This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
Stem canker is a disease caused by the fungus Leptosphaeria maculans and causes significant losses in oilseed rape yields every year, at an estimated £100 million. Additionally, temperature-resilient disease resistance is becoming increasingly important due to warmer summers globally. In Arabidopsis thaliana, the SNC1 gene is a regulator of temperature-sensitive plant immunity, and an ortholog has been identified in Brassica rapa (BrSNC1), which is located next to the FocBr1 gene for resistance against Fusarium oxysporum. Previous tests on TILLING mutants suggested that a BrSNC1 mutant allele provides temperature-resilient resistance against L. maculans. The aims of this project were to 1) confirm the results of previous testing that the BrSNC1 mutant line provides better disease resistance at higher temperatures, and 2) test genotype segregation in mutant lines of B. rapa.
The seeds were pre-germinated and planted in compost with vermiculite. There were four lines in total: the BrSNC1 backcross mutant progeny, a backcross out-segregant wild type, and two segregating FocBr1 mutant lines. The seedlings had a high growth successes rate, as only two of 76 were lost during this stage. Colonies of L. maculans were grown simultaneously on V8 agar for spore production. Once the plants had produced at least 2 true leaves, they were moved into larger pots and their cotyledons collected for DNA extraction and sequencing. Plants were infected at 3 weeks old with conidial spores of L. maculans isolate JN3 at a concentration of 107 spores/ml. This was done through point inoculation, where small wounds were made on both the second and third true leaves, and a 10µl droplet of the spore suspension was added directly on top of each (pictured). Tray covers were misted down and put over the plants to maintain high humidity for the first two days. They were also separated to grow at two different temperatures: half at 20°C and half at 25°C, for two weeks. Disease progression was scored throughout the second week of infection, and samples of surviving leaves were collected for quantification of fungal DNA through qPCR.
Data was analysed using the area under disease progression curve (AUDPC) for each sample. When the BrSNC1 mutant line was compared to the wild type using an ANOVA, a significant effect of both line (p=0.049) and temperature (p= 0.00035) was found. The disease progression for both lines was significantly higher at 25°C compared to 20°C, and the wild type had higher AUDPC scores than the BrSNC1 line especially at the higher temperatures, indicating that the BrSNC1 mutant is more resistant to L. maculans at higher temperatures compared to wild type. The results from the qPCR on pathogen DNA quantity was not conclusive, so more work will be done in the future to confirm the results.
The DNA was extracted from individuals of a segregating backcross F2 generation of FocBr1 mutant lines and amplified using PCR. The products were then checked using gel electrophoresis and quantified with NanoDrop before sequencing. One of these lines was successfully genotyped and analysed using the Kruskal Wallis test, which found that there was a significant effect of temperature on AUDPC scores (p= 0.00088), but no significant differences in scores between genotypes (p= 0.71). The other line will be sequenced later.
During the project, I also had the opportunity to attend the Cereals 2024 event, where we spent the day exploring and learning about the crop strains and new technologies being developed to improve yields and monitor crops. It was a great experience to see what research was occurring across the subject. I was also able to visit Rothamsted Research and was given a tour around the labs and greenhouses by Frederic Beaudoin, which was a great and unique chance to learn about the facilities and research being undertaken at Rothamsted, and to learn about new kinds of equipment I’ve not had access to in the past.
Overall, this project has given me the opportunity to develop my practical skills in the lab alongside learning new techniques. I have enjoyed working on this project at the University of Hertfordshire, and feel much more prepared going into my final year of university. I would like to thank my supervisor Henrik Stotz, the other members of the oilseed rape group, and the lab technicians at the university for the opportunity to work on this project and all the help along the way.
Erin McDonagh
University of York
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
