The Effects of the Fungicide Myclobutanil on Soil Enzyme Activity

: (1) Background: The use of pesticides, although needed to protect crops and increase production, represents an environmental and human health issue. Therefore, measures must be taken in order to develop a better understanding of the risks involved in the overuse of these compounds. Myclobutanil is a chiral triazole fungicide widely used for the protection of crops against fungal diseases. Published data have shown that, although effective in preventing fungal infections, high doses of myclobutanil can affect the soil environment. The aim of this study was to evaluate the effect of different doses of myclobutanil on soil enzyme activity, as well as the possible speciﬁcity of the interactions of the two stereoisomers of myclobutanil with these enzymes. (2) Methods: A combination of experimental and computational approaches was considered. An experimental method was applied in order to assess the effect of different doses of myclobutanil on the activity of dehydrogenase, phosphatase, catalase, urease and protease. The computational approach was based on the molecular docking of the two enantiomers of myclobutanil with the above-mentioned enzymes to assess the possible enantioselectivity of the interactions. (3) Results: High doses of myclobutanil signiﬁcantly affected the enzymatic activity of dehydrogenase and led to a slight increase in the activity of catalase. Molecular docking data showed that both enantiomers of myclobutanil were able to bind to the active sites of dehydrogenase, phsosphatase and protease, with higher interacting energies observed for (S) -myclobutanil, the enantiomer known to be less active against target organisms but have a higher toxicity against non-target organisms. (4) Conclusions: The results of our study conﬁrm the need to implement better management practices regarding the use of myclobutanil (and of pesticides in general) by using the enantiomer that is most effective on target organisms and less toxic to non-target organisms


