Evaluation of the Impact of Flutriafol on Soil Culturable Microorganisms and on Soil Enzymes Activity

Abstract

Fungicides play a role in managing plant diseases but raise concerns about environmental impact, emphasizing the need to understand and minimize their effects on non-target ecosystems. Flutriafol is a fungicide used to combat fungal diseases in crops. It has two enantiomers that exhibit different levels of efficacy and environmental impact. This study focuses on evaluating the effects of different doses of flutriafol on soil microorganism populations and enzyme activity and the possible specificity of enantiomer interactions with soil enzymes by combining experimental and computational approaches. The effects of different doses of flutriafol on the population of microorganism and on the activity of soil enzymes were experimentally assessed. Molecular docking of the enantiomers with soil enzymes was used to assess the possible stereoselectivity of the interactions. Regardless of the dose used (normal dose recommended by the manufacturer for cereal crops, half this dose, and double dose), flutriafol had no significant impact on soil microbial communities or on catalase activity. The half dose of flutriafol produced increases in the activity of dehydrogenases (8%), phosphatases (26%), and urease (33%) during the first 7 days of incubation. Molecular docking showed that both enantiomers were able to bind to the active sites of dehydrogenases and phosphatases. The average value of the interaction energy observed for (R)-flutriafol with dehydrogenases was −7.85 kcal/mol, compared to −7.45 kcal/mol for the interaction of (S)-flutriafol with these enzymes. Similarly, the interaction energy obtained for the interaction of (R)-flutriafol with phosphatase was −9.16 kcal/mol, compared to −9.04 kcal/mol for the interaction of (S)-flutriafol with this enzyme. This study confirms the need to implement optimized application practices when using flutriafol by considering the enantiomer that is most effective on the target organism and less toxic to non-target ecosystems.

1. Introduction

Pesticides are extensively applied in agriculture to improve and maintain crop yields and control pests. Global population growth is the main factor leading to an increasing use of pesticides to provide food for the entire population. Since 1990, the use of pesticides worldwide has increased by approximately 50% [1]. For almost all types of agricultural crops, pesticides are often used several times a year, and the high use of pesticides results in the storage of residues in the soil, especially in groundwater, affecting ecosystems.

To prevent or eradicate fungal infections in crops, fungicides belonging to the azoles and triazoles group have been frequently used. According to data available from Eurostat, the sale of pesticides in the EU in 2021 was estimated to be about 355,000 tonnes. The category of fungicides and bactericides was one of the pesticide groups with the highest sales rate in each year of the reference period (from 2011 until 2021), and imidazole and triazole fungicides accounted for 7% of the total fungicides and bactericides category [2].

Published data show that triazole fungicides can have various effects on soil health depending on the rates used and the types of crop and soil. Typically, triazole fungicides, especially at high doses, affect the structure of soil microorganism populations and disrupt the activities of various enzymes found in soil [3]. Moreover, published data also inform about the enantioselective effects of several triazole fungicides on the environment [4,5,6,7].

Among the triazoles, flutriafol, with the IUPAC name 1-(2-fluorophenyl)-1-(4-fluorophenyl)-2-(1,2,4-triazol-1-yl)ethanol, is a systemic fungicide that is applied to combat diseases in many types of crops, turf, and ornamental plants [8,9]. Flutriafol has one chiral carbon and, consequently, two enantiomers, (R)- and (S)-flutriafol. The marketed product is a racemic mixture of these two enantiomers. The literature reports enantioselective effects of the isomers, with the (R)-isomer revealing greater activity against some fungal species and also greater acute toxicity to earthworms and algae [10].

Flutriafol is considered a very persistent pesticide in soil with a half-life of 1177.3 days and a 90% degradation time of 3911.1 days, both data measured under field conditions [11]. The high persistence of flutriafol in soil can represent a consistent danger to the diversity and functioning of soil microbial communities and the activity of soil enzymes. Only a few studies addressing the effects of flutriafol on soil microbiota and on the activity of soil enzymes have been conducted. A study by Munier-Lamy and Borde showed that high doses of flutriafol reduced the fungal population in favor of bacteria and inhibited cellulolytic fungal activities, but these effects disappeared after 15 days [12]. Other studies have shown that flutriafol had only a small effect on the nitrifying microbial community [13], but when applied in high doses, it resulted in increased cellulolytic activity of microorganisms [14]. The decrease in soil enzyme activities (glucosidase, phosphatase, leucine aminopeptidase) and the populations of some microbial groups (nitrogen-fixing bacteria, ammonia-oxidizing bacteria, cellulose- and chitin-degrading microorganisms) were observed in experiments performed under laboratory conditions when repeated doses of fungicide were applied [15]. Regarding other environmental effects of flutriafol, it showed moderate toxicity to the aquatic organism Lemna minor by inhibiting proteins involved in photosynthesis and cellular detoxification [16] and also to other aquatic organisms: Lemna gibba, Chironomus riparius, Daphnia magna, Pimephales promelas, and Lepomis macrochirus [11].

Little is known about the distinctive environmental effects of flutriafol enantiomers. Consequently, the aim of the present study was to evaluate, under laboratory conditions, the effects of this fungicide on soil microbiota and on the activities of several enzymes found in the soil in correlation with the fungicide dose: dehydrogenase, phosphatase, urease and catalase. These enzymes were selected for analysis because they are considered indicators of soil quality and are among the enzymes that are considered in other studies evaluating the effects of triazole fungicides on soil activity. Furthermore, a molecular docking analysis was implemented to evaluate the possible enantioselectivity of the interactions of the two stereoisomers of flutriafol with soil enzymes.

2. Materials and Methods

2.1. Fungicide

The fungicide used in the present study is flutriafol, which was purchased from the local market as Impact (FMC Agro, Pentre, UK), a commercial product containing 125 g/L flutriafol. Flutriafol (C₁₆H₁₃F₂N₃O) is a chiral molecule that exhibits two enantiomers, (R)- and (S)-flutriafol. The commercial product is a racemate comprising equal quantities of the two enantiomers. The three-dimensional structures of the enantiomers were extracted from the PubChem database [17], visualized using Chimera [18], and illustrated in Figure 1.

The structures of the two stereoisomers were taken into account to implement the molecular docking analysis.

