. However, these species also appear to be considerably represented in cultivated areas.

Fusarium species dominate both grassland and agricultural soils (Hoorman, 2017). Their occurrence and distribution is driven by different biotic and abiotic factors (Bej and Mahbubani, 1992; Nicholson et al. 2003), but may differ in population (Marasas et al. 1988). According to Hoorman (2017), these species are diverse in basic and slightly acidic soils, especially in undisturbed soils. This could be a further explanation for the high occurrence of these species in the grassland soils as observed in this study. Moreover, factors such as temperature, humidity, light intensity, moisture, and organic content may also influence the occurrence and dispersal of different Fusarium species in these grassland soils, species such as F. oxysporum, F. polyphialidicum, F. redolens, F. solani, F chlamydosporum, etc. (Balmas et al. 2010). Some Fusarium species recovered from the present study are not only pathogenic but also have the ability to perform different ecological functions such as nutrient cycling and plant-soil-mycorrhizae fungi interrelationships (Mandeel et al. 1995). These strains may potentially spread disease above and belowground through these interactions (Bej and Mahbubani, 1992). Most Fusarium species in the current study were predominantly associated with the large soil particles rather than small soil particles. The larger soil particles contained debris consisting of roots, dried leaves, and branches that provide suitable environments for pathogenic and saprophytic fungi. Furthermore, the roots provide sources of nutrients and below ground branches provide carbon sources that allows for the growth of fungi (Zakaria, 2011). Fusarium species utilise plant debris as a nutrient resource (Hoorman, 2017; Karim et al. 2016). This suggests that the smaller soil particles are not likely to be as species rich, however this fraction of soil yielded a fairly good representation of species in the current study. A possible reason for this could be that minute pieces of plant debris might have been sieved through along with the fine small particles. As previously stated, the studies by Marasas et al. (1988), Rheeder and Marasas (1998), and also Zakaria (2011) isolated numerous Fusarium species such as the FOSC, FIESC, FSSC, FFSC, and F. nygamai from plant debris. In the present study, the FOSC, FCSC, F. brachygibbosum, and F. nygamai were represented more in the large soil particles whereas the FIESC, FSSC, FCSC, and FFSC were observed slightly

99 more in the small soil particle fraction. Representation of these species at all the four sites varied significantly. The under-representation of species at some of the sites may be due to limited sampling from the various soil fractions. Overall, natural grassland ecosystems such as those studied in the present study provide knowledge on the inherent characteristics as determined by soil formation factors (Schoch et al. 2014). A diversity of Fusarium species were observed in these soils because they had high organic matter, constant nutrient cycling, high nutrient retention, and unaltered microbial communities, features that are not observed in cultivated soils (Laurence et al. 2012).

In our study, most of the isolates identified via nBLAST as species complexes such as the FOSC, FSSC, FIESC, and FCSC received percentage similarity of 99%-100%. Species types identified as F. nygamai, F. commune, and F. redolens also received percentage similarity of 99%-100%. These species complexes and species types are well represented in both databases and may be regarded as conclusive species identification. Geiser et al. (2004) and O’Donnell et al. (2012) concluded that in most instances but certainly not all, a query sequence that obtains an identical match of 99-100% can be elucidated as a conclusive species identification. According to Geiser et al. (2004), the outcomes of previous studies based on genealogical concordance phylogenetic species recognition (GCPSC) present helpful guidance in deciding when a particular percentage similarity equates with cospecificity. Phylogenetic analysis performed in the present study on the FOSC and FIESC substantiated the results obtained via nBLAST results for both these species complexes. These species complexes could then be regarded as conclusive identification. Most formae speciales of the FOSC and MLST isolates of the FIESC clustered accordingly through phylogenetic analysis. Some misidentifications were also resolved through phylogenetic analysis. For example, isolate PPRI 20758 of the FIESC was determined to be closely related to a FIESC MLST-1a. However, phylogenetic analysis clustered this isolate with two previously described species of O’Donnell et al. (2009b), namely, NRRL 20423 MLST-4a (F. lacertarum Subrahm.; host and origin unknown) and NRRL 36123 MLST-4b (host: lizard skin; origin: India). Furthermore, phylogenetic analysis clustered isolates identified as F. nygamai and F. redolens within clades encompassing the FFSC (bootstrap = 85%) and the FRSC (bootstrap = 100%), respectively. Thus, further substantiating results obtained via nBLAST.

