Understanding protoplast technology as a tool to enhance biocontrol : A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Lincoln University

Abstract

Biocontrol of plant pathogens is an integral part of modern pest and disease management. Issues of pesticide residues/resistance and a drive towards sustainability within crop protection systems have created an opportunity for sustainable alternatives to synthetic agro-chemicals. One approach within integrated pest management programmes is the use of biocontrol agents, such as fungi. The development of novel biocontrol strains using protoplast technology provides the opportunity to re-combine or introduce desired features, in particular fungicide tolerance, without the need for sexual reproduction. This present study employed protoplast regeneration to produce novel Trichoderma strains with attributes such as pesticide tolerance for the control of the bacterium causing kiwifruit canker, Pseudomonas syringae pv. actinidae (Psa). To achieve this, leading Trichoderma strains were selected from within current research programmes targeted at kiwifruit health. Using protoplast regeneration, numerous strains were produced that were tolerant separately to copper sulphate and Chief® (a.i. carbendazim). Further bioactivity trials showed that biocontrol potential of the protoplast progeny was not reduced as a consequence of this strain enhancement. However, despite the use of fungal protoplast technology for strain improvement very little is known of the underlying genetic changes responsible for the modified phenotypes. The overall hypothesis was that cytosine methylation may be responsible for the phenotypic plasticity that was observed in these protoplast progeny, with this study being the first to examine the consequences of protoplast regeneration through a multi-layered, integrative ‘omics approach. The differential cytosine mapping through whole genome bisulphite sequencing showed sparse differential methylation between a protoplast regenerant (copper tolerant FCC237/R5 T. sp. “atroviride B”) and its parent (FCC237 T. sp. “atroviride B”). This cytosine methylation did not appear to modulate corresponding gene expression levels to differentially methylated associated genes, as it had been reported in other fungal species. The transcriptome analysis indicated differential gene expression between the parent and regenerant in both sub-lethal levels of copper sulphate (1 mM) (88 DEGs) and also in the control PDB (281 DEGs). The changes in DEGs profiles, especially regarding proteins involved in oxidative stress, secondary metabolites and transporters could suggest that the regenerant may induce a general stress response as opposed to a specific pathway for copper tolerance that has arisen as a putative result of protoplast regeneration. Other epigenetic factors, including histone and chromatin modifications and RNA interference, are likely to play an important in both regulating gene expression states and the phenotypic plasticity observed in the protoplast regenerant. Although other epigenetic modifications have not been tested here, they would be expected to be present due to the interconnectedness of the epigenome and further elucidation is required. This work has contributed to a better understanding of the basic genetic events that occur during protoplasting in Trichoderma, which may allow for greater advances in not only strain enhancement, but also in genetic modification experiments, in both Trichoderma but other filamentous fungi.