A lipophilic cation protects crops against fungal pathogens by multiple modes of action

The increasing resistance of crop pathogens to fungicides presents a significant threat to global food security. The majority of current fungicides target single enzymes, making them vulnerable to resistance development through single point mutations. This resistance has already emerged against azoles, succinate dehydrogenase inhibitors (SDHIs), and QoI inhibitors (strobilurins), jeopardizing agricultural yields. Multi-site fungicides, which disrupt multiple cellular processes, offer a potential solution, but often come with increased toxicity, exemplified by the recent ban of chlorothalonil due to its environmental and ecological impacts. This research focuses on the potential of mono-alkyl lipophilic cations (MALCs) as a novel class of environmentally benign fungicides with multi-site modes of action. Fungal mitochondria, responsible for ATP production through oxidative phosphorylation, represent an attractive target for fungicide development because their composition and respiratory enzyme inventory differ significantly from their mammalian counterparts. Furthermore, fungal mitochondria produce reactive oxygen species (mROS), and deregulation of mROS production can lead to mitochondrial damage and programmed cell death (apoptosis), providing additional targets for antifungal intervention. Lipophilic cations, characterized by a cationic head group and a lipophilic moiety, can accumulate in mitochondria due to the negatively charged mitochondrial matrix, potentially inhibiting respiratory enzymes. While this property has challenged their use in therapeutics, it could be leveraged for antifungal development. MALCs, also known as cationic surfactants, exhibit antibacterial and antifungal activity, but their mode of action is not fully understood. Dodine (C₁₂-G⁺), an existing MALC fungicide, suggests a complex mode of action potentially involving both membrane interaction and intracellular enzyme inhibition. This study aims to investigate the antifungal potential of MALCs against important crop pathogens, elucidate their mode of action, and assess their environmental and mammalian toxicity.

Existing literature highlights the urgent need for novel fungicides with multi-site modes of action (MoA) to combat the growing resistance of fungal pathogens to currently used fungicides. The majority of current fungicides target single sites within the fungal cell, making them susceptible to rapid resistance development. The development of resistance to azoles, SDHIs, and QoI inhibitors, three major classes of fungicides, underscores this vulnerability. Fungal mitochondria have emerged as attractive targets for the development of novel antifungal agents. Their unique features, compared to mammalian mitochondria, offer the possibility of selective toxicity. The role of mitochondria in ATP synthesis, lipid homeostasis, and programmed cell death has highlighted multiple potential targets for disruption by antifungal compounds. Previous studies have indicated that lipophilic cations can accumulate in mitochondria due to the negative charge of the mitochondrial matrix and may inhibit crucial respiratory chain components. However, these studies primarily focused on their effects on mammalian cells and lacked comprehensive investigation into their antifungal potential. MALCs have shown promise as antibacterial and antifungal agents, but the exact mechanism remains debated. While some studies propose membrane disruption as the primary mode of action, other research suggests that MALCs can interfere with various metabolic enzymes. Dodine, a commercially available MALC fungicide, exemplifies this ambiguity, with its mode of action currently classified as “unknown”. This existing ambiguity regarding the mechanism of action of MALCs necessitates further investigation.

This research employed a multi-faceted approach to investigate the antifungal properties and mode of action of MALCs. The primary model organism used was *Zymoseptoria tritici*, a significant pathogen of wheat, for which a range of live-cell imaging marker strains had been previously developed. The study also included *Ustilago maydis* (corn smut) and *Magnaporthe oryzae* (rice blast) to assess the generality of the findings. Initial experiments focused on assessing the effects of C₁₂-G⁺ (a known MALC) on the plasma membrane of *Z. tritici* using live/dead staining, propidium iodide uptake, and DiBAC₄(3) staining to monitor membrane integrity and potential. To investigate the mitochondrial effects, the researchers utilized fluorescent mitochondrial markers and electron microscopy to visualize changes in mitochondrial morphology. Measurements of ATP concentration and oxygen consumption were used to quantify the effects of C₁₂-G⁺ on oxidative phosphorylation. NADH oxidation assays with isolated mitochondria further investigated the specific site of action within the respiratory chain. A series of MALCs with varying alkyl chain lengths and head groups were synthesized and tested for their effects on mitochondrial fragmentation and depolarization in *Z. tritici*. The most effective MALCs were then tested for their ability to induce ROS production using the DHR-123 dye, and the role of ROS in inducing fungal apoptosis was assessed using CaspACE FITC-VAD-FMK and Annexin-V-fluorescein staining. The study further investigated the antifungal activity of the most promising MALCs against *M. oryzae* (rice blast) and *U. maydis* (corn smut), including tests of mitochondrial morphology, membrane potential, and ROS production. Plant protection assays were conducted on wheat and rice plants, assessing the ability of MALCs to protect against infection by *Z. tritici* and *M. oryzae*, respectively. Phytotoxicity assays were performed to evaluate the effects of MALCs on plant growth. Finally, toxicity assays using *Daphnia magna* and human cell lines (C109 fibroblasts and HepG2 hepatoblastoma cells) along with an AMES test for mutagenicity were conducted to evaluate the safety profile of the MALCs. Advanced techniques such as fluorescence microscopy, electron microscopy, and quantitative assays were used to obtain detailed data on cellular responses and biochemical changes.