In contrast to the high percentage similarity observed for species mentioned above, most of the query sequences from this study received low percentage similarities below 99%. Isolates

100 identified as the FFSC, F. brachygibbosum, F. burgessii, F. polyphialidicum, F. sporotrichioides, F. boothii, F. caucasicum, F. beomiforme, F. concolor, F. venenatum, and F. armeniacum were such isolates that received low percentage similarity. There are numerous probable reasons as to why some sequences may not receive a perfect match in any of the databases. Fusarium cf. brachygibbosum, F. cf. sporotrichioides and F. cf. venenatum, F. cf. polyphialidicum, F. cf. sporotrichioides and F. cf. beomiforme, F. cf. burgessii, F. cf. boothii, F. cf. caucasicum, F. cf. concolor, and F. cf. armeniacum have a fair to poor representation in these databases (Geiser et al. 2004). The confer (cf.) indicated that these species were at the time, based on vague to absent phylogenetic support. This may suggest that query sequences identified as these species may produce low percentage similarity that is concurrent with the results observed in the current study. However, in spite of their excellent representation on these databases (Geiser et al. 2004), isolates identified as species of the FFSC and F. hostae received low percentage similarity. The query sequence may possibly be an allele variant from a described species that is absent in the database (Geiser et al. 2004). As a result, a close match will be obtained within the database and the list of the top hits in the nBLAST results would represent previously described related species that belong to a specific species complex. Also, the query sequence may be associated with a species that is poorly circumscribed at the time, for example species such as F. sporotrichioides, F. poae, and F. longipes Wollenw. & Reinking exhibit such patterns (Geiser et al. 2004). Translation Elongation Factor-1α query sequences that display similarity ≤ 99.4% ought to be subjected to further GCPSR analysis and similar queries should also be performed using the RPB1 and/or RPB2 genetic markers (O’Donnell et al. 2015). Phylogenetic analysis performed on a variety of Fusarium species in the present study also helped resolve most of the isolates that received low percentage similarity via nBLAST. Most isolates identified as the FFSC that received low percentage similarity were clustered together and some of these species may represent new undescribed species. A conclusive identification could not be drawn for a clade in the genus-wide dataset consisting of isolates identified via nBLAST as F. burgessii (as per Mycobank results). These isolates were also identified on FUSARIUM-ID as F. beomiforme, F, concolor, and Fusarium sp. and all had low percentage similarity. To solve this uncertainty, more sequences representing a variety of Fusarium isolates not already included in this study will be added in future. Species in these aforementioned clades may potentially be of newly undescribed species. Three query sequences when subjected to nBLAST under the Mycobank database alone came back as not found. Furthermore, a fourth query sequence subjected to nBLAST came back as a restricted item, however a match was obtained in the FUSARIUM-ID database as previously discussed.

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A possible reason for the restricted item matter may be that the isolate was not fully characterised at the time. Moreover, a possible reason for the three sequences whose identities were not found may be that the subject sequence corresponding to these query sequences may not have been submitted to the database at that particular time.

The results of our study displayed substantial variation between F. scirpi Lambotte & Fautrey and F. equiseti species. This confirmed information from O'Donnell et al. (2009b) about clinically significant FIESC encompassing the application of multilocus DNA data. O’Donnell et al. (2009b) determined that the FIESC contains 28 phylogenetically discrete species in which F. scirpi and F. equiseti were both characterised in two separate clusters. These F. scirpi and F. equiseti isolates were also incorporated in the current phylogenetic analysis, and they grouped within seven of the eight clades in the present study. Isolates of the current study clustered with seven of the 28 phylogenetic species, namely, MLST 1, MLST 3, MLST 4, MLST 5, MLST 9, MLST 10 and MLST 12. This suggests that these MLSTs represent the population of FIESC phylogenetic species in the undisturbed grassland soils from Willem Pretorius nature reserve. Grouping of nBLAST results for FIESC 9-b isolates into the F. scirpi clade were strongly supported by the phylogeny and MP bootstrap value of 80%. Interestingly, these were the only isolates grouping with Australian soil F. scirpi pathogens whereas one isolate PPRI 20790 (FIESC 12-c) grouped with a wheat F. scirpi isolate from Germany. This suggests that these TEF-1α sequences may have been subjected to divergence as a result of evolutional separation. According to Summerell et al. (2010), the majority of phylogeographic theories suppose that a species is initiated at a specific time and place and that the dispersal of organisms will then be contingent on changes in both time and space. Furthermore, isolate PPRI 20790 was closely related to three FIESC 12-a, one isolated from Thuja sp. and the other two isolated from Triticum aestivum L. in Germany. It was also related to one FIESC 12-b species isolated from Pinus nigra seedling in Croatia. This finding is important because it suggests that the grassland soils from this current study may house FIESC species that have a potential in posing threat to important commercial plants if they are also found in cultivated ecosystems.

