Fungal diseases can have a devastating effect on agricultural production. By some estimates, these infections account for the loss of as much as a quarter of crops globally each year. For decades, azole fungicides have been used to effectively control infections but exactly how these drugs work has remained a mystery until now. 

In a new study published in Nature Communications, University of Exeter scientists described the cellular mechanisms that azoles use to kill pathogenic fungi. Specifically, their results showed that this class of antifungals causes pathogens to activate self-destructive pathways when they are exposed to treatment. Their paper is titled, “Azoles activate type I and type II programmed cell death pathways in crop pathogenic fungi.”

To date, at least 25 different azoles have been developed and they account for a substantial amount of the global fungicide marketabout 25%. Previous studies found that azoles target enzymes in the pathogen’s cells that produce ergosterol, cholesterol-like molecules that are an important component of cellular bio-membranes. In earlier studies, scientists found that azoles deplete ergosterol levels which ultimately kills the pathogenic cell. They believed that depleting ergosterol levels caused perforations in the outer cell membrane resulting in cell death.

However, the Exeter scientists, led by Gero Steinberg, PhD, a professor of cell biology and director of Exeter’s Bioimaging Center, have found a different explanation. Their research used cells from Zymoseptoria tritici, a fungus that causes septoria leaf blotch in wheat and costs the U.K. more than £250 million (over $318 million) per year due to harvest loss and fungicide spraying

When Steinberg and his team subjected the cells to agricultural azoles and analyzed them using live cell imaging, they found evidence that two cell death pathways were activated in response to treatment. Specifically, azole-induced reduction of ergosterol increased mitochondrial activity. Increasing metabolism resulted in the formation of toxic by-products that kickstart apoptosis. They also found that reduced ergosterol levels triggered a self-destruct pathway which caused treated cells to engage in macroautophagy—a process where cells consume their own cytoplasm including nuclei and organelles.

The research showed that azoles work in the same way in Magnaporthe oryzae or rice-blast fungus, which kills up to 30% of the rice crop. They also tested other types of antifungals that target ergosterol biosynthesis and found that they work in a similar way. All initiated the same response suggesting that cell death pathway activation is a general consequence of inhibiting ergosterol biosynthesis. 

“Our findings rewrite common understanding of how azoles kill fungal pathogens,” said Steinberg. “We show that azoles trigger cellular ‘suicide’ programs, which result in the pathogen self-destructing. This cellular reaction occurs after two days of treatment, suggesting that cells reach a ‘point of no return’ after some time of exposure to azoles.”

However, fungal pathogens like their bacterial and viral counterparts can evolve. In fact, the 48-hour response time “gives the pathogen time to develop resistance against azoles, which explains why azole resistance is advancing in fungal pathogens, meaning they are more likely to fail to kill the disease in crops and humans,” Steinberg said. “We hope that our results prove to be useful to optimize control strategies that could save lives and secure food security.”

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