Introduction
Pesticides are chemicals that include a wide range of compounds, the main categories being represented by herbicides, fungicides and insecticides.These chemicals are widely developed and used to protect crops, but are also found as contaminants in air, water and soil [1].Pesticides affect both their target organisms and numerous non-target species and are recognized for their harmful impact on the environment and human health [2].Thus, new information on their effects is needed to ensure the effectiveness of protective measures.
Among pesticides, the class of triazole fungicides is essential to agriculture and food safety, being used to protect crops against yeast and mildew, their target organisms [3].
The global application amount of triazole fungicides was around 20,000 metric tons in 2020 [4].These fungicides are proven to offer long-lasting control of numerous target plant pathogens and are considered to have moderate risk for developing fungicide resistance [5].The biological action of these chemicals is based on their interaction with the target enzyme 14α-demethylase (a member of the cytochrome 450 family) that results in the inhibition of the synthesis of the fungal-specific sterol, ergosterol [6].Ergosterol is the main sterol component of fungal membranes.It is involved in maintaining the structural integrity, thickness, permeability, and fluidity of the membrane and modulating the activity of several membrane-bound enzymes.The inhibition of ergosterol synthesis has a fungistatic effect because an increase in membrane permeability occurs and adversely affects the activity of several membrane-associated enzymes [7].
The presence of triazoles in the environment may influence not only the target species (fungi) but all kingdoms as well due to their nonspecific interactions with enzymes belonging to the cytochrome P450 family, resulting in the non-target effects of these fungicides [8].
Published data reveal that triazole fungicides usually affect soil enzyme activity, as discussed in a recent review [4].DA was considerably reduced by high doses of difenoconazole, hexaconazole, mycobutanil, paclo-butrazol, tebuconazole, and triadimefon.Urease (UA) and phosphatase (PhA) activities caused strong decreases in the presence of difenoconazole, propiconazole and tebuconazole.Tebuconazole use led to decreased catalase (CA), aryl sulfatase, nitrate reductase, β-glucosidase, and invertase activities.Another study reveals that triticonazole decreased DA, did not affect PhA, but increased UA [9].
Literature data show that both the antifungal activity and non-target toxicity of several triazole fungicides are enantioselective [10][11][12][13][14][15][16][17][18].There was no enantioselectivity of triticonazole stereoisomers in their interaction with soil enzymes, but such selectivity was recorded for the two steroisomers in the interactions with plasma proteins and human cytochromes [9].Enantioselectivity in interactions with plasma proteins and human cytochromes was also recorded for difenoconazole enantiomers [10].Triazole fungicides have also disrupted the structure of soil microbial communities (usually resulting in a decrease in the soil microbial population) [4] and have shown toxicological effects on organisms living in aquatic environments [19].
In the class of triazole fungicides, myclobutanil, with the IUPAC name (RS)-2-(4chlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl) hexanenitrile, is a broad-spectrum systemic fungicide.It is approved for use in many countries worldwide, including Iceland, Norway, Australia, the USA, and countries from Asia and Africa [20].The fungicide may be applied at multiple plant growth stages (from seeds to grown season) but is also used as food additive and wood preservative.The target organisms of myclobutanil include ascomycetes, basidiomycetes and imperfect fungi.Myclobutanil controls the populations of these microorganisms on various crops including almonds, apricots, cereals, cotton, cucumbers, grapes, peaches, plums, strawberries, etc. [21].
Residues of myclobutanil were found in crops, in the soils in which they were grown [22], and in water streams [23,24].The persistence of myclobutanil in various soil types was determined under both laboratory and field conditions, and the reported dissipation half-life (DT 50 ) values ranged from 11.0-19.2days [25][26][27] up to 574 days in anaerobic soil [28].Others studies have discovered that myclobutanil residues are present in air [29] and food [30,31], the accumulation of myclobutanil in the food chain also being due to its ability to persist in the environment.Due to the presence and persistence of the fungicide in the environment, moderate adverse effects have been recorded from myclobutanil on non-target species: aquatic organisms, birds, bees, earthworms and rodents [32].
To the best of our knowledge, there are only several studies published in the literature on the effects of myclobutanil on soil enzyme activity.A small dose of myclobutanil applied to tea orchard soil produced an increase in DA, but higher doses led to decreased DA [33].When using sandy tea garden soil, DA was slightly inhibited in the presence of myclobutanil during the first 30 days, but this effect disappeared over time [34].
Myclobutanil, as a commercial product, is an isomeric equimolar mixture of the (R)and (S)-stereoisomers [35].Literature data show that there are significant differences between the effects on target organisms produced by the racemic mixture and the effects produced by the enantiomers of myclobutanil.(R)-myclobutanil showed a higher antifungal activity than (S)-myclobutanil and also than the racemic mixture [36].The enantioselective effects of myclobutanil isomers were also observed for non-target organisms.In the case of aquatic organisms, racemic myclobutanil showed greater toxicity than its enantiomers, which suggests a toxic synergistic effect between enantiomers.Also, the (S)-myclobutanil isomer revealed slightly greater toxicity than (R)-myclobutanil on these organisms [37].Furthermore, the degradation of the two enantiomers of myclobutanil was distinct in aerobic soils, (R)-myclobutanil being preferentially degraded compared to (S)-myclobutanil [37].
Another study pointed to a preferential degradation of (R)-myclobutanil in cucumber crops, which led to an enrichment of (S)-myclobutanil in plants and soil [38].Despite the proven differences between the biological activities of the myclobutanil enantiomers, this fungicide is still sold as a racemic mixture.
This study aims to evaluate, under laboratory conditions for 28 days, the effects of different doses of myclobutanil on the soil enzyme activity of the following: catalase, dehydrogenase, phosphatase, protease and urease.Numerous other enzymes, such as amidase, β-glucosidase, cellulose and chitinase, are detected in soil; their activities are correlated with soil organic matter and total nitrogen and may be also affected by pesticides [39].The reason for considering dehydrogenase, phosphatase, protease and urease in the present study is that they are the most commonly used enzymes in the assessment of the biological status of soils and are also considered as potential indicators of soil quality [40].Catalase is considered because its activity in soil has been shown to correlate with DA [41].In addition, catalase, dehydrogenase, phosphatase, protease and urease activities are affected by other triazole fungicides [9,42].Last but not least, since published data reveal the distinct biological activities of the two enantiomers of myclobutanil, molecular docking is considered in order to assess the specificity of the interactions of the enantiomers with these enzymes.
Considering the DT 50 values for myclobutanil in soil, 11.0-19.2days [11][12][13], the present study was implemented over a period of 28 days to cover the dissipation half-life.Moreover, numerous studies in the specialized literature mention this period as appropriate for experiments seeking to evaluate the effects of pesticides on the activity of soil enzymes under laboratory conditions.
To the best of our knowledge, this is the first study assessing the effects of myclobutanil on the activities of soil catalases, phosphatases, proteases, and ureases.Furthermore, the molecular docking approach is considered in order to better describe the possible enantioselective effects of the two stereoisomers of myclobutanil on the soil enzyme activities.
For the molecular docking study, the 3D structures of the two enantiomers of myclobutanil were extracted from PubChem database [43].

Soil Sampling
The soil used in experiments was of chernozem type and collected from a field located close to Timisoara city, Romania, (45 • 45 14.54 N, 21 • 18 16.66E) where pesticides were never used.Soil samples were collected from the top layer of soil (about 20 cm) from 5 distinct spots in quantities of 20 kg each.The soil was ground, sieved (2 mm) and scooped via random sampling to obtain sub-samples of 10 kg of soil, which were immediately processed [44].For the molecular docking study, the 3D structures of the two enantiomers of myclobutanil were extracted from PubChem database [43].

Soil Sampling
The soil used in experiments was of chernozem type and collected from a field located close to Timisoara city, Romania, (45°45′14.54″N, 21°18′16.66″E) where pesticides were never used.Soil samples were collected from the top layer of soil (about 20 cm) from 5 distinct spots in quantities of 20 kg each.The soil was ground, sieved (2 mm) and scooped via random sampling to obtain sub-samples of 10 kg of soil, which were immediately processed [44].
SysthaneTM Forte was sprayed onto soil samples at different doses (Table 1) and other soil samples without myclobutanil were used as controls.Samples were maintained in laboratory conditions at 22 °C ± 2 °C and the soil moisture was 19% ± 3%.Soil moisture was adjusted every 3-4 days.