This study revealed several key findings regarding the antifungal properties and mode of action of MALCs: 1. **C₁₂-G⁺ primarily targets fungal mitochondria:** While exhibiting some effects on the plasma membrane at high concentrations, C₁₂-G⁺ primarily inhibits fungal growth by targeting mitochondria, leading to mitochondrial fragmentation, depolarization, and impaired ATP synthesis by reducing NADH oxidation. The EC₅₀ values for mitochondrial fragmentation and depolarization were significantly lower than those for plasma membrane disruption. 2. **Longer alkyl chains enhance MALC activity:** MALCs with longer alkyl chains (C₁₈) demonstrated significantly greater antifungal activity compared to those with shorter chains (C₁₂). The C₁₈-alkyl chain cations (C₁₈-NMe₃⁺ and C₁₈-SMe₂⁺) were more potent in inducing mitochondrial fragmentation and depolarization than C₁₂-G⁺. 3. **C₁₈-SMe₂⁺ induces ROS production and apoptosis:** Unlike other MALCs tested, C₁₈-SMe₂⁺ specifically induced mROS production at respiratory complex I, triggering apoptotic cell death in *Z. tritici*. This effect was dependent on the C₁₈ alkyl chain length, as shorter chain analogues did not induce mROS production. 4. **C₁₈-SMe₂⁺ activates plant defense:** In addition to its direct effects on the pathogen, C₁₈-SMe₂⁺ triggered an oxidative burst in rice leaves, suggesting an activation of plant defense mechanisms. 5. **C₁₈-SMe₂⁺ demonstrates broad-spectrum antifungal activity:** The effects of C₁₈-SMe₂⁺ on mitochondrial function, ROS production, and apoptosis were observed not only in *Z. tritici*, but also in *M. oryzae* and *U. maydis*. 6. **C₁₈-SMe₂⁺ exhibits low toxicity:** C₁₈-SMe₂⁺ demonstrated significantly lower toxicity in *Daphnia magna* and human cells than C₁₂-G⁺ (Dodine). Furthermore, it showed no mutagenic activity in AMES tests and no phytotoxicity in wheat and rice plants at high concentrations (1000 µg/ml). 7. **C₁₈-SMe₂⁺ effectively protects plants against fungal pathogens:** In plant protection assays, C₁₈-SMe₂⁺ effectively reduced the severity of *Septoria tritici* blotch in wheat and rice blast disease in rice, significantly outperforming C₁₂-G⁺ and C₁₈-NMe₃⁺. The multi-site mechanism of action of C₁₈-SMe₂⁺ suggests a lower risk of resistance development.

This study's findings provide compelling evidence for the potential of MALCs, particularly C₁₈-SMe₂⁺, as effective and non-toxic crop fungicides. The multi-site mode of action involving mitochondrial inhibition, ROS induction, apoptosis induction, and plant defense activation makes the development of resistance by fungal pathogens less likely than with current single-site fungicides. The specific activity of C₁₈-SMe₂⁺ on respiratory complex I, inducing mROS production and triggering apoptosis, highlights the value of targeting this essential mitochondrial process in fungal pathogens. The observed synergy between C₁₈-SMe₂⁺'s effects on the pathogen and its activation of plant defense further contributes to its enhanced efficacy. Furthermore, the low toxicity profile of C₁₈-SMe₂⁺ in human cells, *Daphnia magna*, and crop plants indicates its potential for safe and sustainable use in agriculture. The research provides a strong foundation for the development of C₁₈-SMe₂⁺ or related MALCs as next-generation fungicides to address the global threat of crop disease and safeguard food security. The identification of a compound with such a broad spectrum of activity and a low toxicity profile is a major achievement in the search for novel and sustainable pest management tools.

This research demonstrates that C₁₈-SMe₂⁺, a lipophilic cation, exhibits potent antifungal activity against important crop pathogens through a novel multi-site mode of action. Its ability to inhibit oxidative phosphorylation, induce ROS production and apoptosis in the pathogen, and trigger plant defense mechanisms makes it an exceptionally promising candidate for development as a new generation fungicide. The compound's low toxicity profile further enhances its appeal for sustainable agriculture. Further research should focus on optimizing the formulation and delivery of C₁₈-SMe₂⁺, conducting large-scale field trials, and exploring other MALCs with similar properties.

While this study presents compelling evidence for the potential of C₁₈-SMe₂⁺ as a fungicide, several limitations should be considered. The in vitro and greenhouse studies may not fully reflect the complexity of field conditions. Further research is needed to evaluate the long-term efficacy of C₁₈-SMe₂⁺ in diverse environments and under various climatic conditions. Moreover, detailed mechanistic studies are necessary to fully elucidate the molecular mechanisms underlying the interactions of C₁₈-SMe₂⁺ with the respiratory chain and its effect on inducing apoptosis. The study focused on a limited number of fungal species, so testing against a wider range of plant pathogens is necessary to confirm the broad-spectrum activity of C₁₈-SMe₂⁺. Finally, a comprehensive environmental risk assessment needs to be conducted before considering large-scale applications of this compound in agricultural settings.