Antifungal triazoles affect key non-target metabolic pathways in Solanum lycopersicum L. plants.

Several 1,2,4-triazoles are widely used as systemic fungicides in agriculture because they inhibit fungal 14ɑ-demethylase. However, they can also act on many non-target plant enzymes, thereby affecting phytohormonal balance, free amino acid content, and adaptation to stress. In this study, tomato plants (Solanum lycopersicum L. var. 'Cherrola') were exposed to penconazole, tebuconazole, or their combination, either by foliar spraying or soil drenching, every week, as an ecotoxicological model. All triazole-exposed plants showed a higher content (1.7-8.8 ×) of total free amino acids than the control, especially free glutamine and asparagine were increased most likely in relation to the increase in active cytokinin metabolites 15 days after the first application. Conversely, the Trp content decreased in comparison with control (0.2-0.7 ×), suggesting depletion by auxin biosynthesis. Both triazole application methods slightly affected the antioxidant system (antioxidant enzyme activity, antioxidant capacity, and phenolic content) in tomato leaves. These results indicated that the tomato plants adapted to triazoles over time. Therefore, increasing the abscisic and chlorogenic acid content in triazole-exposed plants may promote resistance to abiotic stress.

Triazoles as a Potential Threat to the Nutritional Quality of Tomato Fruits.

Triazole fungicides can threaten plants as abiotic stressors but can also positively affect plant defense by inducing priming. Thus, plant yield is also both protected and endangered by triazoles that may influence several metabolic pathways during maturation processes, such as the biosynthesis of saccharides or secondary metabolites. Here, Solanum lycopersicum L. plants were exposed to foliar and soil applications of penconazole, tebuconazole, or their combination, and their resulting effect on tomato fruits was followed. The exposure to the equimolar mixture of both triazoles influenced the representation of free proteinogenic amino acids, especially Gln, Glu, Gly, Ile, Lys, Ser and Pro, saccharide content, and led to a significant increase in the contents of total phenolics and flavonoids as well as positive stimulation of the non-enzymatic antioxidant system. Among the identified secondary metabolites, the most abundant was naringenin, followed by chlorogenic acid in tomato peel. In turn, all triazole-treated groups showed a significantly lower content of rosmarinic acid in comparison with the control. Foliar application of penconazole affected the fruit more than other single triazole applications, showing a significant decrease in antioxidant capacity, the total content of secondary metabolites, and the activities of total membrane-bound peroxidases and ascorbate peroxidase.

Non-target biotransformation enzymes as a target for triazole-zinc mixtures.

Triazoles inhibit lanosterol 14α-demethylase and block ergosterol biosynthesis in fungal pathogens. However, they also interact with other cytochrome P450 enzymes and influence non-target metabolic pathways. Disturbingly, triazoles may interact with essential elements. The interaction of penconazole (Pen), cyproconazole (Cyp) and tebuconazole (Teb) with Zn2+ results in the formation of deprotonated ligands in their complexes or in the creation of complexes with Cl- as a counterion or doubly charged complexes. Triazoles, as well as their equimolar cocktails with Zn2+ (10-6 mol/L), decreased the activities of the non-target enzymes CYP19A1 and CYP3A4. Pen most decreased CYP19A1 activity and was best bound to its active centre to block the catalytic cycle in computational analysis. For CYP3A4, Teb was found to be the most effective inhibitor by both, activity assay and interaction with the active centre. Teb/Cyp/Zn2+ and Teb/Pen/Cyp/Zn2+ cocktails also decreased the CYP19A1 activity, which was in correlation with the formation of numerous triazole-Zn2+ complexes.

Micellar electrokinetic chromatography in the determination of triazoles in fruit peel.

Triazole fungicides (TAFs) are frequently used fungicides for various antifungal treatments of crops. Tre treatment is provided foliarly. However, some significant amount of TAFs may remain on/in fruits. We have developed a methodology for the determination of penconazole, tebuconazole and cyproconazole in tomato fruit peel. The extraction of TAFs was provided with chloroform (acidified with 0.1% acetic acid). In the electrokinetic chromatography, the mixed micellar pseudo-stationary phase was composed of anionic detergent sodium dodecyl sulphate (15 mM) and randomly highly sulphated gamma-cyclodextrin (17.5 mg/mL). The background electrolyte consisted of 100 mM phosphoric acid and 100 mM Tris in the mixed hydro-organic solvent water/methanol (80/20 v/v), apparent pH 4.8. Complete separation of penconazole, tebuconazole, and two diastereomers of cyproconazole with resolutions higher than 5.1 were achieved within a relatively short time of less than 17 min in the bare fused silica capillary of 425/500 mm total/effective lengths and 50/375 µm I.D./O.D. at separation voltage -15 kV (cathode at injection capillary end) and at constant capillary cassette temperature of 22°C. The TAFs were detected by a UV-spectrophotometric diode array detector set at 200 nm. The limits of detection and limits of quantification were in the range of 71-92 and 214-278 µg/kg of peel, respectively. Analyses of the peel extracts revealed that even 10 days after the last treatment, TAF concentrations were higher than the recommended maximum residue limits in both application ways, as individual as well as in the TAF binary or ternary mixtures.

