Trichoderma species are ubiquitous soil saprotrophs and well-characterized biological control agents. Certain strains have also been shown to stimulate plant growth through the production of bioactive secondary metabolites, and are therefore receiving increased attention as natural plant growth stimulants. Previous research at the University of Exeter (Ryder et al. (2012) Microbiology 158, 84-97) has shown that the Plant Growth Promotion activity of a biocontrol strain of Trichoderma hamatum (strain GD12) can be dramatically enhanced by targeted mutation of the N-acetyl-β-D-glucosaminidase-encoding gene nag. However, due to the loss in chitinase activities, the mutant (ΔThnag::hph) displays loss of saprotrophic competitiveness and reduced fitness as a biocontrol agent. We set out to investigate how we can use genetic manipulation to improve strain GD12 in the context of biological control and plant growth promotion. We approached this by firstly sequencing the whole genome of GD12 and then using the information available from this to produce a targeted deletion mutant in the GD12 background disrupting one of the most down regulated proteins in the ΔThnag::hph, a branched chain amino acid transaminase (bcat), implicated in the production of secondary metabolites. Secondly, we aimed to engineer hyper-secretion and enhanced PGP activities in GD12 without impairing biocontrol activity. Over-expression of the S. cerevisiae gene dolichol-phosphate mannose synthase (dpm1) in T. reesei leads to altered cell wall architecture and increased secretory potential. Using the constitutive promoter ToxA, we over-expressed the dpm1 gene in T. hamatum GD12 and assessed its effects on the biocontrol and PGP activities of the fungus. The data presented herein, shows, that bcat deletion in T. hamatum GD12 results in a detrimental effect of germination of lettuce seedlings grown in the presence of ∆Thbcat::hph. We show that single copy insertions of ToxA-dpm1 leads to improved PGP activities, while biocontrol fitness is unaffected. However, while multiple copy insertions similarly lead to enhanced PGP, such strains display impaired biocontrol of soil-borne pathogens such as the plurivorous damping-off pathogen Sclerotinia sclerotiorum. This work demonstrates that while significant improvements in crop productivity can be achieved through genetic modification of the beneficial rhizosphere fungus T. hamatum GD12, it can have important consequences for other aspects of its biology and ecology and competence as a soil-borne microorganism.

Expanding global populations, unequal food distribution and disease pressure suggest food poverty is increasing. Consequently, much attention is focussed on alternative natural methods in which to increase agricultural yield. Previously, it was observed that Trichoderma hamatum strain GD12 and its respective N-acetyl-β-D-Glucosamine mutant ∆Thnag:hph promoted plant biomass and fitness that, as a result, may provide a credible natural alternative to synthetic fertilisers. However, on a molecular level, the manner in which this is achieved has not been fully elucidated. In this thesis, I report the biofertiliser effect of GD12 and mutant ∆Thnag::hph once applied to autoclaved peat microcosms as sole applications. Furthermore, I demonstrate the biocontrol ability of GD12 when co-inoculated with Sclerotinia sclerotiorum or Rhizoctonia solani and reveal, that once mycelium co-inoculation has occurred, GD12 increase plant biomass and provide protection; whilst ∆Thnag::hph does not. Consequently, I challenged the biocontrol effects of Trichoderma metabolite extract where I validate that both Trichoderma wild type GD12 and mutant ∆Thnag::hph are incapable of suppressing pathogen growth. Subsequently, I characterised the up-regulated signatures associated with GD12 and ∆Thnag::hph using LC-MS techniques where unique compounds were discovered from each strain of Trichoderma. In conclusion, I provide evidence that N-acetyl-β-D-Glucosamine mutation bring about metabolomic changes that affect the fungal secretome which, in turn, alters plant phenotype, fitness and germination. Furthermore, I have shown that these effects are species specific and depend upon pathogen, plant and fungal properties. However, further investigations are needed to fully elucidate the compound(s) responsible for biocontrol and biofertilisation; especially plant-specific effects that take place as a consequence of fungal activity.