Fungal Seed Pathogens of Wild Chili Peppers Possess Multiple Mechanisms To Tolerate Capsaicinoids

Abstract

The wild chili pepper Capsicum chacoense produces the spicy defense compounds known as capsaicinoids, including capsaicin and dihydrocapsaicin, which are antagonistic to the growth of fungal pathogens. Compared to other microbes, fungi isolated from infected seeds of C. chacoense possess much higher levels of tolerance of these spicy compounds, having their growth slowed but not entirely inhibited. Previous research has shown capsaicinoids inhibit microbes by disrupting ATP production by binding NADH dehydrogenase in the electron transport chain (ETC) and, thus, throttling oxidative phosphorylation (OXPHOS). Capsaicinoids may also disrupt cell membranes. Here, we investigate 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 whether fungi possess alternative respiratory enzymes and whether 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 that wild-plant pathogens may be a rich source of alternative respiratory enzymes. We further demonstrate that these fungal isolates are capable of the breakdown of capsaicinoids. Finally, we determine that the OXPHOS theory may describe a weak primary mechanism by which dihydrocapsaicin, but not capsaicin, slows fungal growth. Our findings suggest that capsaicinoids likely disrupt membranes, in addition to energy poisoning, with implications for microbiology and human health.IMPORTANCE Plants make chemical compounds to protect themselves. For example, chili peppers produce the spicy compound capsaicin to inhibit pathogen damage and animal feeding. 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 that 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 rates in the presence of antibiotics with known respiratory targets, we inferred that wild-plant pathogens might be rich in alternative respiratory enzymes. A zone of clearance around the colonies, as well as liquid chromatography-mass spectrometry data, further indicated that these fungi can break down capsaicin. Finally, the total inhibitory effect of capsaicin was not fully explained by its effect on respiratory enzymes. Our findings lend credence to studies proposing that capsaicin may disrupt cell membranes, with implications for microbiology, as well as human health.

Keywords: Alternaria; Colletotrichum; Fusarium; OXPHOS; Phomopsis; capsaicin; capsicum; coevolution; complex I; flavonoid; glycolysis; membrane; mitochondria; secondary metabolites; seed pathogens.

FIG 1

The standard and alternative respiratory enzymes of a fungal electron transport chain. Standard respiratory enzymes are shown in purple. The standard complexes both accept electrons and pump protons to generate proton motive force. The entire standard electron transport chain includes the following: complex I, the NADH dehydrogenase; complex II, the succinate dehydrogenase; membrane-embedded ubiquinone (Q); complex III, the cytochrome bc₁ complex; soluble cytochrome c (Cyt c); and complex IV, the cytochrome c oxidase. Complex V, the ATP synthase, is the site of electron-linked phosphorylation. Alternative respiratory enzymes named from Saccharomyces cerevisiae are shown in green. Three alternative complex I enzymes are known in fungi; these are Nde1 and Nde2, which embed in the external membrane, and Ndi1, in the internal membrane, facing the mitochondrial matrix. These alternative respiratory enzymes accept electrons from NADH but do not pump protons into the intermembrane space. The alternative oxidase (AOX) can directly transfer electrons from ubiquinol to oxygen, reducing oxygen to water, but it bypasses electron transfer through complexes III and IV, reducing overall proton motive force and subsequent ATP production compared to the effects of the standard complexes.

FIG 2

Percentages of inhibition of growth of indicated isolates on glycerol by various drugs. Error bars represent the error propagation standard deviations. (A) Percentages of inhibition on glycerol by rotenone, a complex I inhibitor. Average inhibition across isolates was low at 4.83%, indicating the presence of one or more alternative complex I enzymes. (B) Percentages of inhibition on glycerol by flavone, an inhibitor of multiple targets in the ETC, including alternative NADH dehydrogenases. Average inhibition across isolates was 67.69%. Partial sensitivity confirms the presence of alternative complex I enzyme(s). (C) Percentages of inhibition on glycerol by antimycin, a complex III inhibitor. Average inhibition across isolates was 44.30%, indicating the presence of an alternative oxidase. (D) Percentages of inhibition on glycerol by oligomycin, an ATP synthase inhibitor. Average inhibition across isolates was high at 80.00%, indicating no alternative ATP synthase.

FIG 3

Further evidence of an alternative oxidase. Percentages of inhibition on glycerol by complex III inhibitor antimycin are positively correlated with percentages of inhibition on glycerol by the broad-spectrum ETC inhibitor chloramphenicol, suggesting the presence of an alternative oxidase. Data are denoted by circles for Alternaria isolates, by triangles for Colletotrichum isolates, by squares for Fusarium isolates, and by plus signs for Phomopsis isolates. R² = 0.4254, P = 0.006176.

FIG 4

Effects of the ATP synthase inhibitor oligomycin are carbon dependent. Inhibition by oligomycin was greater on glycerol than on glucose, and percentages of inhibition were 15% higher on glycerol, the carbon source that cannot be fermented (one-sided t test, P = 0.03674; error bars show standard deviations).

FIG 5

The cost of tolerance to flavone. The lower the inhibitory effects of flavone on an isolate, the more slowly that isolate is able to grow on glucose in the absence of drugs (controlling for effects of the drug solvent DMSO). Data are denoted by circles for Alternaria isolates, by triangles for Colletotrichum isolates, by squares for Fusarium isolates, and by plus signs for Phomopsis isolates (R² = 0.2826, P = 0.03406).

FIG 6

LC-MS analysis of capsaicin degradation. LC-MS analysis confirmed that capsaicin was degraded in fungal plates but not in controls (two-sided t test, P ≤ 0.001, n = 3; error bars show 95% confidence intervals).

FIG 7

Fungal growth rates in the absence of drugs. Growth rates on glucose correlate strongly with growth rates on glycerol. Under normal conditions, these fungi use oxidative phosphorylation to produce the majority of their energy. Data are denoted by circles for Alternaria isolates, by triangles for Colletotrichum isolates, by squares for Fusarium isolates, and by plus signs for Phomopsis isolates (R² = 0.8736, P ≤ 0.001).

FIG 8

The effects of capsaicinoids outside the ETC. (A) OXPHOS inhibition alone explains the inhibitory effects of dihydrocapsaicin (DH-Capsaicin) (R² = 0.45, P = 0.0025). (B) OXPHOS inhibition alone does not explain the inhibitory effects of capsaicin (R² = 0.07698, P = 0.2981).