Fungal seed pathogens of wild chili peppers possess multiple mechanisms to tolerate capsaicinoids

The wild chili pepper Capsicum chacoense produces the spicy defense compounds known as capsaicinoids, including capsaicin and dihydrocapsaicin, antagonistic to the growth of fungal pathogens. Compared to other microbes, fungi isolated from infected seeds of C. chacoense possess much higher tolerance to these spicy compounds, having their growth slowed, but not entirely inhibited. Previous research has shown capsaicinoids inhibit microbes by disrupting ATP production via the binding of NADH dehydrogenase in the Electron Transport Chain (ETC), throttling Oxidative Phosphorylation (OXPHOS). Capsaicinoids may also disrupt cell membranes. Here, we investigated capsaicinoid tolerance in fungal seed pathogens isolated from C. chacoense. We selected 16 fungal isolates from four Ascomycete genera (Alternaria, Colletotrichum, Fusarium and Phomopsis). Using relative growth rate as a readout for tolerance, fungi were challenged with ETC inhibitors to infer if fungi possess alternative respiratory enzymes, and if effects on the ETC fully explained inhibition by capsaicinoids. In all isolates, we found evidence for at least one alternative NADH dehydrogenase. In many isolates we also found evidence for an alternative oxidase. These data suggest wild plant pathogens may be a rich source of alternative respiratory enzymes. We further demonstrate these fungal isolates are capable of the breakdown of capsaicinoids. Lastly, we determine the OXPHOS theory weakly explains the primary mechanism by which dihydrocapsaicin slows fungal growth, but not capsaicin. Our findings suggest capsaicinoids likely disrupt membranes in addition to energy poisoning, with implications for microbiology and human health.\n\nImportancePlants make chemical compounds to protect themselves. For example, chili peppers produce the spicy compound capsaicin to inhibit animal feeding and pathogen damage. In humans, capsaicin binds to a membrane channel protein, creating the sensation of heat, while in microbes, capsaicin limits energy production by binding respiratory enzymes. However, some data suggest capsaicin also disrupts membranes. Here we studied fungal pathogens (Alternaria, Colletotrichum, Fusarium, and Phomopsis) isolated from a wild chili pepper, Capsicum chacoense. By measuring growth rate in the presence of antibiotics with known respiratory targets, we infer wild plant pathogens may be rich with alternative respiratory enzymes. A zone of clearance around the colonies, as well as LCMS data, further indicate these fungi can break down capsaicin. Lastly, the total inhibitory effect of capsaicin was not fully explained by its effect on respiratory enzymes. Our findings lend credence to studies proposing capsaicin may disrupt cell membranes, with implications for microbiology as well as human health.