2.2. Soil Sampling

Chernozem soil samples were collected in February 2024 from a field situated near the city of Timisoara (Romania) from a zone where the pesticides were not used. Soil samples were collected from the top layer (about 20 cm) from 5 different locations at 2 kg of soil for each location. The soil was transported to the laboratory and brought to laboratory room temperature. It was then ground and sieved (2 mm) and randomly sampled to obtain sub-samples that were immediately processed. The experiments were carried out under laboratory conditions on soil samples not treated with fungicide and on soil samples treated with three distinct doses of flutriafol: half the normal dose (1/2D), normal dose (1D), and double the normal dose (2D), where D = 0.625 mg/ha flutriafol as recommended by the manufacturer for cereal crops. The fungicide was applied by spraying it on the soil samples. The soil in the container was well mixed before every sampling. The sampling considered several points in the container.

2.3. Assessment of the Effects of Flutriafol on Populations of Soil Culturable Microorganisms

The evaluation of the microbial population in the soil samples (control soil and soil treated with the three different doses of flutriafol: ½D, 1D, 2D) was performed using a standard plate count method [19], as described in our previous work [6]. Basically, the method involves ten-fold dilutions (final dilution of 10⁻³ for bacteria and 10⁻² for fungi enumeration) of soil samples followed by the inoculation of solid growth media. Bacterial counts were done using Plate Count Agar (glucose: 1 g/L, yeast extract: 2.5 g/L, enzymatic digest of casein: 5 g/L, agar: 15 g/L). In the case of fungal counts, Peptone Yeast Extract Agar was used (glucose: 40 g/L, soya peptone: 10 g/L, yeast extract: 5 g/L, streptomycin sulfate: 0.03 g/L, chloramphenicol: 0.05 g/L, agar: 15 g/L). A total of 100 µL of solution was used to inoculate the surface of the solid growth media. The plates were further incubated at 28 °C, and colonies were counted using the SCAN 300 (INTERSCIENCE, Saint Nom la Bretèche, France) automatic colony counter after 25 h for bacteria and after 48 h for fungi. Bacterial and fungal populations were assessed every 7 days for a period of 28 days. All experiments were implemented in triplicate, and the CFU was expressed as CFU/g of soil sample.

2.4. Evaluation of the Effects of Flutriafol on Soil Enzyme Activities

In the present study, the activities of several enzymes that are considered to be soil quality indicators, i.e., dehydrogenases (DA), urease (UA), alkaline phosphatases (PhA), and catalase (CAT), were evaluated in the presence of the three doses of flutriafol. In this study, the activity of alkaline phosphatase was only assessed and not that of acid phosphatase because alkaline phosphatase is known to be more active in neutral to alkaline soils [20], and Chernozem soils generally have a neutral to slightly alkaline pH [21]. The spectrophotometric method was considered for the evaluation of enzyme activities, and the UV–Vis T90 spectrophotometer (PG Instruments, Lutterworth, UK) was used for measurements. The method for determining DA was proposed by Schinner and his coworkers [22]. UA determination followed the method described by Alef and Nannipieri [23], and PhA was evaluated according to the method proposed by Dick et al. [24]. For the evaluation of CAT, the permanganometric method described by Dragan-Bularda [25] was taken into account. The soil samples (control soil and soil treated with flutriafol at the three different doses) were incubated for 28 days under laboratory conditions. The activity of each enzyme was measured every 7 days. The experiment was performed in triplicate, with the same researcher making measurements on the same day.

2.5. Molecular Docking Study

Both enantiomers of flutriafol were docked with the structures of investigated enzymes (dehydrogenase, urease, phosphatase, and catalase) belonging to several microorganisms found in soil: bacteria, fungi, and Rizhobium sp. The Protein Data Bank (PDB) [26] was considered for extracting the spatial structures of the complexes of these enzymes with specific substrates or inhibitors, when available. For the enzymes that did not have solved three-dimensional structures, structural models were extracted from the AlphaFold Protein Structure Database [27]. Table 1 contains information regarding the structural files considered in the molecular docking study.

Table 1. Crystallographic structures of enzymes belonging to soil microorganisms considered in the current study: AF—AlphaFold structural model identifier, PDB ID—structural file identifier in PDB.

Enzyme	Microorganism	Description and PDB/AlphaFold ID
Dehydrogenase	Clostridium beijerinckii	NADP-dependent alcohol dehydrogenase in complex with dihydro-nicotinamide-adenine-dinucleotide phosphate and Zn ion (1KEV chain A) [28]
	Aspergillus fumigatus	Mannitol 2-dehydrogenase in complex with 1,4-dihydronicotinamide adenine dinucleotide (7RK5 chain A) [29]
	Rhizobium leguminosarum	Succinate semialdehyde dehydrogenase in complex with 1,4-dihydronicotinamide adenine dinucleotide (8C54 chain A) [30]
Urease	Bacillus pasteurii	Urease subunit gamma in complex with citrate anion, 1,2-ethanediol, hydroxide, nickel (II), and sulfate ions (4AC7 chain C) [31]
	Aspergillus fumigatus	AlphaFold model of catalase AF-Q6A3P9-F1 [27]
	Rhizobium leguminosarum	AlphaFold model of catalase AF-Q1MCV9-F1 [27]
Phosphatase	Bacillus subtilis	Phosphate phosphatase F in complex with its inhibitory peptide GLN-ARG-GLY-MET-ILE (4I9C chain A) [32]
	Aspergillus niger	Acid phosphatase in complex with oligosaccharides, 2-acetamido-2-deoxy-beta-D-glucopyranose, glycerol, and sulfate ion (1QFX chain A) [33]
	Rhizobium leguminosarum	Alkaline phosphatase in complex with acetate, manganese (II), calcium, and sodium ions (2VQR chain A) [34]
Catalase	Micrococcus lysodeikticus	Catalase complexed with dihydro-nicotinamide-adenine-dinucleotide phosphate and hem (1GWH chain A) [35]
	Komagataella pastoris	Catalase in complex with hem, dihydro-nicotinamide-adenine-dinucleotide phosphate, di(hydroxyethyl)ether, glycerol, sulfate, sodium, chloride and potassium ions (6RJN chain A) [36]
	Rhizobium meliloti	AlphaFold model of catalase AF-Q9X576-F1 [27]

Table 1. Crystallographic structures of enzymes belonging to soil microorganisms considered in the current study: AF—AlphaFold structural model identifier, PDB ID—structural file identifier in PDB.

