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Article

Antifungal Activity of Quaternary Pyridinium Salts Against Fusarium culmorum in Wheat Seedlings

by
Tamara Siber
1,*,
Elena Petrović
2,
Jasenka Ćosić
1,
Valentina Bušić
3,
Dajana Gašo-Sokač
3 and
Karolina Vrandečić
1
1
Department of Phytomedicine, Faculty of Agrobiotechnical Sciences Osijek, University of Josip Juraj Strossmayer in Osijek, Vladimira Preloga 1, HR-31000 Osijek, Croatia
2
Department of Agriculture and Nutrition, Institute of Agriculture and Tourism, Karla Huguesa 8, HR-52440 Porec, Croatia
3
Department of Applied Chemistry and Ecology, Faculty of Food Technology Osijek, University of Josip Juraj Strossmayer in Osijek, Franje Kuhača 18, HR-31000 Osijek, Croatia
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(14), 7889; https://doi.org/10.3390/app15147889 (registering DOI)
Submission received: 11 June 2025 / Revised: 12 July 2025 / Accepted: 14 July 2025 / Published: 15 July 2025
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Abstract

Wheat (Triticum aestivum L.) is a major cereal crop globally, but its production is increasingly threatened by fungal pathogens, particularly Fusarium culmorum (Wm. G. Sm.) Sacc., which causes seedling blight and root rot, leading to yield losses and mycotoxin contamination. Conventional control strategies, such as crop rotation and the use of fungicides, are often inadequate and contribute to the development of resistance, particularly with the overuse of similar modes of action. This study investigated quaternary pyridinium salts—nicotinamide and isonicotinamide derivatives—as potential sustainable antifungal agents. In vivo tests involved treating sterilized wheat seeds grown in sterile sand that had been inoculated with F. culmorum, using compounds previously confirmed to be active in vitro. Disease index, shoot and root length, and fresh and dry biomass were measured. Among the tested compounds, nicotinamide derivatives (2) and (3) showed the lowest disease index (0.9) at a concentration of 10 µg/mL. Most compounds promoted plant and root growth. Isonicotinamide derivatives (6) and (7) at 100 µg/mL increased root dry weight, while compound (6) at 10 µg/mL resulted in the most significant increase in plant length. These findings highlight the dual antifungal and growth-promoting potential of certain eco-friendly derivatives for managing F. culmorum and supporting wheat seedling development.

1. Introduction

Among the most widespread and economically significant fungal genera is Fusarium, which includes numerous species responsible for crop diseases affecting wheat, maize, barley, tomato, cucumber, potato, and many other plants. Diseases caused by Fusarium species are seed rot, seedling blight, root and stem rot, late blight, and wilt disease. The primary sources of infection are contaminated seeds, infected soil, and alternative hosts [1]. Among the Fusarium species, Fusarium culmorum (Wm. G. Sm.) Sacc. is considered one of the most important fungal pathogens, causing significant crop losses, particularly in wheat (Triticum aestivum L.), its primary host [2]. Many researchers have identified F. culmorum, Fusarium graminearum Schwabe, and Fusarium pseudograminearum O’Donnell & T. Aoki as the most critical wheat pathogens globally [3,4,5,6]. Wheat is cultivated at approximately 161.80 thousand ha in Croatia, with an annual yield of around 971.40 thousand t, while globally, it is grown at 219.20 million ha, with an annual production of 99.96 million t [7]. These figures highlight the crucial role of wheat in food security, especially in Croatia, where it is one of the most important agricultural products. According to Ćosić and Jurković [8], F. culmorum is one of the most pathogenic species for wheat in Croatia.
Among the pathogens affecting wheat, F. culmorum causes several economically significant diseases, particularly Fusarium root rot (FRR), which is especially damaging during the early growth stages. FRR symptoms usually appear soon after sowing and can result in seedling death either before or after emergence. Infected plants often show brown lesions on the roots and lower stem, with reddish-pink sporodochia forming under humid conditions due to intense sporulation. In later stages, lesions may extend to the stem internodes, further compromising plant development [9]. Although F. culmorum is also a recognized agent of Fusarium head blight (FHB) [10], this study primarily focuses on FRR because of its impact on early seedling health.
Fusarium species, including F. culmorum, are not only plant pathogens but also potent producers of mycotoxins such as deoxynivalenol (DON), zearalenone (ZEA), and fumonisins. These toxic secondary metabolites can contaminate grains and pose a serious food safety risk due to their potential to disrupt protein synthesis, hormonal balance, and nervous system function [11,12,13]. Beyond visible crop damage, their presence in food and feed poses a hazard to immunocompromised individuals [14]. Beyond food safety concerns, DON also acts as a virulence factor in plants—it promotes disease development in wheat by disrupting normal cellular processes, inducing oxidative stress, and triggering programmed cell death (PCD). This leads to tissue damage and bleaching, which are characteristic symptoms of Fusarium head blight (FHB). Specific wheat genotypes can convert DON into a less toxic form (DON-3-glucoside), contributing to increased resistance to the disease [15,16,17,18]. In addition to DON, other mycotoxins produced by Fusarium, such as fusaric acid, fumonisin B1, and zearalenone, can also harm plants. They can slow down root and shoot growth, damage cell membranes, cause oxidative stress, and lead to cell death, which weakens the plant and helps the disease spread [19].
Effective control of Fusarium diseases requires multiple preventive measures, including crop rotation, selection of healthy seeds, appropriate fertilization, and moisture management. In intensive cereal production systems, chemical fungicides are widely applied. However, excessive use, particularly of fungicides with the same mode of action, promotes the development of resistant pathogen strains and accelerates the emergence of resistance [20,21]. As a result, interest in alternative strategies is growing, including the application of synthetic compounds such as quaternary pyridinium salts.
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 µg/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. culmorum under in vivo conditions, specifically focusing on their ability to mitigate disease symptoms and their effects on plant growth. By evaluating their efficacy in reducing disease symptoms and influencing plant growth, this research contributes to the development of new solutions for controlling phytopathogenic fungi.