Enzymatic Activity Analyses
Dehydrogenase (EC 1.1.),urease (EC 3.5.1.5),phosphatase (EC 3.1.3.2),catalase (EC 1.11.1.6)and protease (EC 3.4) activities were evaluated in this study.For this purpose, the spectrophotometric method and a UV/Vis T90 spectrophotometer (PG Instruments, England) were used.The determination of DA followed the method described by Schinner and his coworkers [45].For the determination of UA, the method proposed by Alef and Nannipieri [46] was taken into account.PhA was obtained according to the method described by Dick [47].CA was determined using the permanganometric method described by Drăgan-Bularda [48].PA was estimated on the basis of the reaction of ninhydrin with the amino acids that resulted from the hydrolysis of the gelatin used as a substrate, which was the method proposed by Drăgan-Bularda [48].These methods have previously been applied in order to evaluate the effects on the activity of these enzymes of the herbicides aclonifen [49], S-metolachlor [50] and oxyfluorfen [51], and on the triazole fungicides triticonazole [9] and difenoconazole [52].The soil treated with myclobutanil at different doses and the control soil were incubated for a period of 28 days under laboratory conditions, and the activity of each enzyme was determined every 7 days.Each experiment was implemented in triplicate and the same researcher performed the measurements on the same day.SysthaneTM Forte was sprayed onto soil samples at different doses (Table 1) and other soil samples without myclobutanil were used as controls.Samples were maintained in laboratory conditions at 22 • C ± 2 • C and the soil moisture was 19% ± 3%.Soil moisture was adjusted every 3-4 days.

Enzymatic Activity Analyses
Dehydrogenase (EC 1.1.),urease (EC 3.5.1.5),phosphatase (EC 3.1.3.2),catalase (EC 1.11.1.6)and protease (EC 3.4) activities were evaluated in this study.For this purpose, the spectrophotometric method and a UV/Vis T90 spectrophotometer (PG Instruments, England) were used.The determination of DA followed the method described by Schinner and his coworkers [45].For the determination of UA, the method proposed by Alef and Nannipieri [46] was taken into account.PhA was obtained according to the method described by Dick [47].CA was determined using the permanganometric method described by Drăgan-Bularda [48].PA was estimated on the basis of the reaction of ninhydrin with the amino acids that resulted from the hydrolysis of the gelatin used as a substrate, which was the method proposed by Drăgan-Bularda [48].These methods have previously been applied in order to evaluate the effects on the activity of these enzymes of the herbicides aclonifen [49], S-metolachlor [50] and oxyfluorfen [51], and on the triazole fungicides triticonazole [9] and difenoconazole [52].The soil treated with myclobutanil at different doses and the control soil were incubated for a period of 28 days under laboratory conditions, and the activity of each enzyme was determined every 7 days.Each experiment was implemented in triplicate and the same researcher performed the measurements on the same day.

Molecular Docking Study
The two stereoisomers of myclobutanil were docked with three-dimensional structures of enzymes found in soil: catalase, dehydrogenase, phosphatase, protease and urease.The three-dimensional structures of these enzymes, which belong to microorganisms found in soil (fungi, bacteria and Rizhobium sp.) in complex with their specific substrates or inhibitors (when available), were extracted from the Protein Data Bank (PDB) [53] and are presented in Table 2.In the case of catalase, AlphaFold [61] structural models, namely, AF-P42234-F1 for Bacillus subtilis and AF-Q1M498-F1 for Rhizobium leguminosarum, were taken into account because these proteins had no three-dimensional structures.To locate the active site regions of the two catalases for which only structural models existed, the AlphaFold models were superposed onto the structure of the catalase belonging to the fungus Komagataella pastoris (PDB ID 6RJN).Very good matches of the superimposed structures were obtained: (i) RMSD = 0.851 Å for 410 alpha carbon pairs out of the 480 total pairs when superposing the structures of catalases belonging to Komagataella pastoris and Bacillus subtillis; and (ii) RMSD = 0.764 Å for 395 alpha carbon pairs out of 480 total pairs when superposing the structures of catalases belonging to Komagataella pastoris and Rhizobium meliloti.
A similar situation was registered for ureases.In the case of urease, AlphaFold models were identified for urease belonging to the fungus Aspergillus fumigatus (AF-P28296-F1) and to bacteria Rhizobium leguminosarum (AF-A0A0Q7ALB8-F1).Very good matches of the superimposed structures were also obtained in these cases: (i) RMSD = 0.585 Å for 544 alpha carbon pairs out of the 565 total pairs when superposing the structures of ureases belonging to Bacillus pasteurii and Aspergillus fumigatus; and (ii) RMSD = 0.550 Å for 550 alpha carbon pairs out of 567 total pairs when superposing the structures of ureases belonging to Bacillus pasterurii and Rhizobium leguminosarum.
The AlphaFold model was available for the protease belonging to Rhizobium leguminosarum, but the structure of this enzyme did not have very good matches with the structures of proteases belonging to Streptomyces griseus and Aspergillus clavatus, respectively.
The three-dimensional structures of the myclobutanil enantiomers and those of the investigated enzymes were prepared for molecular docking using of the DockPrep facility under Chimera 1.16 software [62].SwissDock server [63] was used to implement the molecular docking study.Blind and precise docking were selected.Chimera software was also used to analyze the docking results.
In order to identify the residues that are involved in the non-covalent interactions of myclobutanil enantiomers with the investigated enzymes, the Protein-Ligand Interaction Profiler (PLIP) software was used [64].Starting from the three-dimensional structure of the investigated complex, PLIP allows the detection and visualization of protein-ligand interactions at the atomic level: hydrogen bonds, hydrophobic contacts, pi-stacking and pi-cation interactions, salt bridges, water bridges and halogen bonds [64].