Enzyme	Microorganism	Description and PDB/AlphaFold ID
Dehydrogenase	Clostridium beijerinckii	NADP-dependent alcohol dehydrogenase in complex with dihydro-nicotinamide-adenine-dinucleotide phosphate and Zn ion (1KEV chain A) [28]
	Aspergillus fumigatus	Mannitol 2-dehydrogenase in complex with 1,4-dihydronicotinamide adenine dinucleotide (7RK5 chain A) [29]
	Rhizobium leguminosarum	Succinate semialdehyde dehydrogenase in complex with 1,4-dihydronicotinamide adenine dinucleotide (8C54 chain A) [30]
Urease	Bacillus pasteurii	Urease subunit gamma in complex with citrate anion, 1,2-ethanediol, hydroxide, nickel (II), and sulfate ions (4AC7 chain C) [31]
	Aspergillus fumigatus	AlphaFold model of catalase AF-Q6A3P9-F1 [27]
	Rhizobium leguminosarum	AlphaFold model of catalase AF-Q1MCV9-F1 [27]
Phosphatase	Bacillus subtilis	Phosphate phosphatase F in complex with its inhibitory peptide GLN-ARG-GLY-MET-ILE (4I9C chain A) [32]
	Aspergillus niger	Acid phosphatase in complex with oligosaccharides, 2-acetamido-2-deoxy-beta-D-glucopyranose, glycerol, and sulfate ion (1QFX chain A) [33]
	Rhizobium leguminosarum	Alkaline phosphatase in complex with acetate, manganese (II), calcium, and sodium ions (2VQR chain A) [34]
Catalase	Micrococcus lysodeikticus	Catalase complexed with dihydro-nicotinamide-adenine-dinucleotide phosphate and hem (1GWH chain A) [35]
	Komagataella pastoris	Catalase in complex with hem, dihydro-nicotinamide-adenine-dinucleotide phosphate, di(hydroxyethyl)ether, glycerol, sulfate, sodium, chloride and potassium ions (6RJN chain A) [36]
	Rhizobium meliloti	AlphaFold model of catalase AF-Q9X576-F1 [27]

To locate the active sites for ureases that do not have solved spatial structures in the PDB, their structural models, extracted from the AlphaFold database, were superimposed with the structure of urease belonging to Bacillus pasteurii (PDB ID 4AC7), and very close matches were found: RMSD = 0.585 Å for 544 alpha carbon (CA) pairs out of 565 total pairs for the superimposition of urease structures from Aspergillus fumigatus and Bacillus pasteurii, and RMSD = 0.550 Å for 550 CA pairs out of 567 total pairs for the superimposition of ureases structures from Rhizobium leguminosarum and Bacillus pasteurii (Supplementary Material Figure S1). Similarly, to detect the active site of the catalase belonging to Rhizobium meliloti (705 amino acids), which does not have a solved structure, its AlphaFold model was superimposed on the catalases belonging to Micrococcus lysodeikticus (503 amino acids) and Komagataella pastoris (510 amino acids), but overlaps were not satisfactory, the structural model of catalase from Rhizobium meliloti was superimposed on the structure of catalase from Penicillium vitale (688 amino acids) in complex with the inhibitor aminotriazole (PDB ID 2XF2). A very good match was obtained, RMSD = 0.778 Å for 515 CA pairs out of 688 total pairs when superimposing the structures of catalase from Rhizobium meliloti and Penicillium vitale (Supplementary Material Figure S2). In the case of catalase, with its active site buried deep within the enzyme, its substrate, H₂O₂, reaches the hem through a long channel that cannot accommodate the fungicide. It is known that nicotinamide adenine dinucleotide phosphate (NADPH) can bind to catalase and compensate for the ability of hydrogen peroxide to convert the enzyme to an inactive state [37]. From this point of view, we evaluated the ability of fungicide enantiomers to bind to Micrococcus lysodeikticus and Komagataella pastoris in the NADPH binding sites. For Rhizobium meliloti catalase, the binding of flutriafol enantiomers was assessed considering the binding site of the inhibitor aminotriazole.

The structures of the investigated enzymes were prepared for molecular docking analysis by using Chimera software, DockPrep facility [18]. The SwissDock online server [38] was used to implement the docking by selecting a blind and accurate docking. Analysis of docking results also involved Chimera, version 1.16 software.

The online tool Protein-Ligand Interaction Profiler (PLIP) was considered for identification of enzyme residues associated in non-covalent interactions with (R)- and (S)-flutriafol [39]. The input data for the PLIP computational tool were the enzyme–ligand complexes obtained by molecular docking, and the results allowed for the identification of protein–ligand non-covalent interactions at the atomic level.

2.6. Statistical Analysis

The PAST software, version 4.16 [40], was considered for performing the statistical analysis of the experimental data. The Shapiro–Wilk W test was selected to identify the normality of data distribution. Normally distributed data were further studied by parametric ANOVA tests: (i) Levene’s test to assess homogeneity of variance and (ii) Tuckey’s post hoc test. Not normally distributed data were analyzed by non-parametric ANOVA tests: (i) the Kruskal–Wallis test and (ii) Dunn’s post hoc test.

3. Results

3.1. Assessment of the Effects of Flutriafol on Soil Microbiota

The influence of fungicides on soil microbiota is not unexpected, as they are designed to kill or prevent fungal growth through various mechanisms, such as enzyme inhibition or biosynthesis inhibition [41,42].

In terms of bacteria present in the soil exposed to flutriafol, statistically significant differences were detected between the control, normal, and double doses on day 14. For the 28 study days, differences were observed between days 7 and 14 as well as 7 and 21 in terms of the control; between days 7 and 28 for the half dose; between days 7 and the other test days for the normal dose; and between days 7 and 21 as well as 7 and 28 for the double dose (Figure 2 and

Table 2. Outcomes of Dunn’s post hoc test for soil bacteria (above diagonal) and of Tukey’s post hoc test for soil fungi (below diagonal). Statistically significant data (p < 0.05) are highlighted in bold.