Species of the FIESC are a genetically diverse group (O’Donnell et al. 2009b), which for a long time have been regarded as non-important plant pathogens (Summerell et al. 2011) but rather as those linked with human diseases (Marin et al. 2012). Numerous members of the FIESC incorporated in the Centres for Disease Control and Prevention (CDC) Fusarium keratitis study during 2005 to 2006 were extensively examined at the phylogenetic level

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(O’Donnell et al 2009b; Chang et al. 2006). This indicates that the pathogenicity studies involving this group of fungi have been focused more on human disease. Naturally, studies of the FIESC show that these species are generally isolated on phylogenetically different plant species, plant debris, and in soil (Gerlach and Nirenberg, 1982), which could impose potential risk to agriculture and workers in the agricultural sector. Regardless of their wide occurrence in soil, pathogenicity of species of the FIESC to plants has been questioned. For example, F. equiseti has been reported as a less significant plant pathogen even though it has been associated with few plant diseases such as decays of cucurbit fruits in close contact with soil (Summerell et al. 2011). There are many other reports of this fungus found in association with various plants, however, these records are normally not complemented by evidence of pathogenicity through completing Koch’s postulates and is said to have saprophytic capabilities (Summerell et al. 2011). Likewise, F. scirpi is also postulated to be saprophytic and thus is unlikely to be linked with any plant disease. Generally, F. scirpi is not considered as toxigenic (Desjardins, 2006).

Results of our study for the FOSC show strong similarity to that of Laurence et al. (2014) on the GCPSR in the FOSC. Previously O’Donnell et al. (1998b; 2004b) deduced that the FOSC is identified by four distinct, well supported phylogenetic clades. Some of the FOSC AUST isolates were also incorporated in the current phylogenetic study, and they grouped into two phylogenetic species as identified in a study by Laurence et al. (2014). Laurence et al. (2014) collapsed the four clades into two phylogenetic species (PS1 and PS2) within the FOSC based on the GCPSR methodology developed by Dettman et al. (2003). The results of the analysis indicated that PS1 is proportional to clade 1 and that PS2 is proportional to clades 2, 3, and 4 identified in the O’Donnell et al. (1998b; 2004b) study. Consequently, making our results for the FOSC dataset more coherent with that of Laurence et al. (2014) in that they support the split of the four clades into the two phylogenetic species. In our study, topological inconsistencies within the PS2 clustering were significantly evident. Most AUST isolates were scattered across the phylogenetic tree. This is similar to what Laurence et al. (2012) and other studies reported before the clades were collapsed and all came to a conclusion that these inconsistencies would require further investigation (Laurence et al. 2012; O’Donnell et al. 2009a; Lievens et al. 2009). Two separate clades comprising PS2 isolates were observed in this study and were designated PS2a and PS2b. Phylogenetic species 2b distantly clustered from PS2a, therefore suggesting that there may be a third phylogenetic species. Isolates identified as F. oxysporum f. sp. cubense via nBLAST were observed in both phylogenetic species (PS1 and

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PS2a). Laurence et al. (2014) indicated that this scenario is due to the modern evolutionary synthesis from which distinct species develop unique characteristics as time progresses. Therefore, because these are independent species, there is bound to be some differences between the two phylogenetic species. Laurence et al. (2012) frequently isolated isolates related to clade 1 rather than clade 2. In the present study, contrary to what Laurence et al. (2012) observed, most of our isolates grouped with PS2a isolates previously clustered under clade 2. Whereas, only three PPRI isolates grouped with PS1 isolates previously clustered under clade 1. Several surveys, including that of a South African study by Bushula (2008) also found that amongst their frequently isolated F. oxysporum species, none of them grouped with PS1 isolates. This, combined with our findings reinforces the conclusion that PS1 isolates may be endemic to Australia (Laurence et al. 2012).