Side effects of triazoles on treated crops.

Triazolic fungicides are widely applied in crop production to protect plants against fungal pathogens. However, they may influence the biochemical processes in plants and other non-target species. This paper is aimed at the effect of triazoles (namely tebuconazole, cyproconazole, and penconazole) single/mixed applications on the phenolics production in tomato (Solanum lycopersicum L.) fruit peel, amount of chlorophyll a and b in tomato leaves as well as on basic plant growth parameters. For this purpose, cherry tomatoes were planted in the pot experiment and foliarly-treated weekly, with the same total triazoles dose of 3.52 µmol per plant (in mixtures of 1.71 or 1.17 µmol of each in two or three components, respectively). The treatments increased the weight of fruits in the 1st harvest about 43%, however, this effect was not observed in the next harvest. Increased oxidative stress in the triazoles presence was observed, based on the elevated production of antioxidant phenolics in the 1st harvest. Most alarming is the decrease of the weight of thin stems and foliage and the concentration of chlorophyll a (b) in leaves in all triazoles-treated variants. The non-target impacts on plant biochemical processes (related to the phenolics or chlorophylls production and functionality) were confirmed.

Triazole fungicides in soil affect the yield of fruit, green biomass, and phenolics production of Solanum lycopersicum L.

A part of the fungicides used in foliar treatment penetrates into the soil. This study describes changes in the bioavailability of (essential) elements in soil, fructification, the amount of green biomass and the production of phenolic compounds related solely to the presence of triazoles (penconazole and cyproconazole) in soil, injected as a single compound or their mixture. The triazoles presence has substantially affected the bioavailability of Fe, Cu and Zn in soil. The amount of green biomass has significantly decreased, whereas the chlorophylls a and b have not been affected. As a potential mark of plant stress, the fruits of the treated variants are significantly bigger. The content of phenolics in tomato peel (e.g. quercetin, quercitrin, hesperidin, naringin, and chlorogenic, salicylic and p-coumaric acid) has been quantified. The biggest changes (increase/decrease) have been observed in the contents of p-coumaric and chlorogenic acid, quercetin and quercitrin.

Triazoles and aromatase: The impact of copper cocktails.

Triazoles are used as antifungal agents, they mostly inhibit two enzymes: 14α-demethylase and aromatase. These enzymes are utilised also in other species and therefore the affection in non-target species in the environment is expected as well. Besides, triazoles are often being applied in a mixture and they can also interact with other substances present. This study clarifies how three selected representative triazoles (tebuconazole, penconazole and cyproconazole) interact with each other (group effect) and in mixtures (cocktail effect) with copper, essential/toxic for all organisms. Within the experiments on electrospray and collision-induced dissociations (both ESI-MS), it has been found that the fragments correspond to typical triazole metabolites. For their formation, the presence of copper ions is crucial. The inhibitory effect of Cu cocktails on aromatase enzymatic activity has been studied. The presence of Cu ions together with triazole(s) significantly increases the inhibitory effect on aromatase activity. The highest inhibitory effect (more than 60%) on aromatase activity is produced by cocktails containing penconazole and Cu ions, namely by penconazole/Cu and penconazole/tebuconazole/Cu. The reactivity of triazoles in groups is not significantly affected by the interactions among them. Additionally, the role of triazoles in copper Fenton reaction regulation has been observed and described. These changes may be attributed to the formation and stabilization of the complexes with the central Cu ion, with usually one, two or three triazolic ligands, depending on the mixture. The study demonstrates that the interaction of triazoles and Cu ions is a complex process; their impact on metabolism seems to be rather extensive and must be evaluated in the context of biochemical reactions.

Complexation and stability of the fungicide penconazole in the presence of zinc and copper ions.

RATIONALE: For the risk assessment of penconazole in the environment and the evaluation of the possible consequences of its use, it is important to determine how its reactivity and degradation are influenced by metals commonly found in nature, such as copper and zinc. METHODS: Changes in the reactivity of penconazole in the presence of zinc/copper ions were studied using electrospray ionisation mass spectrometry and density functional theory calculations. RESULTS: Many penconazole complexes with copper and zinc ions were created; a comparison of the elements showed that a few complexes were formed analogously (doubly charged complexes with four penconazole molecules, singly charged complexes with chlorine as a counterion and singly charged complexes with deprotonated penconazole as a counterion). The metal complexes with different structures indicated different reactivity of penconazole with copper and zinc. CONCLUSIONS: The experimental and computational approaches have revealed different changes in the structure of penconazole. In the Zn(II) complex, penconazole deprotonated to stabilise the bond to Zn(II). In the Cu(II) complex, it loses one chlorine atom, creates an additional ring between the triazole ring and the phenyl ring, and/or creates a double bond in the short aliphatic chain.

