Native Fusarium species from indigenous fynbos soils of the Western Cape

Bushula, Vuyiswa Sylvia (Stellenbosch : Stellenbosch University, 2008-12)

Thesis (MSc (Microbiology))--Stellenbosch University, 2008.

Thesis

The genus Fusarium contains members that are phytopathogens of a number of agricultural commodities causing severe diseases such as wilts and rots. Fusarium species also secrete mycotoxins that have devastating effects on humans and animals. The ability of Fusarium species to change their genetic makeup in response to their immediate environment allows these fungi to exist in diverse habitats. Due to the ubiquitous nature of Fusarium, it forms part of the fungal communities in both agricultural and native soils. Fynbos is the major vegetation type of the Cape Floristic Region (CFR), which is a region that is renowned for its high plant species diversity and endemism. In this study, the occurrence and distribution of Fusarium species in indigenous fynbos soils and associated plant debris is investigated. In addition, the phylogenetic relationships between Fusarium species occurring in this particular habitat are evaluated. Fusarium isolates were recovered from soils and associated plant debris, and identified based on morphological characteristics. The morphological identification of isolates was confirmed using Polymerase Chain Reaction (PCR) based restriction fragment length polymorphism (RFLP) analyses of the translation elongation factor 1 alpha (TEF-1α) and internal transcribed spacer (ITS) regions. Furthermore, phylogenetic relationships between Fusarium species were based on the TEF-1α, ITS and β-tubulin gene regions. One-hundred-and-twenty-two (122) Fusarium strains were isolated from the fynbos soils in the Cape Peninsula area (Western Cape). Based on both morphological and molecular identification, the most prevalent Fusarium species in the fynbos soils were F. oxysporum Schlecht. emend. Snyd. and Hans., F. solani (Martius) Appel and Wollenw. emend. Snyd. and Hans., F. equiseti (Corda) Sacc. and an undescribed Fusarium species. Fusarium oxysporum was the dominant species in fynbos soils and strains of this species displayed significant genetic variability. Some strains of both F. oxysporum and F. solani showed close phylogenetic affinities to formae speciales (strains pathogenic to specific plant hosts) in the phylogenetic analyses. However, no diseased plants were observed in and within the vicinity of our sampling sites. In the third chapter, the undescribed Fusarium strains are described as Fusarium peninsulae prov. nom. Morphologically these strains are characterized by falcate macroconidia produced from brown sporodochia. The macroconidia are pedicellate, falcate to curved with hooked apical cells. Also, this fungus produces apedicellate mesoconidia on polyphialides in the aerial mycelium and forms microconidia sparsely. Chlamydospores are formed abundantly on aerial mycelium and submerged hyphae. All these morphological characteristics closely relate this fungus to F. camptoceras species complex in Fusarium section Arthrosporiella. However, phylogenetic analysis based on the ITS sequences differentiate these strains from F. camptoceras and other related species in section Arthrosporiella. Considering the fact that both as phytopathogens and saprophytic fungi, Fusarium species secrete a variety of cell wall degrading enzymes such as cellulases and xylanases. These enzymes allow the fungi to degrade the plant cell wall components to obtain nutrients. In Fusarium, notably endoxylanases play a role in phytopathogenesis of these fungi. Endoxylanase enzymes from F. oxysporum f. sp. lycopersici, F. verticillioides and F. graminearum have been characterized. In this final chapter, the use of the endoxylanase encoding gene, as a molecular marker in phylogenetic analysis was evaluated using F. graminearum (Fg) clade species as model. Degenerated primers were designed and the endoxylanase region amplified by PCR, cloned and sequenced. PAUPgenerated neighbour-joining analysis of the endoxylanase (XYL) region enabled all species to be distinguished and was as informative as the analysis generated with UTPammonia ligase (URA), phosphate permase (PHO), reductase (RED) and trichothecene 3- О-acetyltransferase (TRI101). Furthermore, the results of the phylogenetic analysis of XYL showed better species resolution in comparison to the analysis of the structural genes (TEF-1α and histone H3). Overall, the results demonstrated that phylogenetic analysis of XYL combined with other functional genes (URA, PHO, RED and TRI101) clearly distinguished between the Fg clade species far better than the analysis of structural genes (TEF-1α and histone H3).

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