Dose/ Time (Days)		p Values
		Control				1/2D				D				2D
		7	14	21	28	7	14	21	28	7	14	21	28	7	14	21	28
Control	7		0.98	0.01	0.14	0.91	0.20	0.06	0.01	0.94	0.04	0.02	<0.05	0.78	0.04	0.01	<0.01
	14	1.00		0.01	0.13	0.88	0.19	0.06	0.01	0.97	0.04	0.02	0.04	0.76	0.04	0.01	<0.01
	21	0.96	0.99		0.30	0.02	0.22	0.52	0.98	0.01	0.65	0.85	0.60	0.03	0.63	0.87	0.56
	28	0.02	0.04	0.49		0.18	0.84	0.69	0.31	0.12	0.56	0.40	0.61	0.23	0.58	0.23	0.11
1/2D	7	1.00	1.00	0.97	0.02		0.25	0.08	0.02	0.85	0.05	0.03	0.06	0.87	0.06	0.01	<0.01
	14	1.00	1.00	0.68	<0.01	1.00		0.55	0.23	0.18	0.43	0.29	0.47	0.32	0.45	0.16	0.07
	21	1.00	1.00	1.00	0.28	1.00	0.88		0.54	0.05	0.85	0.65	0.91	0.11	0.87	0.42	0.22
	28	<0.01	<0.01	0.07	1.00	<0.01	<0.01	0.03		0.01	0.67	0.87	0.62	0.03	0.65	0.85	0.54
1D	7	1.00	1.00	1.00	0.04	1.00	1.00	1.00	<0.01		0.03	0.02	0.04	0.73	0.04	0.01	<0.01
	14	1.00	1.00	0.91	0.01	1.00	1.00	0.99	<0.01	1.00		0.79	0.94	0.08	0.98	0.54	0.30
	21	0.17	0.27	0.96	1.00	0.18	0.04	0.84	0.81	0.30	0.12		0.74	0.04	0.77	0.73	0.44
	28	0.02	0.03	0.42	1.00	0.02	<0.01	0.23	1.00	0.03	0.01	1.00		0.09	0.97	0.49	0.27
2D	7	1.00	1.00	0.98	0.03	1.00	1.00	1.00	<0.01	1.00	1.00	0.20	0.02		0.08	0.02	<0.01
	14	1.00	1.00	0.90	0.01	1.00	1.00	0.98	<0.01	1.00	1.00	0.10	0.01	1.00		0.52	0.29
	21	0.39	0.55	1.00	0.99	0.42	0.12	0.98	0.51	0.60	0.29	1.00	0.97	0.45	0.27		0.67
	28	0.01	0.01	0.23	1.00	0.01	<0.01	0.11	1.00	0.01	<0.01	0.99	1.00	0.01	<0.01	0.86

).

In the case of fungi present in soil samples exposed to flutriafol, no statistical differences could be identified between the test doses. However, there was a decrease over time, with statistically significant differences being noticed for the control and flutriafol doses between days 7 and 28 and as well as days 14 and 28, with one exception being the half dose, where day 28 was different from days 7, 14, and 21. This shows the decreased presence of fungi over time (Figure 3 and Table 2).

In general, the fungicide flutriafol did not show a major impact on the microbial communities in the tested soil under laboratory condition. Moreover, our results indicated that the pesticide acted as a source of carbon and nitrogen for the microorganisms, because even at the double dose of flutriafol, the microbial biomass was not inhibited, and in some cases, the microbial population was enhanced by the presence of the pesticide. It is also possible that the total bacterial amount was much higher than the fungal amount, obscuring the change in the result.

3.2. Assessment of the Effects of Flutriafol on Activities of Soil Enzymes

Soil dehydrogenase enzyme activity was affected by the exposure to flutriafol. Statistically significant differences were observed for the control and half-dose on days 14 and 28. Enzyme activity also underwent changes during the test period. For the control, differences were observed between the activities determined on day 7 compared to the other days, while for the flutriafol doses, differences were identified between days 7 and 14 and days 7 and 21 (Figure 4 and

Table 3. Outcomes of Tuckey’s post hoc test for activities of dehydrogenase (above diagonal) and of Dunn’s post hoc test for activities of phosphatase (below diagonal). Statistically significant data (p < 0.05) are highlighted in bold.

Dose/ Time (Days)		p Values
		Control				1/2D				D				2D
		7	14	21	28	7	14	21	28	7	14	21	28	7	14	21	28
Control	7		<0.01	<0.01	<0.01	0.98	0.65	<0.01	1.00	0.83	<0.01	<0.01	0.38	0.98	<0.01	<0.01	0.08
	14	0.02		0.99	0.65	<0.01	<0.01	0.89	<0.01	<0.01	0.23	0.87	<0.01	<0.01	0.08	0.99	0.01
	21	0.08	0.55		0.11	<0.01	<0.01	1.00	<0.01	<0.01	0.86	1.00	<0.01	<0.01	0.55	0.48	<0.01
	28	0.09	0.52	0.97		<0.01	0.38	0.06	0.02	0.23	<0.01	0.06	0.36	0.03	<0.01	1.00	0.87
1/2D	7	0.03	0.87	0.66	0.63		0.04	<0.01	0.25	0.08	<0.01	<0.01	0.01	0.16	<0.01	<0.01	<0.01
	14	0.01	0.69	0.35	0.33	0.59		<0.01	0.99	1.00	<0.01	<0.01	1.00	1.00	<0.01	0.09	1.00
	21	<0.01	0.41	0.17	0.16	0.33	0.70		<0.01	<0.01	1.00	1.00	<0.01	<0.01	0.95	0.29	<0.01
	28	0.20	0.27	0.61	0.63	0.34	0.16	0.07		1.00	<0.01	<0.01	0.93	1.00	<0.01	<0.01	0.40
1D	7	0.36	0.13	0.36	0.37	0.17	0.08	0.03	0.68		<0.01	<0.01	1.00	1.00	<0.01	<0.05	0.98
	14	0.01	0.57	0.27	0.26	0.48	0.88	0.82	0.12	0.05		1.00	<0.01	<0.01	1.00	0.03	<0.01
	21	<0.01	0.46	0.18	0.17	0.37	0.80	0.86	0.07	0.02	0.93		<0.01	<0.01	0.96	0.26	<0.01
	28	0.07	0.61	0.92	0.90	0.73	0.39	0.20	0.55	0.31	0.31	0.22		0.98	<0.01	0.07	1.00
2D	7	0.43	0.10	0.29	0.30	0.13	0.06	0.02	0.58	0.89	0.04	0.02	0.25		<0.01	<0.01	0.55
	14	0.12	0.40	0.81	0.84	0.49	0.25	0.11	0.79	0.49	0.19	0.12	0.74	0.41		0.01	<0.01
	21	0.01	0.77	0.37	0.36	0.66	0.89	0.57	0.16	0.07	0.75	0.66	0.43	0.05	0.26		0.36
	28	0.47	0.08	0.25	0.27	0.11	<0.05	0.02	0.52	0.82	0.03	0.01	0.22	0.94	0.36	0.04

).

