Abstract:
The causative agent of apple scab or black spot is the Ascomycete fungus, Venturia inaequalis. Scab is the most important disease of apple worldwide and is responsible for significant production losses. Controlling this disease requires intensive management throughout the growing season. Declining efficacy of current intervention strategies, concern about environmental impacts of agrochemicals, and, consumer demand for no detectable residues in food mean that alternative control measures are required. Resistance breeding may be the most sustainable control intervention for many agricultural pathogens including V. inaequalis. A durable resistance breeding programme that can deliver to market new cultivars preserving good commercial traits requires knowledge of the host-pathogen interaction at the molecular level. Knowledge about the role of effectors, such as the candidate effector, ViEcp6, is of intrinsic value to any breeding programme. Evaluating the roles of effectors requires robust methods to target mutations to the gene of interest. V. inaequalis was transformed using an Agrobacterium tumefaciens T-DNA vector carrying an inverted repeat designed to silence ViEcp6. Many of the resulting transformants exhibited growth and developmental characteristics that were markedly different from untransformed V. inaequalis. Characterisation of the integration events revealed a range of important developmental and transcription related genes had been disrupted by T-DNA integrations. Multiple integrations were detected in some isolates. Some insight has been gained into the complex mechanisms of T-DNA integration in V. inaequalis and, there is potential to develop a library of knock out mutants for gene characterisation. Given that Agrobacterium delivery is therefore problematic for gene characterisation in V. inaequalis, an alternative strategy to characterise ViEcp6 was adopted and a knock out vector was engineered. This approach necessitated improvements in transformation techniques. A novel adaptation for protoplast generation, using unmelanised stromata grown in vitro, was optimised and a new method of transient cell membrane disruption was piloted. Preliminary results are highly promising; suggesting further development of these methods would be of value for characterising genes in this recalcitrant organism. This will accelerate the rate of discovery about the apple-V. inaequalis pathosystem and, ultimately, the commercialisation of scab resistant cultivars.