Abstract
The ascomycete fungus Fusarium graminearum is a major causal agent of Fusarium head blight (FHB), a devastating disease affecting small grains cereals worldwide. To better understand the pathogenesis of this fungus, we provide here an easy-to-use protocol to examine the sensitivity of the wild-type and mutant strains of F. graminearum to oxidative stress from superoxide anions (O2•-) generated by menadione. Similarly, this assay can also be used to detect other stress responses of different fungal strains to various stress agents. The change in stress response of a mutant can offer a clue for the biological function of mutated genes.
Keywords: Fusarium graminearum, Mutant, Mycelial disc, Sensitivity, Oxidative stress
Background
The ascomycete fungus Fusarium graminearum (previously also called Gibberella zeae for its sexual state) is not only the major causal agent of Fusarium head blight and seedling blight on wheat and barley, but also one of the important causal agents of Gibberella stalk rot on maize (Dal Bello et al., 2002; Bai and Shaner, 2004; Kazan et al., 2012). Apart from causing a huge yield loss of cereals, this fungus also produces mycotoxins which affect human and animal health. Therefore, this fungus has received extensive attention, and ranked the fourth among all investigated plant pathogenic fungi (Dean et al., 2012).F. graminearum overwinters on dead organic matter, particularly on infected crop residues of small grains and corn. To survive such a wide range of environment, F. graminearum has evolved the capacity to confront various stresses. Numerous genes in F. graminearum have been explored for their roles in counteracting stress treatment, and the resulting mutants exhibited diverse responses to the stresses, which indicated the association of the stress responses and pathogenicity (Son et al., 2011). Some of the frequently used stress agents that act on cell wall or cell membrane of fungi include oxidative stress agents (e.g., menadione and H2O2), cell wall-perturbing agents (e.g., Congo red), and membrane stress agents (e.g., SDS). Detection of the stress responses of a fungal mutant strain could provide a clue for further investigating pathogenetic function. Thus, we take oxidative stress treatment as an example to describe a reliable protocol to assess stress response of F. graminearum to a superoxide radical generating agent menadione (Kawamura et al., 2006). This protocol can be used to detect other stress responses of a fungal strain in an analogous procedure.
Materials and Reagents
Equipment
Software
Procedure
Note: This procedure should be carried out following aseptic techniques, working in a biological safety cabinet.
Data analysis
Notes
To obtain enough discs of the same size, prepare at least two V8 agar plates for culturing each strain (Step 1).
Recipes
Acknowledgments
We thank Mr. Gregory Brumberg for English editing. This protocol was modified from the previous method by Yao et al. (2016). This work was supported by the Ministry of Science and Technology of China (Grant 2016YFD0100600), the Natural Science Foundation of China (Grant 31730077) and the Ministry of Agriculture of China (Grant 2016ZX08009-003). The authors declare that there is no conflict of interests.
References
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