I joined the Molecular Plant Pathology group at the University of Bristol for my summer placement. My main project was based on the wheat pathogenic fungus Zymoseptoria tritici, but I also had the opportunity to gain experience in handling and working with plant viruses such as Cassava Brown Streak Virus. Z. tritici is a pathogenic fungus that causes Septoria tritici blotch in wheat, resulting in significant yield losses and necessitating the use of fungicides for crop protection. Despite being a major disease of wheat in the UK little is known about how various strains of septoria impact different wheat cultivars. My project aimed to investigate how symptoms differed in various wheat cultivars when inoculated with different septoria strains, in particular, how the cultivars Bobwhite and Fielder respond, as these are cultivars which are amenable to transformation and CRISPR editing.
In the initial stages of my project comparisons of growth rate and colony morphology were made between the different septoria strains provided; the type strain IPO323 and four local isolates that originated from the Long Ashton Research Station: L951, ST11, ST16 and ST93. The procedure involved inoculating the septoria strains into liquid culture and measuring the optical density of the liquid cultures as a measure of growth rate. Differences in morphology of the septoria strains were recorded through investigation under the microscope.
To investigate the impacts of the different septoria strains on various wheat cultivars the septoria strains were first cultured onto CDV8 plates for a week. A spore suspension was prepared at a concentration of 4×10⁶ spores/ml and this was then applied to the wheat cultivars Bobwhite, Cadenza, Fielder and Riband by gently spreading the spore suspension across the 1st true leaf using a cotton bud (plants at growth stage 12-13). Symptoms of disease were scored for three weeks after inoculation. At the conclusion of the time-course, leaves were analysed for numbers of pycnidiospores that had been made. On harvesting, inoculated leaves were exposed to high humidity overnight and then suspended in water and vortexed to release spores.
The data collected provided key insights into the impact of septoria on wheat cultivars. For example despite Fielder displaying symptoms of chlorosis and developing necrotic lesions for the strains L951, ST11 and ST16 there was a low concentration of pycnidiospores generated. This means that although being susceptible, Fielder was not at major epidemic risk due to the low concentration of pycnidiospores being produced. Notably Fielder was highly resistant to the IPO323 type strain of Z. tritici, whilst Bobwhite was highly susceptible.
During my placement I was also able to design my own experiments investigating how temperature and suspension in liquid affected the viability of septoria. To investigate the effects of temperature on the different septoria strains the same inoculation method was used on the wheat cultivar Riband, a susceptible host. These trays were then placed in rooms with different environments and different types of supplemental lighting, including temperature regulated compartments at 18°C and 20°C and areas with no regulation of temperature. This helped to highlight the parameters needed for effective infection. To investigate the impact of suspension of the spores in water on viability of septoria the number of colonies grown in YPDA plates after suspension in liquid for different amounts of time was assessed. Their virulence on the wheat cultivar Riband was also assessed by inoculating with the spore suspension after different amounts of time in water.
Whilst waiting for plants to develop symptoms for Z. tritici, I was able to be involved in other projects such as the research into gene function in CBSV. This virus is a major threat to the security of Cassava production in East Africa. I was able to help the team by performing routine tasks such as RNA extraction and qPCR to help assess viral titre in various mutant clones, and to set up and score infections of the model host plant N. benthamiana.
During my placement I gained vital insight into what was required to design an experiment and learnt a variety of techniques used in plant pathology such as culturing microbes, plant inoculation and analysis of symptoms.
I am very grateful to the BSPP for providing me with the opportunity to gain valuable experience, that will benefit me in the future, as I hope to pursue a career in research. I would also like to thank my supervisor Dr Andy Bailey for his support and enthusiasm throughout my placement and to the other members of lab 321 for their help and encouragement.
University of Bristol