In the case of soil phosphatase activity, statistically significant differences were observed only between the control and half dose on day 7. However, such differences were observed over time, between days 7 and 14 for the control, days 7 and 21 for the normal dose, and days 21 and 28 for the double dose (Figure 5 and Table 3).

Exposure to flutriafol resulted in statistically significant differences in the tested doses from the control only on day 7 for soil urease activity. However, statistically significant differences could also be observed over time for flutriafol doses between day 7 and the other test days (Figure 6 and

Table 4. Outcomes of Tuckey’s post hoc test for activities of urease (above diagonal) and of Dunn’s post hoc test for activities of catalase (below diagonal). Statistically significant data (p < 0.05) are highlighted in bold.

Dose/ Time (Days)		p Values
		Control				1/2D				D				2D
		7	14	21	28	7	14	21	28	7	14	21	28	7	14	21	28
Control	7		0.97	1.00	0.10	<0.01	1.00	0.99	0.11	<0.01	1.00	0.98	0.07	<0.01	1.00	0.99	0.11
	14	0.02		1.00	0.86	<0.01	1.00	1.00	0.88	<0.01	0.66	1.00	0.78	<0.01	1.00	1.00	0.79
	21	<0.01	0.41		0.38	<0.01	1.00	1.00	0.40	<0.01	0.98	1.00	0.30	<0.01	1.00	1.00	0.35
	28	0.13	0.59	0.20		<0.01	0.63	0.73	1.00	<0.01	0.02	0.84	1.00	<0.01	0.28	0.78	1.00
1/2D	7	0.38	0.15	0.02	0.46		<0.01	<0.01	<0.01	0.02	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
	14	0.03	0.91	0.35	0.66	0.19		1.00	0.65	<0.01	0.99	1.00	0.53	<0.01	1.00	1.00	0.55
	21	<0.01	0.15	0.53	0.07	<0.01	0.12		0.76	<0.01	0.80	1.00	0.63	<0.01	1.00	1.00	0.66
	28	0.28	0.22	0.04	0.57	0.84	0.26	0.01		<0.01	0.02	0.86	1.00	<0.01	0.30	0.80	1.00
1D	7	0.65	0.11	0.02	0.33	0.75	0.13	<0.01	0.62		<0.01	<0.01	<0.01	0.99	<0.01	<0.01	<0.01
	14	0.02	0.99	0.40	0.60	0.16	0.93	0.14	0.23	0.11		0.68	0.01	<0.01	1.00	0.76	0.03
	21	<0.01	0.18	0.60	0.08	0.01	0.14	0.93	0.01	<0.01	0.17		0.76	<0.01	1.00	1.00	0.77
	28	0.83	0.06	0.01	0.23	0.57	0.08	<0.01	0.45	0.82	0.07	<0.01		<0.01	0.21	0.68	1.00
2D	7	0.16	0.37	0.08	0.79	0.60	0.43	0.02	0.74	0.43	0.38	0.02	0.30		<0.01	<0.01	<0.01
	14	0.01	0.64	0.72	0.34	0.06	0.57	0.33	0.09	0.04	0.63	0.38	0.02	0.17		1.00	0.26
	21	<0.01	0.26	0.76	0.12	0.01	0.21	0.76	0.02	0.01	0.25	0.83	<0.01	0.04	0.50		0.71
	28	0.37	0.16	0.03	0.47	0.98	0.20	<0.01	0.86	0.73	0.17	0.01	0.55	0.62	0.06	0.01

).

No statistically significant differences were noticed between the dose variants tested for catalase activity in soil. However, differences were observed over time. For the control, differences were observed between days 7 and 14 and days 7 and 21. For flutriafol doses, differences were noticed between days 7 and 21 and days 21 and 28 (Figure 7 and Table 4).

Overall, the fungicide flutriafol, used at half of the recommended dose, led to minor increases in the activities of several soil enzymes: dehydrogenases, urease, and phosphatase. Regardless of the dose of flutriafol, there were no effects on the activity of soil catalases.

3.3. Molecular Docking Analysis of the Interactions between the Flutriafol Enantiomers and Investigated Soil Enzymes

The results of the molecular docking study on the interactions of the two enantiomers of flutriafol with the soil enzymes considered in the present study are presented in Figure 8, Figure 9, Figure 10 and Figure 11, in

Table 5. Energy values obtained for the best binding modes of flutriafol enantiomers to the investigated soil enzymes.

Enzyme	Organism	ΔG (kcal/mol)
		(R)-Flutriafol	Mean Value	(S)-Flutriafol	Mean Value
Dehydrogenase	Clostridium beijerinckii	−8.41		−8.48
	Aspergillus fumigatus	−7.51	−7.85	−6.89	−7.58
	Rhizobium leguminosarum	−7.63		−7.38
Phosphatase	Bacillus subtilis	−7.54		−7.75
	Aspergillus niger	−7.22	−7.36	it does not bind to the active site	−7.45
	Rhizobium leguminosarum	−7.32		−7.16
Catalase	Komagataella pastoris	−9.16	−9.16	−9.04	−9.04

, and in the Supplementary Materials (Figures S3–S7). Figure 8 shows the binding modes (BMs) corresponding to the highest interaction energies for the interactions of flutriafol stereoisomers with dehydrogenases belonging to Clostridium beijerinckii, Aspergillus fumigatus, and Rhizobium leguminosarum.

Similarly, Figure 9 reveals the BMs corresponding to the best interaction energies for the interactions of flutriafol stereoisomers with phosphatases from Bacillus subtilis, Aspergillus niger, and Rhizobium leguminosarum.

In the case of the phosphatase belonging to Aspergillus niger, only (R)-flutriafol binds to the active site of this enzyme (Figure 9b). Figure 10 shows that BMs of (S)-flutriafol to Aspergillus niger phosphatase do not match the active site of the enzyme.

Figure 11 shows the BMs of the flutriafol stereoisomers to Komagataella pastoris catalase.

