Written by Nicolas Trenk, University of Bath.
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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Take-all disease of wheat is a root disease caused by the soil-borne ascomycete fungus Gaeumannomyces tritici (Gt). It is one of the most important root diseases of cereals, with wheat losses of up to 50 % posing a substantial risk to global food security. The necrotrophic fungus colonises the stele, thereby restricting water and nutrient flow. This causes dark lesions on roots and patches of stunted, prematurely ripening plants. Understanding and finding novel genetic sources of resistance would be beneficial in finding alternative control strategies.
The aim of my project was to explore resistance found in Triticum monoccocum, an ancestral diploid wheat relative, and an ancestral rye relative. A previous study has identified a resistant (MDR031) and a susceptible ancestral wheat line (MDR043). The comparison of a resistant and a susceptible ancestral rye line may give an indication of rye resistance mechanisms. To examine levels of resistance, two 3-week single and combined take-all isolate inoculation pot bioassays were set up for wheat and rye. Both assays compared two ancestral and a cultivated variety, each having 5 replications of 6 combinations of Gt reference strain inoculants. The plants were grown in Vermiculite pots containing 10 seeds and 4 mycelium plugs each for 3 weeks. The seedling root systems were then washed, individually visually assessed for infected roots, and weighed.
Conducting a two-way analysis of variance (ANOVA) on rye root infection showed that there are significant differences in fungal isolates and crop variety. A post-hoc Tukey test revealed that infection of isolate 4 is significantly higher than other isolates. However, this strong increase in infection does not seem to take place in the susceptible rye variety. This result suggests that this take-all isolate has at least partially overcome resistance of rye cultivars. This fact would offer the possibility to identify the fungal genes involved in overcoming resistance. ANOVA of the wheat infection also showed significant differences, however, there is only a small difference in infection between resistant and susceptible lines. This lack of variation may be due to wheat only displaying adult plant resistance, whereas rye displays resistance already at the seedling stage. A post-hoc Tukey test on isolates revealed that the combined isolate inoculation was significantly lower than isolate 2. This could indicate microbial competition between different take-all strains. Analysis of the dry weights revealed no significant differences in wheat or rye. This suggests that three weeks was not enough for the fungus to properly restrict nutrient and water flow.
The second part of the project was concerned with characterising take-all isolates obtained from a previous root core sample obtained in June from mature susceptible and resistant T. monococcum plants growing in field conditions. These were subsequently subcultured and hyphal tipped to obtain pure cultures. They were then tested for sensitivity to the fungicide silthiofam and their DNA extracted to be used for characterisation of type A or B by touchdown polymerase chain reaction. The objective of this experiment was to obtain take-all strains associated with resistant and susceptible lines.
My 6-week summer studentship at Rothamsted Research, which was made possible by the BSPP, provided me with the motivation and excitement to continue my studies with the aims of obtaining a PhD within plant pathology. I also developed an invaluable understanding and confidence in laboratory- and field techniques, including aseptic technique and working with fungal cultures, fungal inoculation techniques, disease assessment, DNA extraction and quantification of mycelium and root samples. It also gave me a deep insight into experimental design, statistical analysis and scientific communication. I would like to thank my supervisors Dr Javier Palma-Guerrero, Prof. Kim-Hammond-Kosack and Jess Spong for their support and guidance during this studentship.
FIGURE 1: Percentage root infection for different combinations of isolate inoculations on ancestral and cultivated rye. Cultivated wheat (Hereward) was included to compare resistance of rye to wheat. Note how the infection of the susceptible variety barely changes for isolates compared to resistant and cultivated varieties. The resistant rye and commercial rye exhibit seedling resistance to three of the four take-all (Gt) isolates tested.
Figure 2: Percentage root infection for different combinations of isolate inoculations on ancestral and cultivated wheat.