2. Materials and Methods

This study’s experiments and data collection were conducted in 2023 at the Faculty of Agrobiotechnical Sciences Osijek, Central Agrobiotechnical Analytical Unit, Croatia. Quaternary pyridinium salts were synthesized at the Faculty of Food Technology Osijek, Laboratory for Organic Chemistry and Biochemistry, Croatia. The isolation and molecular identification of F. culmorum were conducted in 2022 at the Faculty of Agrobiotechnical Sciences Osijek, Central Agrobiotechnical Analytical Unit, Croatia.
Based on preliminary in vitro screening at 10 and 100 μg/mL concentrations, only the most active compounds were selected for in vivo evaluation. Both concentrations were tested in vivo for some of these compounds to assess potential dose-dependent effects. However, for others, only the concentration that exhibited the highest efficacy in vitro was selected for in vivo testing to focus on biologically relevant doses and avoid unnecessary repetition.

2.1. Synthesis of Quaternary Pyridinium Salts

Nicotinamide and isonicotinamide derivatives (Figure 1, Table 1) were employed as nucleophiles in reactions with various electrophiles, including alkyl halides (methyl iodide), alkyl dihalides (dibromopropane, diiodopropane), and differently substituted 2-bromoacetophenones (4-Cl, 4-Br, 4-H, 4-CH3, 4-F, 4-OCH3, 4-Ph, 2-OCH3, 4-NO2). Quaternary pyridinium salts were obtained through conventional quaternization synthesis in ethanol and alternative methods, including microwave-assisted synthesis [39,44] and synthesis in DES [40].
In the conventional approach, the reactants were refluxed in ethanol, yielding the desired products in moderate amounts. In contrast, the microwave-assisted method utilized microwave irradiation in either ethanol or acetone as the solvent, significantly accelerating the reaction. Microwave dielectric heating in this method led to substantially improved yields—up to eight times higher—and reduced reaction times to just 10–20 min [44].
The conventional synthesis of isonicotinamide derivatives involves prolonged heating of the reaction mixture under reflux, while the MW method significantly reduces the reaction time to just 10 min using microwave energy. In both cases, the products are obtained by cooling, washed with ether, and recrystallized from methanol. The MW method enables more efficient and faster synthesis with similar yield and product [39]. The syntheses of quaternary salts were performed in deep eutectic solvents (DES) using three synthetic methods: conventional (2–6 h), microwave (20 min), and ultrasonic (3 h), all at 80 °C. These solvents offer environmentally friendly advantages, including biodegradability, ease of preparation, and simplified purification. The highest yields, up to 98%, were achieved with the microwave method. DES based on choline chloride with urea, oxalic acid, or levulinic acid proved to be the most effective [40].

2.2. Isolation and Identification of Fusarium culmorum

The F. culmorum was isolated from wheat grains, which showed disease symptoms. The procedure followed standard phytopathological methods described by Nelson et al. [45], Leslie and Summerell [46], and Lević [47]. The grains were rinsed under running water, disinfected in 96% ethanol for 30 s, rinsed with sterile distilled water, and dried on filter paper. The grains were then placed in Petri dishes on potato dextrose agar (PDA). The dishes were incubated in a climate chamber (Aralab, Blue Line, Portugal) at 22 °C with a 12 h light/12 h dark cycle. After the development of reproductive structures (mycelium and conidia), pure cultures were obtained by subculturing them in a fresh PDA medium.

DNA Extraction, Amplification, and Sequencing

DNA was extracted from the mycelium at the edges of 10-day-old fungal colonies grown on potato dextrose agar (PDA). Total DNA was isolated using the OmniPrep™ for Fungi PCR Kit (G-Biosciences Co., Overland, MO, USA). Polymerase chain reaction (PCR) was conducted using primers targeting the translation elongation factor 1-alpha (TEF-1α) region: EF1 forward primer (ATG GGT AAG GAR GAC AAG AC) and EF2 reverse primer (GGA RGT ACC AGT SAT CAT G). The PCR mix comprised 12.5 µL EmeraldAmp® PCR ReadyMix, 0.5 µL of each primer, 11 µL of nuclease-free water, and 0.5 µL of genomic DNA. The PCR amplification program for the TEF-1α region is detailed in Table 2 [28]. The PCRs were carried out using a MiniAmp Plus Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The PCR products were visualized under UV light on a 1% agarose gel using the Kodak EDAS 290 system with a UV transilluminator (UVITEC). The PCR products were sequenced by Macrogen Europe (Amsterdam, The Netherlands). The sequences obtained were processed using DNA Dynamo v. 1.629 software (Blue Tractor Software, Bangor, North Wales, UK) and compared with sequences from the GenBank® database. The sequence showed 99.97% similarity to F. culmorum reference sequences in the NCBI database, confirming the isolate’s identity.
After the isolation and identification of F. culmorum, the pure culture was preserved by three methods: lyophilization (freeze-drying), storage at −80 °C in 1.5 mL microtubes with a glycerol–water mixture, and storage at 4 °C in 10 mL tubes containing sterile distilled water. The culture maintained in sterile water is renewed every two years.