Statistical Analysis
The statistical analysis of data was performed using PAST 4.03 software [65].Data normality was assessed through Shapiro-Wilk W test and ANOVA analysis following a distribution test.Parametric tests were used for normally distributed data, these being Levene's test for homogeneity of variance among treatments and Tukey's post hoc test for analysis of variance.Non-parametric tests were used for non-normally distributed data.These tests were Kruskal-Wallis test and Dunn's post hoc test for further analysis of variance.

Effects of Myclobutanil on the Activity of Enzymes Found in Soil
Figures 2-6 present the activities of soil dehydrogenases, urease, phosphatase, catalase and protease over a period of 28 days for the soil samples treated with different doses of myclobutanil, as observed every 7 days after treatment.The error bars in these figures reveal some uncertainty in the measured activities, which can be explained by the variations in soil moisture during the experiment.Statistically significant differences were observed over time during the test period and, in some cases, between effects caused by different doses of myclobutanil (Tables 3-5).
interactions at the atomic level: hydrogen bonds, hydrophobic contacts, pi-stacking and pi-cation interactions, salt bridges, water bridges and halogen bonds [64].

Statistical Analysis
The statistical analysis of data was performed using PAST 4.03 software [65].Data normality was assessed through Shapiro-Wilk W test and ANOVA analysis following a distribution test.Parametric tests were used for normally distributed data, these being Levene's test for homogeneity of variance among treatments and Tukey's post hoc test for analysis of variance.Non-parametric tests were used for non-normally distributed data.These tests were Kruskal-Wallis test and Dunn's post hoc test for further analysis of variance.

Effects of Myclobutanil on the Activity of Enzymes Found in Soil
Figures 2-6 present the activities of soil dehydrogenases, urease, phosphatase, catalase and protease over a period of 28 days for the soil samples treated with different doses of myclobutanil, as observed every 7 days after treatment.The error bars in these figures reveal some uncertainty in the measured activities, which can be explained by the variations in soil moisture during the experiment.Statistically significant differences were observed over time during the test period and, in some cases, between effects caused by different doses of myclobutanil (Tables 3-5).The statistical analysis only reveals statistically significant differences for DA over time in the case of the double dose, with the DA in the soil in the 28th day being significantly different from that found on the other test days.Also, throughout the test period, the double dose showed statistically significant differences from the control and the other two doses (Table 3, above the diagonal).
For soil phosphatases, statistically significant differences were observed over time for both the control and the three doses tested, but no statistically significant differences were observed between the control and the three doses over the entire test period.For both the control and the three doses, significant differences were observed between the day-21 and day-28 activities (Table 3, below the diagonal).A sharp increase in PhA was found to occur on 28 compared to day 21.A possible explanation for this is that the soil moisture on day 21 was lower than that on day 28.After the increase in moisture, the activity of enzyme increased as it is known that phosphatase needs water to cleave the phosphoric acid monoester into phosphate ion and alcohol.Statistically significant differences were observed over time in the case of UA, both for the control and for the entire dose, with significant differences being highlighted between the activity on day 7 and 28.Between the control and applied doses, no statistically significant differences were observed throughout the entire test period (Table 4, above the diagonal).
In terms of CA, some statistically significant differences were observed over time for both control and tested doses, with activity on day 7 being significantly different from activity on day 14.A comparison of the effects produced by the different doses of myclobutanil revealed statistically significant differences between CA for the double dose, CA for the control and, respectively, the other doses on test days 21 and 28 (Table 4, below the diagonal).
Regarding PA, statistically significant differences were observed over time.At a half dose and double dose, significant differences were found between PA on day 7 and day 21.For the full dose, PA on day 7 showed significant differences from the other three test days.No statistically significant differences were observed between PA for the control and the three doses, respectively, over the entire test period (Table 5, above the diagonal).

