# Antifungal Activity of Quaternary Pyridinium Salts Against _Fusarium culmorum_ in Wheat Seedlings
## 2 Materials and Methods
### In Vivo Test on Wheat  
To determine whether quaternary pyridinium salts differ in their antifungal activity against _F. culmorum_ root rot disease and their effect on wheat seedlings, an in vivo test was performed. The experiment was conducted with wheat grains of the Sofru variety. The experiment included selected compounds from the group of nicotinamide and isonicotinamide derivatives.  
The effect of the synthesized compounds on the development of _F. culmorum_ was investigated using a modified method by [PERSON] [52]. The _F. culmorum_ was cultivated on PDA enriched with an antibiotic (streptomycin) and incubated for 10 days in a climate chamber at 22 \(\pm\) 2 \({}^{\circ}\)C under a 12 h light/12 h dark phase. After obtaining a pure culture, a spore suspension was prepared for contamination of the substrate (sand). To prepare the spore suspension, the pure culture's mycelium was scraped with a sterilized scalpel, thoroughly homogenized in distilled water, and filtered to obtain a uniform suspension. The spore concentration was adjusted to 1 \(\times\) 10\({}^{5}\) spores/mL and determined using a Neubauer hemocytometer. Before the experiment, the sand was sterilized in a drying oven at 130 \({}^{\circ}\)C for 5 h.  
The experiment was divided into treatments with three replicates each, with each replicate consisting of 15 wheat grains:  
1. Sterile grains treated with a fungicide and sown in contaminated sand (F/CS);
2. Sterile grains treated with a synthesized compound and sown in contaminated sand (C/CS);
3. Sterile grains sown in contaminated sand (SG/CS).  
The SG/CS treatment (sterile grain/contaminated sand) represents the control variant without applying an agent. In contrast, the F/CS treatment (fungicide/contaminated sand) was a positive control in which the seed was pre-treated with a chemical fungicide and sown in contaminated sand. The fungicide used contained fluidoxonil (25 g/L) and difenoconazole (25 g/L), which were applied as a solution at 16.7%. The containers were covered with aluminum foil and left at room temperature for 24 h to allow the sand to become infected with the pathogen.  
The healthy wheat grains were disinfected in 70% ethanol for 10 min, then rinsed with distilled water and dried. The grains were then soaked for four hours in 49 mL of distilled water with 1 mL of the selected pyridinium compound or 50 mL of distilled water for the control group.  
After treatment, wheat grains were sown in contaminated sand. To maintain moisture, the pots with aluminum foil were covered and left at room temperature for 48 h or until the first grains began to sprout. The aluminum foil was removed, and the pots were moved to a growth chamber set at 22 \({}^{\circ}\)C, with a 12 h light/12 h dark regime and a relative humidity of 70%. The growth of the seedlings was monitored over the next 11 days. On the 11 th day after sowing, healthy and diseased seedlings were evaluated, and the disease index was assessed. Additionally, measurements were carried out, including fresh and lyophilized plant mass and root and shoot length.  
Plants were photographed to obtain accurate growth parameters, and shoot and root lengths (mm) were measured using ImageJ software (version 1.53, National Institutes of Health, Bethesda, MD, USA) [53]. Shoots and roots were carefully separated, and their fresh weight (g) was determined using a precision laboratory balance. Dry weight was measured after lyophilization of the samples for 24 h. Each treatment was performed in triplicate, with 15 wheat grains per replicate (45 seedlings per treatment). These measurements were used to assess the effects of the tested compounds on the growth of wheat seedlings infected with the pathogen. All parameters were measured individually for each seedling, and results are expressed as mean values per replicate (\(n=3\) per treatment).