2.3. Disease Evaluation

The intensity of the seedling infection was rated on a scale of 0 to 5, where 0% infection (score 0) indicated a healthy seedling, 1–10% infected area (score 1), 11–29% infected area (score 2), 30–69% infected area (score 3), 70–89% infected area (score 4), and >90% infected area or rotten grains (score 5) and the disease index was calculated. This index provides information on the severity of the disease. It indicates whether the selected nicotinamide and its derivatives influence the reduction in the intensity of the pathogen F. culmorum infection.
The disease index was calculated according to the method of McKinney [49] using the following formula:
I = ∑(n × k)/(N × K) × 100,
where
n—number of plants in each category; k—rating of each category
N—total number of plants analyzed
K—the highest rating on the scale (in this case, 5)
To make the interpretation and comparison of treatment efficacy easier, Disease Index values were divided into categories based on the range observed in this study and in line with similar approaches from previous research [50,51]. The classification was as follows: most effective (DI ≤ 1.0), very effective (DI = 1.1–3.5), moderate to strong effect (DI = 3.6–6.9), moderate effect (DI = 7.0–8.9), less effective (DI = 9.0–12.0), and least effective (DI > 12.0).

2.4. 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 Mesterházy [52]. The F. culmorum was cultivated on PDA enriched with an antibiotic (streptomycin) and incubated for 10 days in a climate chamber at 22 ± 2 °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 × 105 spores/mL and determined using a Neubauer hemocytometer. Before the experiment, the sand was sterilized in a drying oven at 130 °C for 5 h.
The experiment was divided into treatments with three replicates each, with each replicate consisting of 15 wheat grains:
  • Sterile grains treated with a fungicide and sown in contaminated sand (F/CS);
  • Sterile grains treated with a synthesized compound and sown in contaminated sand (C/CS);
  • 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 fludioxonil (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 °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 11th 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).

2.5. Statistical Analysis

Statistical in vivo data analyses were performed using the SAS statistical program (SAS 9.4 for Windows, 2017, and SAS Enterprise Guide 7.1). Data are expressed as the arithmetic mean (M) and standard error of the mean (SEM). To assess differences in antifungal activity between quaternary pyridinium salts and a commercial fungicide, one-way ANOVA was followed by Tukey’s HSD post hoc test. All tests were two-tailed, and p ≤ 0.05 was considered statistically significant.
A one-way ANOVA was used to compare the disease index between treatments. Due to the limited number of replicates (3 per treatment), statistical significance was assessed using the LSD post hoc test. Values represent the mean disease index calculated from three replicates and are expressed as mean ± standard deviation (SD). Mean values with different letters in the same column indicate significant differences between treatments at p ≤ 0.05.

3. Results

3.1. Disease Index

The data in Table 3 shows the different efficacies of the tested nicotinamide derivatives in reducing disease in wheat seedlings. All nicotinamide derivatives showed significantly better efficacy in reducing disease intensity than the SG/CS control, except for compound (5), which did not differ significantly from the control. The control SG/CS had the highest disease index (14.2). A low disease index of 2.2 was observed for the positive control (F/CS), indicating a high effectiveness of the fungicide. When compounds (1) and (4) were applied at a 100 µg/mL concentration, the disease index was 6.7. This result was lower than the negative control (SG/CS), showing that both compounds had some antifungal effect. Although the disease index was higher than that of the positive control (F/CS), the difference was insignificant. Compound (3), applied at a 100 µg/mL concentration, showed high efficacy with a disease index equal to that of the positive control F/CS. The most effective compounds were (2) and (3), with a disease index of 0.9 at a 10 µg/mL concentration (Figure 2).
The results in Table 4 show the different efficacy of isonicotinamide derivatives in reducing disease severity in wheat seedlings infected with F. culmorum. Compared to the positive control (F/CS), which had a very low disease index of 2.2, the isonicotinamide derivatives were less effective. However, several compounds showed promising antifungal potential and were not significantly different from the positive control.
Among the derivatives, compound (8) at a 100 µg/mL concentration showed the most potent antifungal effect with a disease index of 3.5 (Figure 3). Compounds (6) at a concentration of 10 µg/mL and (7) at 100 µg/mL showed moderate to strong effects with a disease index of 4.9 and 5.3, respectively. Interestingly, compound (6) at a higher concentration (100 µg/mL) showed the weakest efficiency among the tested derivatives, with a disease index of 12.0. Compound (9) was least effective at a lower concentration, with a disease index of 8.9, while its efficacy improved significantly at 100 µg/mL with a disease index of 5.8.