Molecular Docking Study Regarding the Interactions of the Enantiomers of Myclobutanil with Soil Enzymes
The molecular docking results regarding the interactions of the myclobutanil enantiomers with soil enzymes are presented in Figures 7-10, in Supplementary Figures S1-S10 and in Table 6, respectively.Figures 7-9 reveal the binding modes corresponding to the highest interaction energies for the binding of myclobutanil enantiomers to the active sites of Clostridium beijerinckii dehydrogenase (Figure 7), Bacillus subtilis phosphatase (Figure 8) and Streptomyces griseus protease (Figure 9), respectively.For the dehydrogenases belonging to Aspergillus fumigatus and to Rhizobium leguminosarum, the binding modes of the myclobutanil enantiomers to the enzyme active sites are revealed in Supplementary Figures S1 and S2, respectively.In the cases of phosphatases belonging to Aspergillus niger and Rhizobium leguminosarum, the binding modes corresponding to the active sites of enzymes are shown in Supplementary Figures S3 and S4, respectively.Supplementary Figures S5 and S6, respectively, show the binding modes corresponding to the active sites of Aspergillus clavatus and Rhizobium leguminosarum proteases.
Agriculture 2023, 13, x FOR PEER REVIEW 10 of 20 significant differences were observed throughout the entire test period (Table 4, above the diagonal).
In terms of CA, some statistically significant differences were observed over time for both control and tested doses, with activity on day 7 being significantly different from activity on day 14.A comparison of the effects produced by the different doses of myclobutanil revealed statistically significant differences between CA for the double dose, CA for the control and, respectively, the other doses on test days 21 and 28 (Table 4, below the diagonal).
Regarding PA, statistically significant differences were observed over time.At a half dose and double dose, significant differences were found between PA on day 7 and day 21.For the full dose, PA on day 7 showed significant differences from the other three test days.No statistically significant differences were observed between PA for the control and the three doses, respectively, over the entire test period (Table 5, above the diagonal).

Molecular Docking Study Regarding the Interactions of the Enantiomers of Myclobutanil with Soil Enzymes
The molecular docking results regarding the interactions of the myclobutanil enantiomers with soil enzymes are presented in Figures 7-10, in Supplementary Figures S1-S10 and in Table 6, respectively.Figures 7-9 reveal the binding modes corresponding to the highest interaction energies for the binding of myclobutanil enantiomers to the active sites of Clostridium beijerinckii dehydrogenase (Figure 7), Bacillus subtilis phosphatase (Figure 8) and Streptomyces griseus protease (Figure 9), respectively.For the dehydrogenases belonging to Aspergillus fumigatus and to Rhizobium leguminosarum, the binding modes of the myclobutanil enantiomers to the enzyme active sites are revealed in Supplementary Figures S1 and S2, respectively.In the cases of phosphatases belonging to Aspergillus niger and Rhizobium leguminosarum, the binding modes corresponding to the active sites of enzymes are shown in Supplementary Figures S3 and S4, respectively.Supplementary Figures S5  and S6, respectively, show the binding modes corresponding to the active sites of Aspergillus clavatus and Rhizobium leguminosarum proteases.Table 6 contains the values of the interaction energies for the binding modes corresponding to the binding of myclobutanil enantiomers to the active sites of the enzymes resulting from molecular docking.
Figure 7 illustrates that the orientations of (R)and (S)-myclobutanil are distinct when binding to the active site of Clostridium beijerinckii dehydrogenase.The orientations of the myclobutanil enantiomers are similar when bound to the catalytic site of Aspergillus fumigatus dehydrogenase (Supplementary Figure S1), but there are distinct orientations for the two enantiomers bound to the active site of Rhizobium leguminosarum dehydrogenase (Supplementary Figure S2).There are also small differences in the interaction energies for each enantiomer with investigated dehydrogenases (Table 6).The interactions of myclobutanil enantiomers with Clostridium beijerinckii dehydrogenase are stronger than those with Aspergillus fumigatus dehydrogenase, which are also stronger than those with Rhizobium leguminosarum dehydrogenase (Supplementary Table S1).The binding of myclobutanil to the active site of all investigated dehydrogenases is in good correlation with the experimental results, revealing a significant decrease in DA in the presence of the high dose of fungicide.In addition to the distinct orientations, there are also small differences in the interaction energies for the binding modes of the two enantiomers of myclobutanil to soil enzymes (Table 6).
The interactions between the two myclobutanil enantiomers and the soil enzymes were analyzed using PLIP software and the results are shown in Table 7.This table also contains the enzymes residues involved in the interactions with the ligands that are present in the crystallographic structures, when available: NADPH-dihydro-nicotinamideadenine-dinucleotide phosphate, signaling peptide GLN-ARG-GLY-MET-ILE, inhibitory tetrapetide ACE-PRO-ALA-PRO-PHE, and NAG-2-acetamido-2-deoxy-beta-D-glucopyranose.
Table 7. Enzyme residues involved in the interactions with the enantiomers of myclobutanil obtained using Protein-Ligand Interaction Profiler (PLIP) software and compared with the enzymes residues involved in the interactions with the ligands that are present in the crystallographic structures: NADPH-dihydro-nicotinamide-adenine-dinucleotide phosphate, NAG-2-acetamido-2-deoxy-beta-D-glucopyranose.