The two stereoisomers of flutriafol did not bind to the active site of ureases and the active sites of Micrococcus lysodeikticus and Rhizobium meliloti catalases (Supplementary Figures S3–S8).

Table 5 shows the binding energies for the two stereoisomers of flutriafol to soil dehydrogenases, phosphatases, and Komagataella pastoris catalase.

The results of the PLIP software on the existing non-covalent interactions for each enantiomer and enzyme residues are shown in

Table 6. Noncovalent interactions established by flutriafol enantiomers and residues of the enzymes considered in this study. For comparison, the noncovalent interactions for the substrates and enzyme residues are also presented: NADPH—1,4-dihydronicotinamide adenine dinucleotide, NAG—2-acetamido-2-deoxy-beta-D-glucopyranose.

Enzyme	Ligand	Residues Associated with the Non-Covalent Interactions
Clostridium beijerinckii dehydrogenase	(R)-flutriafol	GLY176, ALA177, VAL178, GLY179, ARG200
	(S)-flutriafol	THR38, ILE175, GLY176, ALA177, VAL178, TYR267, ILE345
	NADPH	THR38, SER39, ILE175, ALA176, VAL178, GLY179, SER199, ARG200, GLU247, ASN266, TYR267, LYS340
Aspergillus fumigatus dehydrogenase	(R)-flutriafol	GLY44, PHE45, THR139GLN244
	(S)-flutriafol	GLY44, PHE45, THR137, THR139, GLU140
	NADPH	GLY44, PHE45, GLN74, ASP77 (2), ASP199, ASN200, VAL238, THR242
Rhizobium leguminosarum dehydrogenase	(R)-flutriafol	PRO161, TRP162, LYS186, SER240
	(S)-flutriafol	TRP162, LYS186, ALA188, SER240
	NADPH	THR160, PRO161,LYS186, PRO187, SER240, GLU261
Bacillus subtilis phosphatase	(R)-flutriafol	TYR66, TYR152, TYR153, ARG223, TYR226, LEU230, GLN263
	(S)-flutriafol	-
	Inhibitory peptide	TYR66, TYR152, TYR153, LYS155, GLN183, LEU187, ASP194, ARG223, TYR226 ASN227, SER260, GLN263, PHE266, TYR300, GLU303, ALA334, ASP335, ASP338
Aspergillus niger phosphatase	(R)-flutriafol	ALA292, ASN296, LEU415, ASP417, TYR418, THR419, LEU432
	NAG	ASN296, THR300, ASN439, LEU432
Rhizobium leguminosarum phosphatase	(R)-flutriafol	TYR105, PHE138, ASN141
	(S)-flutriafol	TYR105, LEU125, PHE138, ASN141, TYR215
	Calcium	ASP12, ASP324, HIS325

. For each enzyme, the residues associated in non-covalent interactions with its substrate were also extracted from the PDB files and are also shown in Table 6 for comparison.

The data presented in Table 5 and Table 6 and in Figure 8, Figure 9 and Figure 11 show that the investigated stereoisomers are able to bind to dehydrogenases, phosphatases, and the Komagataella pastoris catalase, as the binding energies are quite different from one stereoisomer to another and the orientations of the stereoisomers in the binding site are also distinct. Usually (R)-flutriafol binds more strongly to enzymes. Amino acids associated with the interactions between (R)-flutriafol and enzymes are distinct compared to those associated with the interactions between (S)-flutriafol and these enzymes. This emphasizes the distinct orientation of the enantiomers in the active sites of the enzymes. Enzyme amino acids that form noncovalent interactions with flutriafol enantiomers are among those involved in noncovalent interactions of enzymes with their substrates/ligands, which underlines the inhibitory potential of flutriafol stereoisomers.

4. Discussion

4.1. Evaluation of the Effects of Flutriafol on Populations of Soil Culturable Microorganisms

Our findings are in good agreement with the results of other studies on the effect of triazole fungicides on soil microbial biomass. Munier-Lamy and Borde reported that flutriafol applied in the recommended dose and even ten times higher did not affect the ability of soil microflora to decompose cellulose, but higher doses of flutriafol initially inhibited the cellulolytic activity before further stimulating this activity [12]. Sim and coworkers tested the effect of flutriafol on soil microorganisms and reported that flutriafol increased cellulolytic activity in soil when applied at five times the recommended dose [14]. In another study, Sim and coworkers showed that flutriafol application did not significantly affect the abundance and the composition of bacterial and fungal groups in the three tested soils, with only a small decreasing effect on the nitrifier microbial community [13]. Literature data also show that the effects of flutriafol are more prominent in field studies compared to the effects obtained in laboratory scale tests, the effects of pesticides are transient, and only repeated application of flutriafol significantly affected soil microbial communities [15]. Under laboratory conditions, flutriafol had an inhibitory effect only on several microbial groups, such as ammonia-oxidizing bacteria, chitin- and cellulose-degrading microorganisms, and nitrogen-fixing bacteria [15].

For another triazole fungicide, propiconazole, at a concentration of 5 kg/ha (about ten times the recommended dose), a stimulatory effect on the bacterial population was observed, while a decrease in the bacterial population occurred at a concentration of 10 kg/ha. Also, a decrease in the fungal population was recorded for high concentrations (15 and 20 kg/ha of propiconazole), while the concentration of 10 kg/ha represented the modulatory dose for fungal growth. Similar to our results, the fungal population showed a gradual decrease during the third and fourth weeks of incubation [43]. In the case of tritizonazole, another triazole fungicide, in the recommended and double doses, it did not significantly affect the bacterial population in the soil during the 28 days of incubation, but after 14 days, the total soil population of microorganisms decreased [6]. Triticonazole also had a nonsignificant effect on microorganisms associated with carbon and nitrogen mineralization [11]. It was revealed that the presence of tebuconazole and triticonazole (both triazole fungicides) increased the population of soil bacteria [44]. The presence in soil of other sterol-targeting fungicides, propiconazole and fenpropimorph, inhibited overall soil bacterial activity [45].