3.2. The Effect of Nicotinamide Derivatives on the Fresh and Lyophilized Root Mass of Wheat Seedlings

The results in Figure 4 show the effect of nicotinamide derivatives (at concentrations of 10 and 100 µg/mL) on wheat seedlings’ fresh and lyophilized root mass. Most nicotinamide derivatives resulted in a statistically significant increase in fresh root mass compared to the control (sterile grain). The exceptions were compounds (1) and (5) at a 100 µg/mL concentration, for which no statistically significant differences were observed compared to the control (sterile grain). However, all tested compounds showed significantly lower fresh root mass values than the fungicide treatment. Compounds (1) and (5) at 100 µg/mL exhibited similarly low effects on the fresh root mass of wheat, while compound (2a) at 10 µg/mL showed a moderately higher value. Compound (3), at both concentrations (10 and 100 µg/mL), resulted in similarly high fresh root mass values.
A statistically significant increase in lyophilized root mass compared to the control was observed only for compound (3) at 100 µg/mL. No significant differences were found for the remaining compounds. All tested derivatives had a lower effect on lyophilized root mass than the fungicide. Among the compounds, similar values for lyophilized root mass were observed for compounds (1) and (5) at 100 µg/mL and compound (2a) at 10 µg/mL. Additionally, compounds (3) and (4), both at 100 µg/mL, had comparable effects on lyophilized root mass (Figure 4).

3.3. The Effect of Nicotinamide Derivatives on the Fresh and Lyophilized Shoot Mass of Wheat Seedlings

A comparison of nicotinamide derivatives and the control (sterile grain) regarding the fresh shoot mass showed that three compounds − (4) and (5) at a 100 µg/mL concentration and compound (3) at concentrations of 10 and 100 µg/mL—exhibited a statistically significant higher influence on fresh shoot mass compared to the control treatment (sterile grain).
When comparing the mutual influence of individual nicotinamide derivatives on the fresh shoot mass, similar effects were observed between compound (1) at a 100 µg/mL concentration and compound (2) at a 10 µg/mL concentration, as well as between compound (3) at both concentrations (10 and 100 µg/mL) and compounds (4) and (5) used at a 100 µg/mL concentration (Figure 5).
Comparison of nicotinamide derivatives and the control (sterile grain) concerning the lyophilized shoot mass showed a statistically significant higher value of lyophilized seedling mass for compounds (4) and (5) applied at a 100 µg/mL concentration and compound (3) used at both concentrations (10 and 100 µg/mL). Furthermore, all tested compounds resulted in significantly lower lyophilized shoot mass values than the fungicide treatment.
When comparing the mutual influence of nicotinamide derivatives on the lyophilized shoot mass, similar effects were observed between compound (1) at 100 µg/mL and compound (2) at a 10 µg/mL concentration. A similar effect was noted for compound (3), regardless of concentration, and for compounds (4) and (5) at a 100 µg/mL concentration (Figure 5).

3.4. The Effect of Nicotinamide Derivatives on the Shoot and Root Length

A comparison of the effect of nicotinamide derivatives and the control (sterile grain) on shoot length showed a statistically significant increase in shoot length compared to the control (sterile grain). No statistically significant difference in plant length was observed between the tested compounds and the positive control. The exception was treatment with compound (3) at a 100 µg/mL concentration, where the shoot was statistically significantly longer than the positive control (fungicide) (Figure 6). Furthermore, the analysis has a positive effect on shoot length.
The effect of nicotinamide derivatives on root length showed a statistically significant increase compared to the negative control. Compared to the positive control (fungicide), most compounds did not show significant differences, including compound (3) at both concentrations. All other nicotinamide derivatives showed a similar effect on root length. Compound (5) at a 100 µg/mL concentration showed a slightly lower value but without a statistically significant difference compared to most other compounds.

3.5. The Effect of Isonicotinamide Derivatives on the Fresh and Lyophilized Root Mass of Wheat Seedlings

As shown in Figure 7, all tested isonicotinamide derivatives significantly increased the fresh root mass in wheat seedlings compared to the control treatment (sterile grain). However, all tested compounds had a statistically significantly lower influence on fresh root mass than the fungicide treatment. When comparing the effects of each compound on fresh root mass, compounds (6) and (8) at a 100 µg/mL concentration showed statistically similar values. These were significantly lower than the values observed for compound (9) at 100 µg/mL and compound (6a) at 10 µg/mL, both of which resulted in significantly higher fresh root mass.
A comparison with the control treatment (sterile grain) showed that only compounds (6) and (7) at 100 µg/mL resulted in a statistically significant increase in lyophilized root mass. In contrast, the lyophilized root mass in all isonicotinamide-treated seedlings was significantly lower than that in the fungicide treatment.
When comparing the effects of different isonicotinamide derivatives on lyophilized root mass, most compounds resulted in statistically similar values. However, compound (7) showed significantly higher lyophilized root mass than compounds (6a), (9), and (9a), which had significantly lower values, comparable only to the sterile grain control (Figure 7).