Our analyses of some of the strains obtained in the present study were based on the dataset published by Laurence et al. (2016). This analysis produced a phylogenetic tree with sixteen clades presenting four species complexes and one species. Isolates from the current study grouped in the FFSC, FOSC (initially identified as Fusarium spp. and phylogenetically resolved as members of the FOSC), FRSC, FSAMSC, and one identified as F. commune and were also observed in the study by Laurence et al. (2016). Our results signify a wide diversity of the genus Fusarium in undisturbed grassland soils of the Willem Pretorius Nature reserve. Evidently so, the current study identified ten potentially new species of the genus corresponding to F. burgessii, F. polyphialidicum, F. sporotrichioides, F. armeniacum, F. boothii, F. venentum, F. hostae, F. concolor, F. beomiforme, and the FFSC that also includes the F. nygamai isolates. Further characterisation will be performed on these species in a future study. Surveys of undisturbed environments are essential for revealing the diversity of Fusarium species (Summerell et al. 2010). Moreover, they are also important for identifying phylogeographical signs that may possibly be affected by human activity. This is also true for the natural ecosystems of South Africa that accommodate a diverse number of species belonging to the genus Fusarium. However, a study by Marasas et al. (1988) deduced that Fusarium populations in both cultivated and un-cultivated soils in South Africa varied qualitatively and quantitatively. They indicated that the occurrence of F. oxysporum, F. equiseti, F. solani, and other Fusarium species were higher in cultivated ecosystems than in natural ecosystems. They believed that this pattern may be due to the dispersal of Fusarium propagules through cultivation and the escalated degree of infectious strains found in coexistence with suitable hosts. Therefore, the occurrence of such species is more likely to be

104 accelerated in cultivated ecosystems. Similarly, our study also recovered these species in high numbers, only these were obtained in undisturbed soils that are generally known to house a diversity of Fusarium species (Laurence et al. 2016). This suggests that agricultural ecosystems promote the occurrence of these species and further signifies the cosmopolitan prevalence of these species in all soil types.

Several PPRI isolates from the current study grouped within the FFSC in the Laurence et al. (2016) dataset. These isolates may represent new species in the African lineage clade that is rich and diverse in species and encompasses most mycotoxin producers (O’Donnell et al. 1998b; Proctor et al. 2004; Fourie et al. 2013). This assumption is supported by the fact that a previously described F. nygamai isolate, a member of the African clade (Proctor et al. 2004) is found in the clade encompassing the FFSC in the current study. Members the FFSC are recognised as important plant pathogens as they are accountable for numerous plant diseases of economic importance (Leslie, 1995; Moretti et al. 2007). According to Herron et al. (2015), members of the African, American and Asian clades are normally linked with host plants originating in a particular geographic region. For example, F. verticillioides that is a member of the FFSC in the African (and Asian) clades, is known to be pathogenic to cereal plants on both these continents (Ma et al. 2010; Proctor et al. 2004). The species complex comprise of at least 50 Fusarium species that are closely related (O’Donnel et al. 2013). Two PPRI isolates (PPRI 19149 and PPRI 22756) of the current study, both belonging in the FFSC were phylogenetically closely related to a Fusarium sp. (AF160268) isolate that originates in Zimbabwe and was isolated from Zea mays L. This isolate was described as F. cf. dlamini Marasas, P.E. Nelson & Toussoun (O’Donnell et al. 2000). Moreover, isolate PPRI 19224 was related to a previously described F. dlaminii (AF160277; host: Zea mays L; origin: South Africa) isolate. Therefore, suggesting that these three PPRI isolates are endemic to South Africa. Furthermore, PPRI 21033 and PPRI 21055 were phylogenetically related to two newly described F. mundugurra sp. nov. (KP083255 and KP083256) isolates of the FFSC, both of which were isolated from soil in an Australian study by Laurence et al. (2016). Thus suggesting another possible occurrence of divergence caused by evolutional segregation as mentioned previously. Isolate PPRI 21574 and PPRI 19124 identified via nBLAST as F. redolens and F. commune respectively, phylogenetically corresponded to a described F. redolens (AF331816; source unknown) and a described F. commune (HM804942; origin: USA), respectively. Similarly, this also suggests a possible occurrence of divergence caused by evolutional segregation. Isolate PPRI 19183 identified phylogenetically as F. palustre Elmer & Marra

