This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
This summer I joined Dr Nichola Hawkins at NIAB, Cambridge with the aim of establishing patterns of fungicide resistance in Zymoseptoria tritici over the last 20 years as well as determining any cross-resistance patterns present.
Z. tritici is the causative agent of Septoria Leaf Blotch. Characterised by brown, necrotic regions on the leaves, this pathogen primarily affects wheat, however also occurs in rye and some grass species, highlighting its global impact on agriculture. In addition to necrotic regions, black pycnidia can be seen on infected leaves. Z. tritici spreads between individual plants via air-borne ascospores and within a season, spreads between leaves of individual plants by rain-splash. In the UK, wheat farmers can lose up to 30% of their crop to Septoria infections. Wheat is a high value crop: UK production in 2021 equaled £2.1 billion. As a staple crop, wheat consumption is high, with predictions that the world will need 840 million tonnes per year by 2050. In a world where food insecurity is of growing concern, tackling crop pathogens is of increasing importance.
Currently, the most effective control measure for Septoria Leaf Blotch is fungicide use. However, over time Z. tritici has developed resistance against multiple classes of fungicide. Predicting developments and identifying potential cross-resistances provides potential to reduce its impact. Over the course of my project, I tested Septoria isolates collected from 2003 to 2023 against a range of fungicides used during this time. This longevity study observed shifts in sensitivity to these compounds across 20 years.
This study was completed using a concentration assay. Septoria isolates from 2003, 2008, 2013, 2018 and 2023 were taken from cold storage and grown on agar plates at 15°C. Once grown samples were scaped from the agar plates and added to sterilised water. The number of spores present were counted and diluted to the same concentration for the assay. A serial dilution of each fungicide was completed before adding to 96 well plates. No fungicide was added in column 1 as a control to ensure samples could be grown, then increasing concentrations of each fungicide were added across the plates. Septoria spores were added, then the plates were incubated for 4 days before reading using a microplate reader. This process was repeated for around 50 isolates for each of the 5 years tested, then data was analysed.
Initially EC50 values – the point the isolate is regarded as resistant to that particular fungicide – were plotted. This showed an increase in resistance to all of the tested fungicides across the 20-year period. This highlighted the ability of Septoria to develop resistance to multiple classes of fungicide and the importance of finding alternatives to reduce its impact.
Next potential cross-resistance patterns were explored. Again, EC50 values for two different compounds were plotted against each other to observe any correlations. Cross-resistance patterns are key for understanding how different compounds contribute to resistance development as well as to inform scientists and farmers about potential to use compound combinations or if this would drive further resistance development.
Towards the end of this project EC50 values for each fungicide for each year were ranked. Isolates with a range of EC50 values were grown again and DNA extracted. Next PCR and gel electrophoresis were completed to assess if known causative mutations of increased resistance were present. This work is on-going.
I enjoyed the freedom of this project in comparison to University practical sessions. It has been nice to see the data come together and look forward to hearing where the research goes. This project has improved my confidence and independence around the lab as well as given me the opportunity to practise techniques I had previously only learnt about in lectures. It has confirmed that lab work is for me and has opened up the field of plant pathology. Working with fungal pathogens has reaffirmed this is an area of interest for me and it has been great to research them further as this is an area we cover less of at University.
I would like to thank the team at NIAB in particular Dr Pooja Pandey for helping me get to grips with the practical techniques, and Dr Nichola Hawkins for supervising the project as well as the data analysis and DNA extractions. Additionally, I would like to thank the BSPP for funding this summer placement.
Natalie Shores
University of Birmingham
Zymoseptoria tritici viewed at x250
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