3.6. The Effect of Isonicotinamide Derivatives on the Fresh and Lyophilized Shoot Mass of Wheat Seedlings

Figure 8 shows that applying isonicotinamide derivatives showed that most tested compounds significantly increased the fresh shoot mass in wheat seedlings compared to the control (sterile grain). Exceptions were compounds (6) and (8) at a 100 µg/mL concentration, where no significant difference was observed compared to the control. All tested compounds, except compound (9), resulted in significantly lower shoot fresh mass than the fungicide treatment.
All tested compounds significantly increased the lyophilized shoot mass compared to the control (sterile grain). However, lyophilized shoot mass remained significantly lower for all isonicotinamide treatments than for the fungicide treatment. The only exception was compound (7) at 100 µg/mL, which resulted in a significantly higher lyophilized shoot mass than the other isonicotinamide derivatives. As shown in Figure 7, all tested isonicotinamide derivatives significantly increased the fresh root mass of wheat seedlings compared to the control treatment (sterile grain). However, all tested compounds had a statistically significantly lower influence on fresh root mass than the fungicide treatment.

3.7. The Effect of Isonicotinamide Derivatives on the Shoot and Root Length

Figure 9 shows that the effect of isonicotinamide derivatives on the shoot length of wheat compared to the control (sterile grain) showed a statistically significant increase in shoot length for compound (6) at a 10 µg/mL concentration and compound (9) at a 100 µg/mL concentration. When comparing the effects of different isonicotinamide derivatives on shoot length, most compounds showed similar results, except compound (6) at a 10 µg/mL concentration, which showed a statistically significant increase in shoot length compared to the fungicide treatment.
On the other hand, the effect of isonicotinamide derivatives on the root length of the shoot showed a statistically significant increase in root length compared to the control for most compounds, except for compounds (6) and (8) at a 100 µg/mL concentration.
Compared to the fungicide treatment, a statistically significant increase in root length was only observed after treatment with compound (6) at a 10 µg/mL concentration. In contrast, the other compounds showed no statistically significant differences (also presented in Figure 9). When comparing individual compounds based on their effect on shoot root length, similar values were observed for compounds (6) and (8) at a 100 µg/mL concentration and for compound (9) at 10 µg/mL and 100 µg/mL concentrations.

4. Discussion

This study aimed to evaluate the in vivo antifungal efficacy of nicotinamide and isonicotinamide derivatives in reducing F. culmorum infections and their effects on the growth parameters of wheat seedlings. The results show that the tested compounds have varying degrees of efficacy, with some showing similar or better performance than the positive control. However, further research is needed to understand their applicability and long-term effects on plant development and health. The disease index is an important indicator of disease control effectiveness, quantifying symptom intensity on individual plants. Its relevance is highlighted in studies on fungicide efficacy, where a high disease index indicates low treatment effectiveness [54]. These results confirm substantial variation in antifungal efficacy among nicotinamide and isonicotinamide derivatives, even within the same structural group, suggesting that specific molecular features strongly influence their biological activity. Compounds (2) and (3) contain phenacyl substituents, likely improving interaction with fungal enzymes such as succinate dehydrogenase through π–π stacking and hydrogen bonding. Carboxamide-containing compounds inhibit succinate dehydrogenase by forming hydrogen bonds with histidine and interacting with aromatic residues through π–π and hydrophobic forces [55,56]. Structural features, such as substituent type and flexibility, influence binding: phenacyl groups enhance inhibition, while rigid or hydrophobic groups like methyl or bromopropyl reduce activity by limiting proper alignment within the active site. Some nicotinamide and isonicotinamide derivatives with different natures of substituents preferentially bind to the active site, increasing fungicidal efficiency, while others, due to their specific substituents, favor binding to peripheral sites, ultimately reducing their inhibitory effect. These structural and interaction-based differences likely explain the variations in antifungal activity observed in this study.
According to the data, the control group (sterile grain) exhibited the highest disease index (14.2), which was expected due to the absence of any protective treatment. In contrast, the positive control (fungicide) demonstrated strong efficacy, with a significantly lower disease index of 2.2 (Table 3 and Table 4). Most nicotinamide and isonicotinamide derivatives also resulted in a notable reduction in disease severity compared to the untreated control, indicating their potential to reduce F. culmorum infection in wheat seedlings. Compounds (2) and (3) at 10 µg/mL showed the strongest antifungal effect, with a disease index of 0.9, even lower than the positive control (Table 3). These results are consistent with previous studies on vitamin B3 derivatives. For example, nicotinamide mononucleotide and nicotinamide reduced late blight incidence in wheat, indicating their ability to suppress fungal infections [57]. Similarly, the application of nicotinic acid to fenugreek plants infected with Agroathelia rolfsii (Sacc.) Redhead & Mullineux significantly reduced disease symptoms [58]. These studies, in line with our results, support that B3-based compounds act as elicitors of early plant defense responses, potentially enhancing both pathogen resistance and physiological resilience under stress.
In our study, some nicotinamide derivatives improved the growth parameters of wheat seedlings, particularly root biomass. However, not all compounds were effective, and a lower disease index did not always result in increased biomass. This indicates that the antifungal activity of the compounds and their effects on plant growth may be governed by different mechanisms (Table 4, Figure 4). Similar findings have been reported for nicotinamide mononucleotide (NMN), which activated plant defenses against Fusarium without significantly influencing plant growth [59]. Some derivatives also showed a positive effect on shoot development, and all tested compounds improved shoot and root length under pathogen stress (Figure 5 and Figure 6). These effects may be related to healthier root systems, improved nutrient uptake, or reduced infection intensity. Similar findings have been reported for vitamin B3-based compounds (nicotinic acid), where treated plants showed better growth and vitality under stress conditions [58]. In barley (Hordeum vulgare L) exposed to lead stress, nicotinamide treatment also improved shoot and root development, higher pigment levels, and reduced oxidative damage. Although the stress factor was abiotic, these outcomes further highlight the potential of vitamin B3 derivatives in enhancing plant tolerance to various stress conditions [60].
All applied isonicotinamide derivatives positively affected fresh root and shoot mass compared to the control (Figure 7 and Figure 8), although their effects on dry biomass varied. In particular, some compounds led to lower fresh mass but higher lyophilized mass, which may indicate an impact on the plant’s water content. The results suggest that compounds (6) and (7) may lead to higher dry biomass while reducing the water content in plant tissue. This could indicate that the compounds affect how plants regulate internal water balance. A similar pattern has been described as part of a stress-priming response, where isonicotinamide was shown to enhance plant defense mechanisms and stimulate the production of secondary metabolites under stress conditions [61]. Similar patterns (lower fresh mass but higher dry weight) have been reported in drought-stressed plants as part of a physiological strategy to conserve water. A study on maize (Zea mays L.) showed that, under drought conditions, the plant reduces water content in aboveground tissues and reallocates resources to root development to improve water-use efficiency [62]. Although our study did not focus on drought stress, these observations may indicate a similar adaptive response.
Furthermore, previous research indicates that nicotinamide derivatives can help alleviate the effects of soil salinity, a stress that often reduces plant height, root length, leaf area, and overall biomass production [63]. In our study, several isonicotinamide derivatives stimulated shoot and root elongation, with the most pronounced effects observed at lower concentrations, while higher doses were generally less effective (Figure 9). For instance, compound (6) at 10 µg/mL significantly increased shoot length compared to the fungicide, whereas this effect was not observed at 100 µg/mL. These results show that vitamin B3-based compounds may promote plant growth but also highlight the importance of dose optimization. Compound (6) promoted plant growth at 10 μg/mL but not at 100 μg/mL, suggesting a hormetic effect—a biphasic response where low doses stimulate and high doses inhibit growth [64]. Such non-monotonic responses are known for many plants’ bioactive compounds [65,66].
Although this study did not investigate the underlying mechanisms, lower concentrations may enhance physiological responses such as hormone-like activity or stress protection. At the same time, higher doses may disrupt cellular balance. These results emphasize the importance of dose optimization when evaluating the dual role of bioactive compounds in pathogen control and plant development.
Similar concentration-dependent responses have been reported for nicotinamide mononucleotide (NMN) [59]. Given the promising effects of certain nicotinamide and isonicotinamide derivatives in reducing pathogen infections and improving plant development, it is important to consider their potential toxicity. Previous studies have investigated the cytotoxicity of nicotinamide derivatives by evaluating their effects on human neuronal and renal cells, using IC50 values as a toxicity measure [67]. The results showed that only two halogenated derivatives with chlorine and bromine substituents exhibited notable toxicity, with the lowest IC50 values of approximately 85 μM. Other derivatives demonstrated no significant cytotoxic effects on the tested cell lines. These findings underscore the importance of comprehensive toxicity profiling, especially if these derivatives are intended for therapeutic or agricultural applications.