Enzyme
Ligand/Substrate Interacting Residues
Distinct orientations and different interaction energies (Table 6) of myclobutanil enantiomers are observed for their interactions with proteases belonging to Streptomyces griseus (Figure 9), Aspergillus clavatus (Supplementary Figure S5) and Rhizobium leguminosarum (Supplementary Figure S6).
Even though the two enantiomers of myclobutanil are able to bind to the active sites of phosphatase and protease, the activities of these enzymes were not significantly affected by the presence of the fungicide.The interaction energies between myclobutanil and phosphatase and protease, respectively, were lower than those corresponding to interactions with dehydrogenase.
Enantiomers of myclobutanil with distinct orientations bound to the active site of Komagataella pastoris catalase (Supplementary Figure S7) but did not bind to the active sites of catalases from Bacillus subtilis (Figure 10a) and Rhizobium meliloti (Supplementary Figure S8).They also did not bind to any of the ureases belonging to Bacillus pasteurii (Figure 10b), Aspergillus fumigatus (Supplementary Figure S9) and Rhizobium leguminosarum (Supplementary Figure S10).This is in good correlation with experimental results indicating that UA was not significantly affected by myclobutanil and that CA registered a slight increase.
In addition to the distinct orientations, there are also small differences in the interaction energies for the binding modes of the two enantiomers of myclobutanil to soil enzymes (Table 6).
The interactions between the two myclobutanil enantiomers and the soil enzymes were analyzed using PLIP software and the results are shown in Table 7.This table also contains the enzymes residues involved in the interactions with the ligands that are present in the crystallographic structures, when available: NADPH-dihydro-nicotinamide-adeninedinucleotide phosphate, signaling peptide GLN-ARG-GLY-MET-ILE, inhibitory tetrapetide ACE-PRO-ALA-PRO-PHE, and NAG-2-acetamido-2-deoxy-beta-D-glucopyranose.
Table 7. Enzyme residues involved in the interactions with the enantiomers of myclobutanil obtained using Protein-Ligand Interaction Profiler (PLIP) software and compared with the enzymes residues involved in the interactions with the ligands that are present in the crystallographic structures: NADPH-dihydro-nicotinamide-adenine-dinucleotide phosphate, NAG-2-acetamido-2-deoxy-beta-D-glucopyranose.

Enzyme
Ligand/Substrate Interacting Residues

Experimental Approach
The results presented in this study complete our knowledge of the spectra of the effects of triazole fungicides on soil enzyme activities with information on the effects of myclobutanil.Additionally, they emphasize that high doses of myclobutanil produce decreases in the DA for a period of 28 days.Regarding the PhA, myclobutanil does not significantly affect it regardless of the dose and this is also true for UA and PA.On the contrary, an increase in CA is registered for the double dose of myclobutanil.It should be noticed that, during the period of investigations, there are changes in all the enzymes activities, both for the control and for the soil samples treated with myclobutanil.This can be due to the variations in soil moisture correlated with changes in the physicochemical parameters and bacterial community of soil and underlying the high complexity of soil systems.
Some of these results are in quite good agreement with other published data concerning the triazole fungicides, as is presented in the following.Regarding the effects of triazole fungicides on DA, in a study performed by Zhang et al., the application of a small dose of myclobutanil to tea orchard soil induced, in the first 10 days, an increase in the DA, but higher doses induced decreased DA [33].Another study revealed that the DA in sandy loam tea orchard soil was inhibited to some extent in the presence of myclobutanil for the 30 days [34].DA also registered decreased values in the presence of other triazole fungicides: triticonazole [9], difenoconazole, hexaconazole, paclobutrazole, tebuconazole and triadimefon [42].In the case of UA, a slight decrease was registered in the presence of the triazole fungicides difenoconazole, propiconazole and tebuconazole [42].Among the triazole fungicides, difenoconazole produced a slight increase in the PA [42].PhA seemed to be unaffected by the presence myclobutanil and a similar result wasobserved for triadimefon; however, several other triazole fungicides (difenoconazole, propiconazole and tebuconazole) produced a decrease in PhA [42].
Little is known about the effects of triazole fungicides on CA.The present study emphasized that the presence of myclobutanil slightly increased the CA, but other studies revealed that tebuconazole produced a decrease in CA [66,67].These dissimilar finding may be due to the different experimental approaches.This study was performed in laboratory conditions on soil without crop, while that produced by Saha and coworkers [66] was implemented in the field in peanut soil [66,67].Additionally, the study made performed Baćmaga and coworkers [67] considered the sandy clay soil cultured with spring barley [66,67].
Specific literature data reveal that triazole fungicides may also affect the activity of other soil enzymes such as chitinases [68], glucosidases, arylsulfatases and invertases [67].All these outcomes confirm the high sensitivity of soil enzymes to the presence of triazole fungicides.
One of the limitations of this study is that the physicochemical properties of soil and the bacterial community of soil were not assessed.The activities of enzymes found in soil, the soil physicochemical properties and the soil microbial communities are closely related, with soil physicochemical properties determining the composition of communities of soil microorganisms [44].These microorganisms directly influence soil enzyme activities, with some of enzymes operating intracellularly in living microbial cells and others being secreted extracellularly by the living microorganisms [69].Furthermore, soil dehydrogenase is an enzyme occurring in the viable microbial cells and its decrease in the presence of myclobutanil suggests a diminution of soil microbial population.Consequently, the effects of myclobutanil on the investigated enzymes indirectly emphasizes that there are interactions between myclobutanil, the bacterial community, and the physical and chemical characteristics of the soil and that myclobutanil may lead to modified soil physicochemical properties and affect the populations of the microorganisms living in soil, the population of fungi as target organisms and the population of yeasts and bacteria as non-target organisms.The decrease in DA suggests the reduction in microbial biomass in the presence of myclobutanil, but the slight increase in CA suggests that some bacteria may use the fungicide as a source of nutrients.This underlines that myclobutanil may also alter the structure of microorganism communities in soil.

