# Antifungal Activity of Quaternary Pyridinium Salts Against _Fusarium culmorum_ in Wheat Seedlings
## 1 Introduction
Given the environmental and health risks associated with conventional fungicides, there is an increasing need for alternatives with a smaller ecological footprint. Many conventional fungicides contain harmful chemicals that persist, contaminating soil and water and negatively affecting non-target organisms, including beneficial insects, aquatic ecosystems, and plants. Their accumulation can disrupt ecological balance and reduce biodiversity [22; 23; 24]. In response to these challenges, research has focused on developing more sustainable antifungal compounds that maintain or enhance efficacy while minimizing ecological risks. One such approach relies on synthetic compounds with antifungal potential, such as quaternary pyridinium salts, pyridine derivatives, and aromatic heterocyclic compounds containing a nitrogen atom. These salts are synthesized by reacting pyridine or its derivatives,such as nicotinamide and isonicotinamide, with suitable electrophiles [25]. Nicotinamide and isonicotinamide, derivatives of vitamin B3, are of particular interest due to changes in their chemical and physical properties during quaternization, which can enhance their biological activity. These compounds exhibit various biological activities, including antifungal properties [26; 27]. However, while quaternary pyridinium salts show promise as alternative antifungal agents, their synthesis often involves the use of toxic solvents, such as dimethylformamide, anhydrous benzene, and dichloromethane, in quaternization reactions of tertiary amines with organic halides [28; 29; 30; 31]. These solvents pose significant environmental and health hazards, contradicting the broader goal of developing more sustainable fungicide alternatives. Therefore, in addition to identifying effective antifungal agents, improving their synthetic routes to minimize ecological impact is equally important. Green chemistry principles have been increasingly applied to synthesizing quaternary pyridinium salts to address these challenges. A promising approach involves using deep eutectic solvents (DES), which offer an environmentally friendly alternative due to their biodegradability, nontoxicity, and biocompatibility [32; 33; 34]. Replacing hazardous organic solvents with DES enhances the sustainability of the production process while maintaining or even improving the biological activity of the synthesized compounds [35]. Nicotinamide-derived fungicides, such as boscalid, primarily exert their antifungal activity through the inhibition of succinate dehydrogenase (SDH), a key enzyme in both the tricarboxylic acid (TCA) cycle and the mitochondrial electron transport chain. SDH catalyzes the oxidation of succinate to fumarate, simultaneously facilitating the reduction of ubiquinone to ubiquinol via a series of electron transfers through flavin adenine dinucleotide (FAD) and iron-sulfur (Fe-S) clusters [36; 37; 38]. Different synthesis methods were explored to investigate the potential of quaternary pyridinium salts as environmentally friendly antifungal agents, and their effectiveness against phytopathogenic fungi was assessed. In our earlier in vitro studies, several compounds were synthesized using conventional methods, microwave-assisted synthesis [39; 40], and synthesis in DES [40; 41], all recognized in the literature as sustainable and efficient approaches for producing bioactive quaternary salts [41; 42; 43; 44]. In vitro activity was tested on four phytopathogenic fungi, including _F. culmorum_, using 22 compounds derived from nicotinamide and isonicotinamide at two concentrations (10 and 100 \(\upmu\)g/mL). Mycelial growth (mm) was measured 48 h after inoculation, relative to control (pure PDA) and a reference chemical fungicide. The most active compounds were selected for in vivo testing to evaluate their effectiveness under biological conditions. This study aims to assess the impact of quaternary pyridinium salts on the growth of wheat seedlings infected with _F.