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(GQ856949; origin: USA) and two other isolates, PPRI 20541 and PPRI 20791, were closely related to F. kyushuense O'Donnell & T. Aoki (AJ427274; origin: Japan). Both these described species were grouped in the FSAMSC. Substantial TEF-1α sequence divergence was also observed in these associations. Most species of the FSAMSC are also soil borne-fungi, produce tricothecene mycotoxins (Laurence et al. 2016), and are phytopathogenic to several plant species (Desjardins and Nelson, 1995). Some species of this complex have been linked with seedling rot and fusariose of ear of grain (Nelson et al. 1983; Armstrong and Armstrong, 1981), and dry rot of potato tubers (Desjardins and Nelson, 1995).

Recent studies on Fusarium using molecular phylogenetics has uncovered species that would not have been identified though morphological characterisation (Ko et al. 2011). As true as this is, identification of Fusarium species using characterization of morphological characters in culture is still necessary and crucial as it can provide additional information for identification. Cultural characteristics may benefit both the phylogenetic and biological species concepts. Several studies, especially those making use of the GCPSR concept to identify Fusarium, incorporate both morphological and molecular characters for comparison. This aims to strengthen the quality of research and necessitate the use of single spore cultures (Goh and Hanlin, 1997). Similarly, isolates obtained from single spores are important for testing the biological species concept through mating studies (Choi et al. 1999). Consequently, studies that utilise a single method to characterise Fusarium species are likely to be critically received, therefore, a dual species concept approach is essential for the precise identification of Fusarium (e.g., Cai et al. 2009; Tejesvi et al. 2011). The ecological and geographical species complexes are also recognised in Fusarium studies. These complexes assist in delimiting intra-specific variation that is vital for strengthening species descriptions (Leslie et al. 2001). These two species concepts will be put to a test in future studies as a continuation of the current study.

The ML and MP bootstrap trees of the FIESC and FOSC dataset were least congruent. This was also reflected by the genus-wide analyses but to a lesser extent. A possible reason for this could be that phylogenetic incongruence may be due to variations in processes of evolution or history (Planet, 2006). However, it is also indicated that this incongruence may also simply occur as a result of a sampling errors or random chance, all which are highly plausible. According to Nadler (1995), most molecular phylogenetic studies generally made use of a single molecular marker for sequence comparison, and the same was done in the current study through the use of the TEF-1α gene. However, there may have been a potential limitation that arose from using this approach and as a result the evolutionary history of these species

106 disagreed with the genealogy of the single TEF-1α gene used (Nadler, 1995). Therefore, in addition to the TEF-1α sequence, other protein coding molecular markers are recommended for use (Geiser et al. 2004).

Conclusion and future work Molecular identification of Fusarium species using the TEF-1α gene served as a critical tool for identification of Fusarium species from the Willem Pretorius nature reserve. This study revealed that there is a high degree of Fusarium species diversity in the undisturbed soils of this nature reserve. This study alone has recovered five species complexes and several species of the genus of which most are important plant pathogens. The identification of new species as potential plant pathogens can have far-reaching impacts on South African agriculture in future. The phylogenetic analysis results based on the TEF-1α gene provided a vivid picture towards the phylogenetic relationships of the FIESC, the FOSC, and various species of the genus. The development of the dependable phylogenetic species concept has allowed more precise species definitions in the genus Fusarium that have inadequate distinct morphological or biological characters. However, the morphological and biological characters should not be overruled in the process of defining species as these may provide more information for describing and distinguishing new species. This study contributes information about the composition of Fusarium species in uncultivated ecosystems of South Africa. Indeed, it will further assist in determining risk evaluation methods for farmers and peri-urban families. Future work on this study will include characterization of new species based on detailed morphological characterisation and further phylogenetic analysis. The phylogenetic analysis will be based on RPB1 and RPB2 molecular markers so as to establish deep level phylogeny of these isolates. The phylogeography of these species will also be examined through the acquisition of additional data with an expansion of the study’s scope. Additional sampling sites from other natural reserves within the grassland biome of South Africa will be incorporated in the continuation of this project.