Computational Approach
The outcomes of the molecular docking complete the information derived from the experimental approach.The highest binding energies were recorded for the two enantiomers of myclobutanil to dehydrogenases, which is in good correlation with the significant registered decrease in DA recorded for the soil treated with the double dose of myclobutanil.Even though both enantiomers of myclobutanil were able to bind to the active sites of phosphatases and proteases, experimental data showed that the activities of these enzymes were not significantly affected by the presence of myclobutanil.Phosphatases and proteases were enzymes secreted into the soil by some microorganisms, while dehydrogenases are intracellular enzymes.Moreover, the energies were higher for the interactions of myclobutanil with dehydrogenases compared to the energies corresponding to the interactions with phosphatases and proteases.
Usually, the orientations of the enantiomers in the interactions with the active sites of the enzymes are different.There are also differences between the binding energies for the two enantiomers and this emphasizes their enantioselectivity.(S)-myclobutanil, the enantiomer that revealed lower antifungal activity and higher toxicity against non-target organisms, presents relatively higher interaction energies with these enzymes in many cases, suggesting its higher inhibitory potential.
The enantiomers of myclobutanil could only bind to the active site of catalase from Komagataella pastoris.However, this was not the case for the other investigated catalases.The interaction energies for these bonds were lower compared to those recorded for dehydrogenases.The two enantiomers did not bind to the active sites of the other investigated catalases.This was potentially due to the structures of these enzymes, which revealed long and narrow channels before reaching the active site to prevent the binding of numerous chemicals [70].This was in good agreement with the experimental result that revealed myclobutanil did not lead to CA decrease and with published data revealing catalase activity to be considered relatively stable in soil [71].
Myclobutanil enantiomers did not bind to urease.This was is agreement with the experimental results, which showed that myclobutanil produced no changes in UA.A similar result was observed for the triticonazole enantiomers [9] and for aclonifen and its metabolites [49] that could not bind to the active site of this enzyme.This was explained by the obtuseness, rigidity and hydrophilicity of the catalytic site of urease [60].
The limitation of this computational study is that the dose of myclobutanil cannot be taken into account.The advantage of using a molecular docking study is reflected by the fact that it allows researchers to take into account the racemic structure of the commercial product.

Conclusions
The outcomes of the present study reveal that the triazole fungicide myclobutanil negatively affected the activity of soil dehydrogenase when used in high doses.Furthermore, both enantiomers of myclobutanil were able to bind to the active sites of dehydrogenase, phosphatase and protease, the interacting energies being to some extent higher for the (S)myclobutanil, the enantiomer that is known to be less active against target organisms and to manifest higher toxicity against non-target organisms.This underlines the enantioselective effects of the triazole fungicide myclobutanil on the activities of enzymes found in soil.It also emphasizes that the racemic mixtures that are used in crop and food management practices should be replaced by the enantiomer that reveals the highest activity against target organisms and the lowest toxicity against non-target organisms.

Figure 2 .
Figure 2. Soil dehydrogenases activity (DA) for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).

Figure 2 .
Figure 2. Soil dehydrogenases activity (DA) for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).Agriculture 2023, 13, x FOR PEER REVIEW 7 of 20

Figure 3 .
Figure 3. Soil phosphatase activity (PhA) for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).

Figure 4 .
Figure 4. Soil urease activity (UA) for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).

Figure 3 .
Figure 3. Soil phosphatase activity (PhA) for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).

Figure 3 .
Figure 3. Soil phosphatase activity (PhA) for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).

Figure 4 .
Figure 4. Soil urease activity (UA) for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).

Figure 4 .
Figure 4. Soil urease activity (UA) for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).

Figure 4 .
Soil urease activity (UA) for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).

Figure 5 .
Figure 5. Soil catalase activity (CA) for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).

Figure 5 . 20 Figure 6 .
Figure 5. Soil catalase activity (CA) for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).Agriculture 2023, 13, x FOR PEER REVIEW 8 of 20

Figure 6 .
Figure 6.Soil protease (PA) activity for every experimental variant and determined every 7 days for a period of 28 days: C-control soil, D-normal dose of myclobutanil (0.1 L/ha).

Figure 7 .
Figure 7. Binding of (R)-myclobutanil (red sticks) and of (S)-myclobutanil (green sticks) to the active site of Clostridium beijerinckii dehydrogenase (solid hydrophobicity surface, blue regions are hydrophilic and orange regions are hydrophobic).The substrate dihydro-nicotinamide-adenine-dinucleotide phosphate (yellow sticks) illustrates the position of the active site.

