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

Fungal pathogens are a major threat to plant health and food security, and resistance to current fungicides—most of which target single enzymes—has become widespread. There is an urgent need for fungicides with multi-site modes of action that reduce resistance risk while remaining environmentally benign and safe for mammals. Mitochondria are attractive antifungal targets because fungal mitochondrial composition and respiratory enzymes differ from those of mammals. Lipophilic cations accumulate in the negatively charged mitochondrial matrix and may perturb oxidative phosphorylation. Mono-alkyl lipophilic cations (MALCs), including the fungicide dodine (C12-guanidinium), have antifungal activity, though their precise mode of action is unclear. This study investigates MALCs as crop fungicides, testing their effects on mitochondrial function, reactive oxygen species (ROS), fungal viability, and plant defense, and evaluates their efficacy and safety in crops and model organisms.

The authors contextualize the work by noting: (1) increasing resistance to azoles, SDHIs, and strobilurins threatens crop protection; (2) multi-site fungicides can reduce resistance but often have toxicity issues (e.g., chlorothalonil bans due to aquatic toxicity and effects on pollinators); (3) fungal mitochondria differ from mammalian mitochondria and are targeted by current fungicides that disrupt respiration; (4) lipophilic cations possess positive LogP values enabling membrane permeation and mitochondrial accumulation; (5) MALCs (cationic surfactants) have known antibacterial and antifungal activities, often attributed to plasma membrane or cell wall disruption, yet older studies on dodine suggest intracellular targets and metabolic enzyme inhibition, leaving MoA unclear. These points motivate exploring MALCs’ mitochondrial effects and multi-site antifungal potential.

- Fungal strains: Zymoseptoria tritici (IPO323 and fluorescent organelle/membrane marker strains), Magnaporthe oryzae (Guy11), Ustilago maydis (FB1 and marker strains). Growth on standard media with defined temperatures and conditions. - Compound panel: Commercial MALCs and synthesized variants differing in head group (guanidinium, trimethylammonium, triethylammonium, dimethylsulfonium) and alkyl chain length (C6, C12, C18); anionic controls and a symmetric bis-sulfonium control. Lipophilicity (LogP) estimated via SwissADME from SMILES. - Plasma membrane assays (Z. tritici): Live/dead staining; propidium iodide uptake; membrane potential changes using DiBAC4(3); fluorescence microscopy; electron microscopy for membrane ultrastructure. - Mitochondrial assays: Morphology via fluorescent markers and transmission EM; inner membrane potential by TMRM staining; ATP quantification in cell extracts using luciferase assay; oxygen consumption via modified Winkler titration; NADH oxidation measured in isolated mitochondria with colorimetric assay, including rotenone and diphenyleneiodonium controls. - ROS and apoptosis: Mitochondrial ROS detected with dihydrorhodamine-123 (DHR-123); pharmacological modulation with rotenone and antimycin A; apoptosis markers via CaspACE FITC-VAD-FMK (caspase/metacaspase activity) and Annexin-V-fluorescein (phosphatidylserine exposure) with propidium iodide counterstain. - Fungal development: M. oryzae conidial germination and appressorium formation assays on hydrophobic coverslips. - Plant studies: Phytotoxicity on wheat (Triticum aestivum cv. Galaxy) and rice (Oryza sativa cv. CO39) by high-dose leaf spray; protective efficacy measured by pre-spraying MALCs and subsequent inoculation with Z. tritici or M. oryzae, quantifying pycnidia/lesion areas; plant defense activation via DAB staining for H2O2 after MALC or salicylic acid treatment; rice biomass following repeated C18-SMe2+ sprays. - Safety assays: Human cells—C109 skin fibroblasts and HepG2 hepatoblastoma cells—assessed for mitochondrial morphology, membrane potential (TMRM), and viability (MTT assay). Daphnia magna toxicity: mitochondrial potential (TMRM) and 24 h immobilization EC50. Genotoxicity: Ames plate incorporation test (OECD 471) on multiple Salmonella/E. coli strains with and without S9 metabolic activation. - Imaging: High-resolution fluorescence microscopy (Olympus IX81/IX83), EM (JEOL JEM 1400). Statistical analyses used Prism (dose-response, ANOVA, t-tests, Mann–Whitney). EC50s corrected for counter-ion mass.