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SUMMARY

Fusarium is a genus initially defined by Link in the year 1809 as Fusisporium and belongs to the phylum Ascomycota. The genus includes a large number of species and is globally distributed in soils and plants as saprobes and endophytes. Members of the genus Fusarium are well known for their ability to cause plant diseases and are toxic to both domestic animals and humans. The kinds of plant diseases caused are relatively diverse and may include seedling blight, seed rot, ear rot, stalk rot, kernel rot, head blight, wilts, leaf diseases, and cankers. The Translation Elongation Factor 1-α gene (TEF 1-α) has been the most broadly recognised throughout the genus and provides the most robust level of species discrimination. The RNA (Ribonucleic Acid) Polymerase II (RPB 1 & 2) is also a gene of choice in the classification of Fusarium and have been used to establish deep level phylogeny in Fusarium. Three species concepts, the morphological, biological, and phylogenetic, have been widely used to demarcate Fusarium species. The aim of the study was to determine the diversity of Fusarium species and to characterise these species using the morphological and phylogenetic species concept. The Fusarium species were obtained from undisturbed soils in the grassland biome from the Willem Pretorius nature reserve (Free State Province, South Africa). The soils were obtained from four different sites of the nature reserve and the localities mainly represented three vegetation types (Thornveld and riverbank communities, Grassland communities and Hygrophilous communities) within the reserve. Pure cultures of Fusarium species were obtained through processing of these soils. Polymerase chain reaction and cycle sequencing were performed using the TEF-1α gene using DNA obtained from the individual cultures. The resulting 354 sequences were edited and subjected to nBLAST analysis on the Mycobank and FUSARIUM- ID databases. The Mycobank BLAST results of the partial TEF-1α sequences for the 354 Fusarium isolates identified five species complexes (FFSC, FIESC, FOSC, and FSSC) and different species (F. acuminatum, F. brachygibbosum, F. boothii, F. burgessii, F. caucasicum, F. inflexum, F. nygamai, F. polyphialidicum, F. redolens, F. sporotrichioides, and a group of unknown species designated as Fusarium spp.). The FUSARIUM-ID BLAST results also identified the five species complexes and included F. brachygibbosum, Fusarium spp., F. acuminatum, and F. redolens. Fusarium venenatum, F. beomiforme, F. armeniacum, F. hostae, F. commune, and F. concolor were species only identified using FUSARIUM-ID. The morphological characters of five Fusarium species (F. solani, F. equiseti, F. oxysporum. F. fujikuroi, and F. brachygibbosum) were evaluated. Morphological characters of the latter four

128 species were in line with previous descriptions by Leslie and Summerell (2006a). Those of the fifth species, identified via nBLAST as F. brachygibbosum did not resemble any previously described species. This implied that they may represent a new undescribed species which requires further investigation. Phylogenetic analysis of the FIESC, FOSC, and genus-wide datasets produced eight, two phylogenetic species, and 16 clades, respectively. Phylogenetic relationships of most of the FIESC, FOSC, and genus-wide datasets were successfully inferred. The results of our study displayed substantial variation between F. scirpi and F. equiseti species for the FIESC dataset. This confirmed information from O'Donnell et al. (2009b) about clinically significant FIESC encompassing the application of multilocus DNA data. Results of our study for the FOSC indicated strong similarity to that of Laurence et al. (2014) on the GCPSR in the FOSC. Consequently, supporting the split of the four clades into the two phylogenetic species. The results also indicated that a third phylogenetic species may exist. Furthermore, our results of the genus-wide dataset showed a wide diversity of the genus Fusarium in undisturbed grassland soils of the Willem Pretorius Nature reserve. Evidently so, the current study identified ten potentially new species of the genus corresponding to F. burgessii, F. polyphialidicum, F. sporotrichioides, F. armeniacum, F. boothii, F. venentum, F. hostae, F. concolor, F. beomiforme, and the FFSC that also includes the F. nygamai isolates. This study will contribute on the information about the composition of Fusarium species in uncultivated ecosystems of South Africa. Furthermore, it will assist in determining risk evaluation methods for farmers and peri-urban families. The information will educate them on the most prevalent Fusarium species found in certain soil types. However, it is not enough for the grower to only identify the type of Fusarium connected to a disease that may potentially harm their crops, therefore certain control practices will have to be taken. A follow up on this study can also focus on implementing effective control measures for Fusarium species that affect their crops, such as the use of specific fungicides.

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