Abstract
The filamentous ascomycete Fusarium graminearum is the causal agent of Fusarium head blight, a devastating disease of cereals with a worldwide distribution. Fusarium graminearum infections result in a quantitative yield reduction by impairing the growth of the kernels, and a qualitative reduction by poisoning the remaining kernels with mycotoxins toxic to animals and humans. The colonization of wheat florets by phytopathogenic fungus requires high-efficiency energy generation in the mitochondria (Bönnighausen et al., 2015). Mitochondrial activity in microorganisms can be measured using the oxygen consumption rate (OCR) method. Here we describe a method for the assessment of fungal respiration using an XF24 extracellular flux analyzer. The Seahorse XF Analyzer is a microplate-based respirometer which measures oxygen consumption by changes in the fluorescence of immobilized fluorophores (Gerencser et al., 2009). Multiple mitochondrial parameters can be measured by the application of mitochondrial substrates and inhibitors which are injected automatically during the assays via ports (Divakaruni et al., 2014). The experimental work-flow involves the inoculation with conidia and the application of specific inhibitors of mitochondrial functions. The analysis of fungal respiration represents a valuable tool that complements classical phenotypic screenings.
Materials and Reagents
Equipment
Software
Procedure
All steps of OCR measurement are summarized in Figure 1. This section describes the setup of an appropriate program on the Seahorse controller. The injection compounds will be injected subsequently in all wells by air pressure.
Representative data
A measurement of fungal respiration according to the procedures described above should typically result in an OCR as depicted in Figure 3. We recommend 5 replicates per experimental group. Figure 3. Oxygen consumption rate of mycelia of Fusarium graminearum grown in liquid minimal medium
Notes
The measurement of oxygen consumption rates in axenic culture described here was highly reproducible.
Recipes
Acknowledgments
The authors thank the “Kompetenzzentrum Nachhaltige Universität” of the University Hamburg for funding.
References
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