- Plasma membrane effects of C12-G+ in Z. tritici were minor at sublethal doses: PI uptake rose to ~30% only at 100 µg/ml; DiBAC4(3) indicated depolarization primarily in dead cells, suggesting the plasma membrane is not the primary target. - Mitochondrial targeting by C12-G+: EC50 for mitochondrial fragmentation 4.12 µg/ml; ultrastructure showed swollen/disorganized cristae; strong depolarization (TMRM loss) at <0.5 µg/ml; ATP levels significantly decreased at 5 µg/ml; dissolved oxygen consumption dropped markedly (control consumed 57.9% O2; C12-G+ samples reduced O2 by only 18% over the same period); NADH oxidation in isolated mitochondria significantly inhibited, comparable to rotenone+DPI controls. - Selectivity: Human fibroblasts required ~6.3× higher concentration for mitochondrial fragmentation and ~46× higher for depolarization than Z. tritici, indicating fungal selectivity of mitochondrial effects. - SAR and identification of improved MALCs: Long-chain cations (C18-NMe3+, C18-SMe2+) were most potent at inducing mitochondrial fragmentation (EC50 0.11 and 0.10 µg/ml, respectively) and depolarization (EC50 0.11 and 0.20 µg/ml) versus C12-G+ (0.28 µg/ml for depolarization). Both C18 compounds reduced ATP by ~54–59% at 5 µg/ml in 2 h and inhibited NADH oxidation in isolated mitochondria. Neither caused significant plasma membrane depolarization in live cells at high multiples of EC50. - Fungal killing: After 24 h at 10 µg/ml, mortality was 32.29% (C12-G+), 66.47% (C18-SMe2+), and 89.27% (C18-NMe3+). - Unique ROS/apoptosis with C18-SMe2+: Unlike C12-G+ and C18-NMe3+, C18-SMe2+ increased mitochondrial ROS at complex I (blocked by rotenone) after 0.5 h and 24 h. Chain-length specificity: C12-SMe2+ and C16-SMe2+ did not increase mROS. C18-SMe2+ significantly induced early apoptosis markers (CaspACE FITC-VAD-FMK; Annexin-V) in Z. tritici, whereas the other MALCs did not. - Cross-pathogen generality: In M. oryzae and U. maydis, MALCs disrupted mitochondrial ultrastructure and depolarized mitochondria; all inhibited M. oryzae germination (EC50 ~0.76–0.83 µg/ml), but only C18-SMe2+ increased mROS in both pathogens. - Plant efficacy and phytotoxicity: No chlorosis/necrosis in wheat or rice at 1000 µg/ml for any MALC. Pre-treatment protected against Septoria tritici blotch and rice blast in a dose-dependent manner; C18-SMe2+ achieved near-complete protection at 75–100 µg/ml and outperformed C12-G+ and C18-NMe3+ at higher doses. C18-SMe2+ induced a stronger DAB-detectable H2O2 burst at 6 h than other MALCs, indicating plant defense activation; no adverse effect on rice biomass over 28 days and slight growth promotion observed. - Safety profile: In human cells, C18-NMe3+ and C18-SMe2+ were less cytotoxic than C12-G+ by MTT (C109 EC50: 2.32 µg/ml for C12-G+, 4.13 for C18-NMe3+, 3.53 for C18-SMe2+; HepG2 EC50: 0.99, 2.42, 3.67 µg/ml, respectively). In Daphnia magna, 24 h immobilization EC50s: 0.31 µg/ml (C12-G+), 1.61 (C18-NMe3+), 2.42 (C18-SMe2+); TMRM showed acute mitochondrial effects primarily with C12-G+. Ames tests were negative for mutagenicity for C18-SMe2+ (±S9), though bactericidal at >35 µg/ml.

The study demonstrates that MALCs primarily target fungal mitochondria, inhibiting oxidative phosphorylation by reducing NADH oxidation and depolarizing the inner membrane. These mitochondrial effects underlie fungal selectivity, likely due to fungal-specific components of complex I and alternative NADH dehydrogenases, and possibly membrane biophysical perturbations. Among MALCs, C18-SMe2+ exhibits a multi-pronged antifungal mechanism: inhibition of respiration, induction of complex I-linked mitochondrial ROS, and activation of fungal apoptosis, alongside elicitation of a plant oxidative burst indicative of defense activation. This combination accounts for strong in planta protection against Septoria blotch and rice blast with no detectable phytotoxicity and a favorable safety margin relative to dodine in human cells and Daphnia. The multi-site mode of action reduces the likelihood of resistance emergence. While direct molecular interactions (e.g., binding within complex I) are proposed, additional mechanisms such as altering IMM fluidity or protonophoric effects may contribute and warrant further elucidation.

MALCs, particularly the dimethylsulfonium long-chain cation C18-SMe2+, are promising crop-protective antifungals. They act mainly on fungal mitochondria to inhibit oxidative phosphorylation and, uniquely for C18-SMe2+, elevate complex I-dependent ROS, triggering apoptosis in pathogens and priming plant defenses. C18-SMe2+ effectively protects wheat and rice without phytotoxicity and displays lower toxicity than the established MALC fungicide dodine in human cells and Daphnia, with no detectable mutagenicity. Future work should clarify precise molecular targets within the respiratory chain, assess environmental fate (stability and biodegradability), and expand toxicological profiling to support development as a sustainable fungicide.

- Mechanistic details remain to be fully resolved: the precise binding site(s) and molecular consequences of MALCs on respiratory complexes (especially complex I) and membrane biophysics require further study. - Toxicology and environmental assessments were limited in scope; broader and longer-term studies (mammalian toxicity, ecotoxicology, biodegradability, environmental persistence) are needed. - Efficacy concentrations vary across assay formats (in vitro cells vs. leaf surfaces), likely influenced by plant cuticle absorption; translation to field conditions will require formulation optimization and field trials.