Thielaviopsis basicola, a phytopathogenic filamentous fungus, is the causative agent of black root rot in a variety of host plants, including the economically important crop, cotton. The method of Agrobacterium tumefaciens mediated transformation (ATMT) was chosen to investigate the molecular interactions that exist between T. basicola and cotton. ATMT has long been used to generate transgenic plants and has more recently become a popular method for random insertional mutagenesis in the transformation of filamentous fungi. Generation of a large number of reduced pathogenicity mutants using this technique will aid to elucidate the identification of key pathogenic genes providing a better understanding of the molecular interactions between T. basicola and cotton, governing the pathogenesis of black root rot.

Development of an efficient ATMT protocol, designed specifically for transforming T. basicola, required optimisation of the experimental conditions prior to, during and after transformation. Transformation efficiency was found to be dependent upon the duration and temperature of pre­ cultivation, co-cultivation and selection. The number of A. tumefaciens cells and the status of the T. basico/a cells were also found to have significant influence on the efficiency of transformation. A consistently high rate of transformation efficiency was achieved by employing the hypervirulent strain AGLI, carrying the binary vector p8Ht2, which contains the modified bacterial Hygromycin B phosphotransferase hph gene under the control of the Aspergillus nidulans trpC promoter. The

media used during co-cultivation and the method of selection also played an important role in

optimising the ATMT protocol for T. basicola. Optimal conditions of transformation led to the

production of 300-770 Hygromycin B resistant (HygR) putative transformants per I x 106 conidia of

T. basicola.

All I 0 HygR putative transformants tested remained mitotically stable, maintaining their Hygromycin B resistance after five generations on non-selective medium. Primary pathogenicity screenings indicated that three of the I 0 mitotically stable HygR putative transformants had reduced pathogenicity, showing decreased virulence towards infected cotton seedlings when compared to the WT. Vegetative growth tests of these same 10 HygR putative transformants, displayed varying growth by comparison to the WT; with six showing reduced growth and four growing at a similar rate to the WT. Colony morphology also indicated that at least seven of the HygR putative transformants differed in colour, texture, and number of chlamydospores compared to the WT.

Further genetic testing will be required to confirm that single and random insertion of the T-DNA

occurs in the T. basicola genome.

Southern blot analysis on three of the five T. basicola reduced pathogenicity mutants generated by PEG/CaC(z, revealed that in p737 and p888, more than one insertion of pGpdGFP took place at multiple loci in the fungal genome; a common occurrence when using this method of transformation. The reduced pathogenicity mutant p 16 instead had a single insert of the plasmid pGpdGFP integrated at a locus in the fungal genome, which suggests that further attempts could be made to recover the tagged pathogenicity gene from this mutant. Phenotypic analyses of all five PEG mutants, as well as 20 HygR putative ATMT transformants, indicated that T. basicola most likely has some pathogenicity genes that are similar to those found in other filamentous fungi; including genes involved in the formation of infection structures and hydrophobins, spore development and germination, regulation and biosynthesis of melanin, cuticle and cell wall degrading hydrolytic enzymes, and regulatory proteins, including transcription factors, receptors, G proteins, and enzymes.