This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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Phoma stem canker is a disease that causes stem base cankers and upper stem lesions in oilseed rape (Brassica napus). It is caused by Leptosphaeria maculans (Lm) and L. biglobosa (Lb). This disease causes a loss of more than £80 million a year in the UK alone. While both fungal pathogens are affected by azole fungicides, studies show that Lb is less sensitive to some azole fungicides. Concerning other fungal pathogens, research has shown that mutations in the sterol 14a-demethylase (CYP51) gene are the main cause of fungicide resistance. This project aims to investigate the differences in CYP51 expression in Lm and Lb as a contributing factor to fungicide sensitivity.
During this project, I was introduced to many molecular biology and plant pathology techniques. To begin, I performed DNase treatment and cDNA synthesis on 224 RNA samples that had been extracted from oilseed rape cotyledons inoculated with different Lm and Lb isolates. The DNase treatment was performed to remove any genomic DNA from the sample so that it wouldn’t also be amplified later on. The cDNA synthesis produces DNA strands that are complimentary to the RNA. Also, I learned how to prepare PCR primers and to do end-point PCR assays. End-point PCR was performed on selected cDNA samples with Lm/Lb CYP51 and actin (selected as a reference gene) primers. PCR products were analysed using gel-electrophoresis to validate the primers. Following this, the Lm, Lb and control samples were separated, and RT-qPCR was performed on all the samples using the light-sensitive fluorescent dye SYBR Green for both the target and the reference genes. All the samples were tested in duplicate and individual CT values were determined to calculate and compare the relative transcript levels between different treatments/isolates. A dissociation curve was performed, which had only a single peak indicating no contaminations or primer-dimer formation.
Through the vacation bursary, I have had the opportunity to apply the knowledge I have gained so far in my degree to real research. Along with this, I have been able to expand my knowledge of plant pathology as well as learn and practice new laboratory skills. I learnt to carry out experiments under controlled-environment conditions, along with how to culture fungal isolates and prepare spore suspensions. I also had the opportunity to do disease assessments in the field. I am very grateful to have been given this opportunity. I also had the chance to shadow and discuss with PhD students about their experiences who along with my supervisor inspired me to pursue post-graduate education. I am very thankful to BSPP for funding this project and my supervisor Dr Chinthani Karandeni Dewage as well as the students and staff I worked with during my placement.
University of Hertfordshire
Gbemi in the field collecting oilseed rape samples for disease assessment