Written by Araminta Gray, University of Kent. This is the report from a BSPP Undergraduate Summer Vacation Bursary. Click here to read more/apply for one yourself.
My project was based at NIAB-EMR working alongside Thomas Heaven. The project was focused upon Venturia inaequalis, an ascomycete fungus which is responsible for causing apple scab; one of the most economically important diseases affecting commercially grown apples. Apple scab is characterised by the presence of scab-like lesions on the fruit and leaves of apples trees. The current treatment for apple scab outbreaks involves spraying the orchard up to twenty times a season with fungicides, most commonly sterol demethylation inhibitors (DMI). However, the frequent use of fungicides has placed selection pressure on the pathogen resulting in the evolution of resistant strains. My project was part of an investigation into those resistant strains.
Isolates of V. inaequalis that demonstrated resistance and susceptibility to fungicides were crossed and grown on agar plates in order to produce mapping populations with recombinant genomes. Throughout the project the progeny from successful crosses were isolated by transferring sexual spores (ascospores) contained within asci to new agar plates in order to grow colonies. A key part of the project was extracting DNA from the parent V. inaequalis isolates from which genes of interest were amplified using PCR, specifically the CYP51A1 gene that codes for the target of DMI class fungicides. The main technique used throughout the project was PCR to identify wild type or mutant alleles within a gene or section of a gene, which was achieved by using a range of primers. Resulting PCR products were visualised using gel electrophoresis. This process taught me how to troubleshoot by giving me experience of optimising protocols. The concentration of DNA was measured using a spectrophotometer in order to determine if the concentration of purified PCR products was high enough to be sent off for diagnostic sequencing.
The results of my project have implications for research into scab fungicide resistance, in the future the genomes from the crosses will be sequenced as part of Thomas’ PhD. By using results looking at the dose response to fungicides in the different progeny isolates it will be possible to map the resistance trait and identify the QTL for fungicide resistance in V. inaequalis. Ascertaining the genetic basis of the resistance trait will lay down the foundations for identifying fungicide resistant samples in the field, which would be valuable to commercial growers.
This summer project has been a fantastic opportunity during which I have learnt a variety of techniques and skills, including DNA extraction and purification, which I am looking forward to applying during my final year lab project when I return to the University of Kent. Additionally, this project has provided me with an invaluable insight and understanding of working in a research environment and the perseverance required as part of scientific investigations. As a result of the experience I have been inspired to pursue a PhD after I graduate. I would like to thank Thomas along with everyone else in the GGB lab for making me feel welcome and helping me throughout the summer.
