Undergraduate Student Report 2022

Sporulation of Zymoseptoria tritici

Ella Vettori, University of Bristol

Over the summer I had the pleasure of assisting Dr Andy Bailey and his PhD student, Rosie Ford, in their investigation of the fungal pathogen, Zymoseptoria tritici. The project was very hands-on, and my involvement in the investigation ranged from molecular work in the lab through to spending hours in the greenhouses inoculating wheat plants with the fungus, scoring plants for infection and later on scanning their leaves to quantify lesions and pycnidia.

Zymoseptoria tritici is a fungal pathogen that causes necrosis in wheat leaves. Wheat is the second most cultivated crop globally and is thus a paramount part of food security. To date, commercial fungicides have been used in an attempt to control the spread of the fungus, however Z. tritici has developed resistance to multiple fungicides, rendering this control method problematic. Plant breeding can deliver resistant cultivars of wheat, but the sexual cycle of the fungus means there are high levels of genetic reassortment in the pathogen population, so resistance can be short-lived, as was recently seen with the cultivar Cougar. This calls for further research into the relationship between the pathogen and current global cultivars. Recent literature suggests that Z. tritici has undergone strong adaptive evolution, suggesting how different cultivars of wheat affect effector gene expression. Therefore, over the course of my studentship, I set out to investigate the interaction between different isolates of Z. tritici and different wheat cultivars of diverse global origins, and to embed image analysis methods within the research group to better quantify infection and sporulation.

Initially a collection of 14 different strains of Z. tritici were investigated, the majority were kindly provided by Prof. B. McDonald (ETH Zurich) as well as some strains of local origin. These were grown on PDA and CZ-V8 media and strain TN26 (from Tunisia) gave a yellow coloured colony compared to the more usual salmon-pink sporidial cultures for the other strains. To look for interactions or competition between strains, paired cultures were prepared to cover all possible combinations. The cultures were assessed visually for any interactions on agar plates, but other than some minor changes in the balance between sporidial vs mycelial growth, no significant changes were observed to morphology of the mixed cultures compared to pure culture, despite these pairings including strains of diverse geographic origin and representing different mating types. Genomic DNA was extracted from all of these isolates for us in a different study.

Six of the Z. tritici of different global origin were taken through to in planta study against a panel of wheat cultivars. Sporidia of each strain were grown on plates of CDV8 agar for 5 days, harvested from the plates and resuspended in 0.05% tween 80 at a concentration of 5x106ml-1. Trays of 12 wheat cultivars were grown for 3 weeks from seed before being inoculated with the different isolates. Whilst still attached to the plant, the first true leaf of each seedling was held horizontal by sticking to a cardboard sheet using double-sided tape and brushing the isolates onto the surface of the leaf with a cotton bud (Figure 1). A minimum of four leaves were scored for each strain:cultivar interaction. The plants were then kept in clear plastic bags for 3 days to create adequate humidity for infection. The bags were removed and the plants were left to grow for 3 weeks before the infected leaves were scored for infection referring to a 0-4 disease scale (Figure 2).

Vettori Figure 1

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1 Preparation of wheat leaves for inoculation.

 

Vettori Figure 2

 

 

 

 

 

 

 

 

 

 

Figure 2 Disease scale used to assess the level of infection.

 

All the strains tested were capable of causing disease with isolates ISY92 (Israel) and 3D7 (Switzerland) observed to cause the most symptoms. The wheat cultivar Synth 46 was found to be most resistant to infection for all of the Z. tritici strains tested here as no symptoms were observed (Figure 3), whilst Paragon appeared to be universally susceptible.

Vettori Figure 3

 

 

 

 

 

 

 

Figure 3 Disease assessment of the different wheat cultivars infected with various strains of Z. tritici scored on a 0-4 scale.  Wheat Synth 46 appears to exhibit fewest symptoms, while Paragon is most sensitive both to handling and infection. Data are averages of a minimum of 4 leaves per interaction.

 

There were differences between specific strain:cultivar combinations. As expected, Z. tritici IPO323 was avirulent on Riband, likely due to stb6 resistance.

The infected leaves were then harvested and mounted on paper with respective QR codes for automated image analysis (Figure 4). The scans were saved and uploaded to ImageJ for further analysis. This technique was new to the lab and it took a while to get the system working with the available scanner to ensure correct resolution of image and recognition of the QR code and page coordinates. Sporulation analysis using the imaging software ImageJ will be carried out by Rosie Ford as my time came to an end before I was able to complete it myself. However, using disease assessment and scanning equipment, it is clear that Synth 46 is resistant to infection from all strains evaluated here. Whether this would hold true in a field situation is yet to be determined as lab resistance and field resistance are not directly comparable.

Vettori Figure 4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4. Mounting of wheat leaves for scanning and image analysis.

 

The image analysis has since been successful and in the future we hope to quantify pycnidial production from infected lesions, as this is likely a more relevant indication of ability of the fungus to reproduce and to cause epidemics in the field rather than simply the severity of the lesions.

Acknowledgements

I would like to thank the British Mycology Society for providing me with this opportunity to gain experience in hands-on research and working as part of a group in a lab. I feel that I have learnt valuable skills such as plant inoculation, experiment design and gained valuable experience of handling fungi, observing their growth and applying them to plants.  It has given me insight into things like time management and planning of work, especially where fungi and plants grow at different rates but need to be ready at the same time. In addition the molecular techniques I have performed have helped my understanding of the theory I have learnt through my undergraduate studies. I would also like to thank Dr Andy Bailey and Rosie Ford for taking me under their wing, and for their support and belief in my capabilities. I have learnt how to apply my knowledge, for which I am most grateful. My curiosity in mycology and plant-microbe interactions has grown deeper roots and I look forward to the research of the future!

Ella Vettori was supervised by Dr Andy Bailey (University of Bristol).