Furthermore, there are studies that reveal the biodegradation capacity of bacterial strains toward three triazole fungicides. For example, Luong and coworkers demonstrated that several species of bacteria (Klebsiella sp., Pseudomonas sp., and Citrobacter sp.) isolated from agricultural soils can degrade triazole fungicides (hexaconazole, difenoconazole, and propiconazole), achieving a degradation rate ranging from 86 to 96% at a concentration of 50 mg/kg of each fungicide in 45 days in a soil–plant system. Moreover, the synergistic combination of the three bacterial isolates improved plant growth and promoted nitrogen fixation, phosphate dissolution, and cellulose degradation, amongst others [46]. Satapute and coworkers also reported that a strain of Pseudomonas aeruginosa isolated from a paddy soil adulterated with propiconazole was able to utilize the fungicide up to a concentration of 8 μg/L after 72 h incubation [47].

Fungicides are considered to be effective only against fungi, and not against bacteria, but it is commonly recognized that the two soil microbial communities are closely related. Some studies have found negative correlations due to competitive exclusion, while others have identified positive correlations in environments where mutualistic interactions are prominent. The products of pesticide decomposition are also known to function as carbon sources for soil microorganisms [48].

The limitations of this study are that the total population of microorganisms was measured only based on the estimate of culturable microbes, and the analysis was not completed at the taxon level. Soil health and ecosystem functions (like nutrient cycling, organic matter decomposition, etc.) are driven by the entire microbial community. Focusing only on culturable microorganisms provides an incomplete understanding of how flutriafol might affect these critical processes. Moreover, this study might underestimate the negative effects of flutriafol because it does not account for unculturable microorganisms that could be more susceptible to this fungicide. These limitations underscore the need for more comprehensive approaches that include both culturable and unculturable microorganisms and an analysis at the taxon level to accurately assess the impact of flutriafol on soil health.

4.2. Evaluation of the Effects of Flutriafol on Soil Enzyme Activities

Data from the experimental evaluation show that flutriafol influences soil DA with dose-dependent effects, with a half dose of flutriafol producing a significant increase in DA in the first 14 days after treatment. However, the other doses appeared to have no effect on DA because changes in DA were not significant when compared to the control samples. This was also true for PhA and UA, which were significantly increased by a half dose of flutriafol in the first 7 days after treatment. Regardless of dose, flutriafol did not significantly affect CAT in soil. Taking into account the results of the docking analysis and other published data on the effects of other triazole fungicides on the activity of soil enzymes [3,6,7], we consider that the recorded effects of flutriafol on activities of the investigated enzymes are mainly due to the changes in the microbial population. The lack of effect of flutriafol on catalase activity may be due to the fact that catalase is a relatively stable and robust enzyme [49] and cannot be easily denatured or inhibited by the fungicide. Another reason could be that flutriafol cannot interfere with hydrogen peroxide and has no effect on catalase activity. Furthermore, soil microbial communities are diverse and can compensate for the loss of activity in some members by increasing the activity of others.

These results inform that, at the doses considered in this study, flutriafol only has a small effect on soil enzyme activity.

Published data reveal that the effects of triazole fungicides on soil enzyme activities are dependent on dose, soil type, and fungicide, some of the published data being in agreement with the findings presented in this study. In the case of flutriafol used in the recommended dose and five times this dose, it has an inhibitory effect on the activity of β-1,4-N-acetyliglucosaminidase in loam soil, but the effect of flutriafol on this enzyme was not significant in loamy-sand and sandy-loam soils [13]. The effects of a repetitive soil treatment with flutriafol revealed strong inhibitory effects on several soil enzymatic activities: phosphatase, α-1,4-glucosidase, β-1,4-N-acetyliglucosaminidase, β-1,4-glucosidase, leucine aminopeptidase, β-xylosidase, and β-D-cellobiohydrolase [15].

Another triazole fungicide, propiconazole, incubated under laboratory conditions in two soil types, led to increased phosphatase and urease activities when applied at low doses (1.0, 5.0 and 10 kg/ha) for 4 weeks post-treatment. The increase was more pronounced in the first 2 weeks after treatment, but the increase in enzyme activities was steadily reduced after 2 to 4 weeks post-treatment. Soil phosphatase and urease showed higher activities in black soil compared to red soil. High propiconazole concentrations (15 and 20 kg/ha) led to decreased phosphatase and urease activities in both soil types [43].

Under laboratory conditions, for the Cernoziom soil in which barley and wheat seeds treated with several doses of triticonazole (minimum, medium, and maximum) were sown, there was a decrease in the activity of dehydrogenases and phosphatases and an increase in the activity of urease [6]. A decrease in dehydrogenase activity and an increase in catalase activity over a 28-day period were also observed for Chernozem samples treated with twice the recommended dose of myclobutanil. Regarding the activities of phosphatases, urease, and protease, they were not affected by the presence of myclobutanil regardless of the dose used [7]. Triadimefon, applied at a dose of 1 mg/kg of molisol and inceptisol, led to a decrease in the activity of dehydrogenases, but triadimefon did not affect the activity of phosphatases, regardless of the type of soil [50].

It should be noted that, during the incubation period, there were fluctuations in the activities of all enzymes considered in the present study. Enzyme activities did not show a regular trend but increased or decreased at different durations and concentrations. These changes were not only recorded for the soil treated with different doses of flutriafol but also for the control soil samples. Since we did not take measurements of soil physicochemical parameters, we can only assume that these changes may be due to the variations in moisture and soil physicochemical properties that are also correlated with changes in the structure of soil microorganisms.

4.3. Molecular Docking Analysis

Flutriafol enantiomers are able to bind to the active sites of investigated dehydrogenases and phosphatases and to the active site of the Komagataella pastoris catalase. The higher interaction energies recorded for the interactions of the two enantiomers with soil dehydrogenases compared to the interaction energies with soil phosphatases correlate with the experimental data, which reveal the inhibition of dehydrogenase activity in the presence of flutriafol. The results of the molecular docking analysis revealing that the two enantiomers of flutriafol bind to the active sites of investigated phosphatases do not correlate with the experimental outcome that shows a slight increase in the activity of soil phosphatases in the presence of flutriafol. This inconsistency can be explained by the fact that there are various microorganisms in the soil that produce different types of phosphatases, not all of which are affected by the presence of flutriafol. In addition, experimental data indicated that the fungicide represented a source of carbon and nitrogen for the microorganisms, in some cases the microbial population was enhanced by the presence of the fungicide, and the composition and abundance of soil microorganisms is known to significantly influence the soil phosphatase activity.

The two enantiomers of flutriafol bind to the catalytic site of Komagataella pastoris catalase but not to the active sites of the other catalases. This result is correlated with experimental data that do not reveal significant differences in soil CAT and microorganism populations for different doses of flutriafol.