5. Conclusions

This study showed that nicotinamide and isonicotinamide derivatives could reduce F. culmorum infection and help wheat seedlings grow better. Some compounds were even more effective than the chemical fungicide, especially at lower concentrations. However, not all compounds that reduce disease also improve plant growth, which means their effects depend on the type and dose of the compound. The results suggest that concentration may influence the effectiveness of some compounds, but further research is needed to confirm this due to the limited number of treatments tested at multiple concentrations.
Future research should examine how these compounds affect plants over a more extended period to determine whether they can be used in agriculture. It is also essential to study their environmental safety, especially how they might affect soil, beneficial organisms, and the overall ecosystem.

Author Contributions

Conceptualization, T.S., K.V. and J.Ć.; methodology, T.S., K.V. and V.B.; investigation, T.S.; resources, V.B. and D.G.-S.; data curation, T.S.; writing—original draft preparation, T.S.; writing—review and editing, T.S., K.V., E.P., J.Ć., V.B. and D.G.-S.; visualization, T.S. and E.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are available in the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Applsci 15 07889 g001
Figure 1. Structure of the selected derivatives: R1 = -CH3 (1), -CH2CH2CH2Br (5); R2 = 4-Cl (2), -4-H (3), -4-NO2 (4); R3 = -4-OCH3 (6), -4-Ph (7), -2-OCH3 (8), -4-NO2 (9).
Figure 1. Structure of the selected derivatives: R1 = -CH3 (1), -CH2CH2CH2Br (5); R2 = 4-Cl (2), -4-H (3), -4-NO2 (4); R3 = -4-OCH3 (6), -4-Ph (7), -2-OCH3 (8), -4-NO2 (9).
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Figure 2. Effect of treatment with nicotinamide derivatives on disease intensity: (a) compound (2) at 10 µg/mL/contaminated sand, (b) compound (3) at 10 µg/mL/contaminated sand; (c) sterile grain/contaminated sand; (d) fungicide/contaminated sand.
Figure 2. Effect of treatment with nicotinamide derivatives on disease intensity: (a) compound (2) at 10 µg/mL/contaminated sand, (b) compound (3) at 10 µg/mL/contaminated sand; (c) sterile grain/contaminated sand; (d) fungicide/contaminated sand.
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Figure 3. Effect of treatment with isonicotinamide derivatives on disease intensity: (a) compound (7) at 100 µg/mL/contaminated sand; (b) compound (8) at 100 µg/mL/contaminated sand; (c) sterile grain/contaminated sand; (d) fungicide/contaminated sand.
Figure 3. Effect of treatment with isonicotinamide derivatives on disease intensity: (a) compound (7) at 100 µg/mL/contaminated sand; (b) compound (8) at 100 µg/mL/contaminated sand; (c) sterile grain/contaminated sand; (d) fungicide/contaminated sand.
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Figure 4. The effect of nicotinamide derivatives on the fresh and lyophilized root mass. The columns (fresh root mass) and lines (lyophilized root mass) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (1), (3), (4), (5)—compounds at a 100 µg/mL concentration; (2a), (3a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in fresh root mass; capital letters indicate differences in lyophilized root mass.
Figure 4. The effect of nicotinamide derivatives on the fresh and lyophilized root mass. The columns (fresh root mass) and lines (lyophilized root mass) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (1), (3), (4), (5)—compounds at a 100 µg/mL concentration; (2a), (3a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in fresh root mass; capital letters indicate differences in lyophilized root mass.
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Figure 5. The effect of nicotinamide derivatives on the fresh and lyophilized shoot. The columns (fresh shoot mass) and lines (lyophilized shoot mass) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (1), (3), (4), (5)—compounds at a 100 µg/mL concentration; (2a), (3a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in fresh shoot mass, and capital letters indicate differences in lyophilized shoot mass.
Figure 5. The effect of nicotinamide derivatives on the fresh and lyophilized shoot. The columns (fresh shoot mass) and lines (lyophilized shoot mass) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (1), (3), (4), (5)—compounds at a 100 µg/mL concentration; (2a), (3a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in fresh shoot mass, and capital letters indicate differences in lyophilized shoot mass.
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Figure 6. The effect of nicotinamide derivatives on the shoot and root length. The columns (shoot length) and lines (root length) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (1), (3), (4), (5)—compounds at a 100 µg/mL concentration; (2a), (3a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in shoot length, and capital letters indicate differences in root length.
Figure 6. The effect of nicotinamide derivatives on the shoot and root length. The columns (shoot length) and lines (root length) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (1), (3), (4), (5)—compounds at a 100 µg/mL concentration; (2a), (3a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in shoot length, and capital letters indicate differences in root length.
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Figure 7. The effect of isonicotinamide derivatives on the fresh and lyophilized root mass of shoots. The columns (fresh root mass) and lines (lyophilized root mass) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (6), (7), (8), (9)—compounds at a 100 µg/mL concentration; (6a), (9a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in fresh root mass, and capital letters indicate differences in lyophilized root mass.