Figure 7 .
Figure 7. Binding of (R)-myclobutanil (red sticks) and of (S)-myclobutanil (green sticks) to the active site of Clostridium beijerinckii dehydrogenase (solid hydrophobicity surface, blue regions are hydrophilic and orange regions are hydrophobic).The substrate dihydro-nicotinamide-adeninedinucleotide phosphate (yellow sticks) illustrates the position of the active site.

Figure 8 .
Figure 8. Binding of (R)-myclobutanil (red sticks) and of (S)-myclobutanil (green sticks) to the active site of Bacillus subtilis phosphatase (mesh hydrophobicity surface, blue regions are hydrophilic and orange regions are hydrophobic).The position of the active site is illustrated by the signaling inhibitory peptide (yellow sticks).

Figure 9 .
Figure 9. Binding of (R)-myclobutanil (red sticks) and of (S)-myclobutanil (green sticks) to the active site of Streptomyces griseus protease (solid hydrophobicity surface, blue regions are hydrophilic and orange regions are hydrophobic).The position of the active site is illustrated by the inhibitor tetrapeptide ACE-PRO-ALA-PRO-PHE (yellow sticks).

Figure 8 . 20 Figure 8 .
Figure 8. Binding of (R)-myclobutanil (red sticks) and of (S)-myclobutanil (green sticks) to the active site of Bacillus subtilis phosphatase (mesh hydrophobicity surface, blue regions are hydrophilic and orange regions are hydrophobic).The position of the active site is illustrated by the signaling inhibitory peptide (yellow sticks).

Figure 9 .
Figure 9. Binding of (R)-myclobutanil (red sticks) and of (S)-myclobutanil (green sticks) to the active site of Streptomyces griseus protease (solid hydrophobicity surface, blue regions are hydrophilic and orange regions are hydrophobic).The position of the active site is illustrated by the inhibitor tetrapeptide ACE-PRO-ALA-PRO-PHE (yellow sticks).

Figure 9 .
Figure 9. Binding of (R)-myclobutanil (red sticks) and of (S)-myclobutanil (green sticks) to the active site of Streptomyces griseus protease (solid hydrophobicity surface, blue regions are hydrophilic and orange regions are hydrophobic).The position of the active site is illustrated by the inhibitor tetrapeptide ACE-PRO-ALA-PRO-PHE (yellow sticks).

Figure 10 .
Figure 10.Binding of (R)-myclobutanil (red sticks) and of (S)-myclobutanil (green sticks) to the: (a) Bacillus subtilis catalase (mesh hydrophobicity surface, blue regions are hydrophilic and orange regions are hydrophobic), the position of the active site being illustrated by the cofactor hem (yellow sticks); (b) Bacillus pasteurii urease (mesh hydrophobicity surface, blue regions are hydrophilic and orange regions are hydrophobic), the position of the active site being illustrated by the ligand citrate ion (yellow sticks).

Figure 10 .
Figure 10.Binding of (R)-myclobutanil (red sticks) and of (S)-myclobutanil (green sticks) to the: (a) Bacillus subtilis catalase (mesh hydrophobicity surface, blue regions are hydrophilic and orange regions are hydrophobic), the position of the active site being illustrated by the cofactor hem (yellow sticks); (b) Bacillus pasteurii urease (mesh hydrophobicity surface, blue regions are hydrophilic and orange regions are hydrophobic), the position of the active site being illustrated by the ligand citrate ion (yellow sticks).

Table 1 .
Experimental variants considered in this study: D-normal dose of myclobutanil.

Table 1 .
Experimental variants considered in this study: D-normal dose of myclobutanil.

Table 2 .
Crystallographic structures of enzyme found in soil and considered in the present study: PDB ID-identifier of the structural file in Protein Data Bank, AF-AlphaFold structural model.

Table 3 .
Results of Dunn's post hoc test for enzymatic activities of soil dehydrogenases (above diagonal) and of enzymatic activities of soil phosphatases (below diagonal).Statistically significant data (p < 0.05) are written in bold.

Table 4 .
Results of Tukey's post hoc test for enzymatic activities of soil urease (above diagonal) and of enzymatic activities of soil catalases (below diagonal).Statistically significant data (p < 0.05) are written in bold.

Table 4 .
Results of Tukey's post hoc test for enzymatic activities of soil urease (above diagonal) and of enzymatic activities of soil catalases (below diagonal).Statistically significant data (p < 0.05) are written in bold.

Table 5 .
Results of Tukey's post hoc test for enzymatic activities of soil protease (above diagonal).Statistically significant data (p < 0.05) are written in bold.

Table 6 .
Interaction energies for the best binding modes of the myclobutanil enantiomers to soil enzymes.

Table 6 .
Interaction energies for the best binding modes of the myclobutanil enantiomers to soil enzymes.

Table 6 .
Interaction energies for the best binding modes of the myclobutanil enantiomers to soil enzymes.