The results of the molecular docking analysis also show that the two enantiomers of flutriafol are not able to bind to the catalytic sites of the ureases. This correlates with the experimental results that do not reveal statistically significant changes observed for urease activities in the soil treated with different doses of flutriafol. In addition, enantiomers of other triazole fungicides, triticonazole [6] and myclobutanil [7], did not bind to the active site of ureases.

Distinct orientations, different interaction energies, and noncovalent interactions with different amino acids were observed for the two stereoisomers of flutriafol in their interactions with dehydrogenases and phosphatases. The interaction energies were usually higher for enantiomers that bind to dehydrogenases than to phosphatases. These distinct interactions suggest distinct effects of flutriafol enantiomers on soil health and quality. This result is not unexpected, as it is known that stereoisomers, due to their distinct interactions with proteins, can exhibit significantly different bioactivity towards both target and non-target organisms. Furthermore, the results of the present study are in strong agreement with literature data reporting that the (R)-flutriafol enantiomer revealed higher activity than the (S)-flutriafol enantiomer against some species, and (R)-flutriafol showed higher environmental toxicity [10]. Moreover, distinct biological activities have been noticed for the isomers of numerous pesticides, including triazole fungicides: difenoconazole [4,50,51], metconazole [52], myclobutanil [7], prothioconazole [53], tebuconazole [54], and triticonazole [6]. All these data highlight that the effectiveness and environmental impact of stereoisomers can vary significantly.

4.4. Factors Affecting the Impact of Pesticides on Soil and Crops and Remediation Solutions

The effects of pesticides on soil and crops are influenced by a variety of factors, including chemical properties of the pesticide, environmental factors, soil characteristics, and crop management practices. The most important soil characteristics affecting pesticide toxicity include soil pH, soil-to-water ratio, organic matter content, soil structure, coexisting substances, and soil microbial activity, as soil microorganisms can break down pesticides, influencing their persistence and impact on the soil ecosystem [55]. Understanding the complex interactions among these factors is crucial to developing sustainable pesticide management practices that minimize adverse effects on soil health and crop productivity.

Pesticides can affect crop plants that may be sensitive to these chemicals. Triazole fungicides, such as flutriafol, can have phytotoxic effects on a wide range of plant species, even those crops that are intended to be protected by fungicide application, as triazole fungicides have been found to affect the gibberellins and sterols synthesis and can reduce the transpiration of crop plants [56,57].

The modernization of agriculture is also based on the use of various methods of remediation of contaminated soil. Numerous physical and/or chemical techniques have been established for the remediation of pesticide-contaminated soils. To our knowledge, there are no published articles disclosing methods for remediation of soil contaminated with flutriafol, but there is information on remediation of soil and water contaminated with other triazole fungicides. Soil management techniques such as solarization and ozone application have been shown to improve the degradation rates of triazole fungicides in soil, with the removal efficiency of these fungicides also being improved by longer exposure time [58]. Another method of remediation of soil polluted with triazole fungicides was the use of bacteria that have been identified as active for the degradation of these fungicides, such as Klebsiella sp., Citrobacter sp., and Pseudomonas sp. [46].

The use of porous biological media in the remediation of pesticide-polluted soil is a promising approach that harnesses natural processes and materials to remediate contaminated environments. Porous biological media usually involve the use of materials that have high porosity and are favorable to microbial activity, which can enhance the breakdown of pesticides. Porous media improve soil aeration, provide a habitat that supports high microbial activity and diversity, and are able to adsorb pesticides, reducing their mobility and bioavailability in soil. For example, four types of structured wetlands were used to effectively treat agricultural wastewater contaminated with the triazole fungicides triticonazole and myclobutanil, and the significant factor determining the dissipation of the fungicides was the type of porous media added and the plant species used [59]. The use of Fe-biochar composites also proved to be a promising and ecofriendly carbon-supported iron catalyst for remediation of organic-polluted soil [59]. Others studies have shown that the ball-milled combination of sepiolite and phosphate rock was efficiently used as a passivator in cadmium-polluted soil [60], and magnetic biological pellets were effective to remediate atrazine-polluted soil [56].

Consequently, effective remediation of pesticide-polluted soil requires a comprehensive understanding of the type of contamination and the selection of appropriate techniques. By integrating physical, chemical, and biological methods, it is possible to restore soil health and mitigate the adverse effects of pesticide pollution in a sustainable manner.

5. Conclusions

The present study evaluated the effects of flutriafol, a triazole fungicide, on the microbial population and enzyme activity in soil, as well as the possible enantioselectivity of the interactions of the two enantiomers of flutriafol with several enzymes considered as indicators of soil quality.

The fungicide flutriafol did not show a major impact on microbial communities in the tested soil, as the microbial biomass was not inhibited. On the contrary, in some cases, the presence of flutriafol increased the microbial population. It should be noted that fungicides are specifically designed to target and control fungal infections and diseases and are not effective against bacteria, as bacteria and fungi are different types of organisms with distinct biological structures and mechanisms. It is also generally accepted that there is a correlation between fungal and bacterial populations that is influenced by a multitude of biotic and abiotic factors. Understanding this correlation is important, and further analysis is needed to establish if the pesticide acted as a nutrient for the microorganisms.

The fact that the fungicide did not impact the soil microbial communities was in good correlation with the observation that, used at half the recommended dose, the fungicide resulted in a weak enhancement of the activity of soil dehydrogenases, phosphatases, and urease, and it had no effect on soil catalase activity regardless of the flutriafol dose.

Molecular docking analysis highlighted distinct interaction energies between the flutriafol enantiomers and soil dehydrogenases, phosphatases, and one of the catalases, with (R)-flutriafol providing higher interaction energies. Distinct spatial orientations of the two enantiomers in the catalytic sites of these enzymes were also observed. These findings emphasize the distinct bioactivity of flutriafol enantiomers and the need to use the most active stereoisomer against fungi in practice so as to maximize efficacy and minimize the side effects of the fungicide.

To our knowledge, this is the first study on the interactions of flutriafol enantiomers with enzymes found in soil. In our opinion, understanding the distinct bioactivities of fungicide enantiomers is important for the development of sustainable and ecological pest management strategies. Monitoring the effect of fungicides, both individually and their enantiomers, on non-target ecosystems provides information on the toxicity and consequences of fungicide use.