Figure 7. The effect of isonicotinamide derivatives on the fresh and lyophilized root mass of shoots. The columns (fresh root mass) and lines (lyophilized root mass) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (6), (7), (8), (9)—compounds at a 100 µg/mL concentration; (6a), (9a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in fresh root mass, and capital letters indicate differences in lyophilized root mass.
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Figure 8. The effect of isonicotinamide derivatives on the fresh and lyophilized shoot. The columns (fresh shoot mass) and lines (lyophilized shoot mass) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (6), (7), (8), (9)—compounds at a 100 µg/mL concentration; (6a), (9a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in fresh shoot mass, and capital letters indicate differences in lyophilized shoot mass.
Figure 8. The effect of isonicotinamide derivatives on the fresh and lyophilized shoot. The columns (fresh shoot mass) and lines (lyophilized shoot mass) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (6), (7), (8), (9)—compounds at a 100 µg/mL concentration; (6a), (9a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in fresh shoot mass, and capital letters indicate differences in lyophilized shoot mass.
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Figure 9. The effect of isonicotinamide derivatives on the shoot and root length. The columns (shoot length) and lines (root length) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (6), (7), (8), (9)—compounds at a 100 µg/mL concentration; (6a), (9a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in shoot length, and capital letters indicate differences in root length.
Figure 9. The effect of isonicotinamide derivatives on the shoot and root length. The columns (shoot length) and lines (root length) represent the mean of three replicates per treatment. Error bars represent the standard error (SE) of the mean. Differences between treatment means are indicated by different letters according to the Tukey HSD test (p ≤ 0.05). Labels: (6), (7), (8), (9)—compounds at a 100 µg/mL concentration; (6a), (9a)—compounds at a 10 µg/mL concentration. Small letters indicate differences in shoot length, and capital letters indicate differences in root length.
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Table 1. Selected derivatives: chemical names and labels.
Table 1. Selected derivatives: chemical names and labels.
Chemical NameLabel
Nicotinamide derivatives
3-carbamoyl-1-methylpyridinium iodide(1)
3-carbamoyl-1-(2-(4-chlorophenyl)-2-oxoethyl)pyridinium bromide(2)
1-(2-phenyl-2-oxoethyl)-3-carbamoylpyridinium bromide(3)
3-carbamoyl-1-(2-(4-nitrophenyl)-2-oxoethyl)pyridinium bromide(4)
1-(3-bromopropyl)-3-carbamoylpyridinium bromide(5)
Isonicotinamide derivativesLabel
4-carbamoyl-1-(2-(4-methoxyphenyl)-2-oxoethyl)pyridinium bromide(6)
1-(2-([1,1′-biphenyl]-4-yl)-2-oxoethyl)-4-carbamoylpyridinium bromide(7)
4-carbamoyl-1-(2-(2-methoxyphenyl)-2-oxoethyl)pyridinium bromide(8)
4-carbamoyl-1-[2-(4-nitrophenyl)-2-oxoethyl]pyridinium bromide(9)
Table 2. PCR amplification program for TEF1-α region (primers EF1 and EF2) [48].
Table 2. PCR amplification program for TEF1-α region (primers EF1 and EF2) [48].
PhaseTemperature (°C)Duration
Initial denaturation95 °C5 min
35 cycles:
Denaturation94 °C30 s
Annealing55 °C45 s
Elongation72 °C1 min 30 s
Final elongation72 °C10 min
Table 3. Effect of nicotinamide derivatives (10 and 100 µg/mL) on disease intensity caused by Fusarium culmorum in wheat seedlings.
Table 3. Effect of nicotinamide derivatives (10 and 100 µg/mL) on disease intensity caused by Fusarium culmorum in wheat seedlings.
TreatmentConcentration (µg/mL)Disease Index
(± SD) *		Notes
(1)1006.7 ± 2.03 bModerate effect
(2)100.9 ± 0.78 cMost effective with the lowest disease index among this group
(3)100.9 ± 0.95 cMost effective with the lowest disease index among this group
(3)1002.2 ± 1.91 bcVery effective
(4)1006.7 ± 2.39 bModerate effect
(5)10011.6 ± 4.91 aLess effective
SG/CS-14.2 ± 3.73 aNegative control, least effective
F/CSFludioxonil + Difenoconazole 25 + 25 g/L (16.7%)2.2 ± 1.54 bcPositive control, highly effective
* Values represent the mean disease index calculated from three replicates and are expressed as mean ± standard deviation (SD). Results were analyzed using one-way analysis of variance (ANOVA). Mean values with different letters within the same column are significantly different according to the LSD post hoc test at p ≤ 0.05. Labels of treatments: C/CS—compound/contaminated sand (treatments (1)–(5)), SG/CS—sterile grain/contaminated sand, F/CS—fungicide/contaminated sand.
Table 4. Effect of isonicotinamide derivatives (10 and 100 µg/mL) on disease intensity caused by Fusarium culmorum in wheat seedlings.
Table 4. Effect of isonicotinamide derivatives (10 and 100 µg/mL) on disease intensity caused by Fusarium culmorum in wheat seedlings.
TreatmentConcentration (µg/mL)Disease Index (± SD) *Notes
(6)104.9 ± 1.64 bcModerate to strong effect
(6)10012 ± 8.33 abLess effective
(7)1005.3 ± 5.29 bcModerate to strong effect
(8)1003.5 ± 3.11 cVery effective, with the lowest disease index among this group
(9)108.9 ± 5.37 abcLess effective
(9)1005.8 ± 2.37 bcModerate to strong effect
SG/CS-14.2 ± 3.73 aNegative control, least effective
F/CSFludioxonil + Difenoconazole 25 + 25 g/L (16.7%)2.2 ± 1.54 cPositive control, highly effective
* Values represent the mean disease index calculated from three replicates and are expressed as mean ± standard deviation (SD). Results were analyzed using one-way analysis of variance (ANOVA). Mean values with different letters within the same column are significantly different according to the LSD post hoc test at p ≤ 0.05. Labels of treatments: C/CS—compound/contaminated sand (treatments (6)–(9)), SG/CS—sterile grain/contaminated sand, F/CS—fungicide/contaminated sand.
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MDPI and ACS Style

Siber, T.; Petrović, E.; Ćosić, J.; Bušić, V.; Gašo-Sokač, D.; Vrandečić, K. Antifungal Activity of Quaternary Pyridinium Salts Against Fusarium culmorum in Wheat Seedlings. Appl. Sci. 2025, 15, 7889. https://doi.org/10.3390/app15147889

AMA Style

Siber T, Petrović E, Ćosić J, Bušić V, Gašo-Sokač D, Vrandečić K. Antifungal Activity of Quaternary Pyridinium Salts Against Fusarium culmorum in Wheat Seedlings. Applied Sciences. 2025; 15(14):7889. https://doi.org/10.3390/app15147889

Chicago/Turabian Style

Siber, Tamara, Elena Petrović, Jasenka Ćosić, Valentina Bušić, Dajana Gašo-Sokač, and Karolina Vrandečić. 2025. "Antifungal Activity of Quaternary Pyridinium Salts Against Fusarium culmorum in Wheat Seedlings" Applied Sciences 15, no. 14: 7889. https://doi.org/10.3390/app15147889

APA Style

Siber, T., Petrović, E., Ćosić, J., Bušić, V., Gašo-Sokač, D., & Vrandečić, K. (2025). Antifungal Activity of Quaternary Pyridinium Salts Against Fusarium culmorum in Wheat Seedlings. Applied Sciences, 15(14), 7889. https://doi.org/10.3390/app15147889

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