Does resveratrol retain its antioxidative properties in wine? Redox behaviour of resveratrol in the presence of Cu(II) and tebuconazole.

Resveratrol is antioxidant naturally occurring in wine grapes. It is thought to have a preventive biological activity against number of diseases. However, it has been recently shown that in the presence of metal ions, such as Cu2+, resveratrol forms oxidative radicals. Cu2+ is usually present in wine due to former usage of bluestone in vineyards. Fungicide tebuconazole has substituted bluestone and is presently one of the most widely used agrochemicals in wine industry; wine thus may contain traces of tebuconazole. Here, we study the ternary system of resveratrol, Cu2+, and tebuconazole experimentally and theoretically (using mass spectrometry, antioxidant capacity assay and quantum-chemical calculations) to model the redox behaviour of resveratrol in wine. We show that tebuconazole prevents formation of oxidative resveratrol radicals (induced by Cu2+ reaction with resveratrol) via preferential Cu2+ capture and protection of the binding sites of resveratrol. This positive effect of tebuconazole has not been observed before.

Mimicking of cyproconazole behavior in the presence of Cu and Zn.

RATIONALE: The frequently used pesticide cyproconazole (Cyp) interacts with the essential elements commonly present in soil which play important roles in various enzymatic processes. These interactions predetermine the Cyp degradation pathways. We suggest a simple experimental and theoretical approach for the prediction of pesticide behavior. METHODS: Cu/Cyp complexes are explored because of the typical Cu(II) reduction in complexes. Its level and the stability of the Cu-ligand bond depend on the type and the number of the surrounding ligands. Zn/Cyp complexes were compared as it is not expected that Zn(II) will reduce. The complexations were studied by means of electrospray ionization ion trap mass spectrometry and MS/MS collision-induced dissociations with comparative and explicative density functional theory calculations. RESULTS: The Cyp ligand allows both Cu(II) reduction as well as, in specific cases, it protects the higher Cu oxidation state. The reduction is observed in the complexes with solely neutral Cyp where the number of ligands is below 3; a higher number protects the Cu(II) state. The metal atom binds to Cyp via N2 of the triazole ring as well as via π-electrons of the benzene ring; additional stabilization brings an interaction with the deprotonated OH group. CONCLUSIONS: The character of Cyp interactions with doubly charged metals (Cu(II), Zn(II)) clarified the creation of Cyp metabolites. The phenyl and triazole rings are bound to the metal cation and enable access for the isopropyl ring to be cleaved leaving the common metabolite (CAS Number: 58905-19-4).

Biomass of speckled alder on an air-polluted mountain site and its response to fertilization.

The article summarizes outcomes of a biomass study conducted in a young speckled alder plantation on a cold mountain site. At this location, the previously existing old forest was clear felled because of damage from air pollution, and present-day surface humus is in need of restoration. The intention of this study was to quantify the biomass and nutrients accumulated by alders and their components and assess whether the initial fertilization resulted in increased biomass production and nutrient accumulation in the biomass. Besides the control, two fertilized treatments were installed. In the surface treatment (SUT), the amendment was applied as a base dressing in small circles around trees. In the planting-hole treatment (PHT), the amendment was incorporated into soil inside the planting holes. Five growth seasons after planting and fertilization, six alders from each treatment were harvested including roots. Their biomass was quantified and analyzed for macroelements. The greatest pool of dry mass (DM) was branches in the control and stem wood in the fertilized treatments. The greatest pools of macroelements were leaves and branches. The most pronounced effects of fertilization were recorded in the DM and consequently in the absolute quantities of nutrients. The DM of an average tree in the control, SUT, and PHT was 85, 226, and 231 g, respectively. The absolute contents of nutrients per tree in the fertilized treatments showed the following increases, as compared with the control: (N) 2.5-2.6 times; (P) 1.6-2.4 times; (K) 1.8-2.1 times; and (Mg) 1.8-2.0 times, respectively. Speckled alder responded positively to fertilization.

Complexation of malic acid with cadmium(II) probed by electrospray ionization mass spectrometry.

Electrospray ionization was used as a technique for the characterization of the interactions between cadmium(II) ions and malic acid (1) in aqueous solution. Particular attention was paid to the nature of the species formed, which generally correspond to complexes of CdX(+) cations with neutral malic acid, where X either is the counterion of the metal salt used as a precursor (i.e. X=Cl, I) or corresponds to singly deprotonated malic acid. In pure water solutions, also highly coordinated complexes [Cd(1-H)(1)(2)](+) and [CdCl(1)(2)](+) were detected, whereas the most abundant complexes detected in a sample of soil solution were: [Cd(1-H)(1)](+) and [CdCl(1)](+). With respect to possible application in environmental analysis, the effects of (i) metal salts present in solution, (ii) modest mineralization, and (iii) the matrices of real soil solutions were probed. While the presence of other metals leads to additional complexes, the characteristic species containing both cadmium(II) and malic acid can still be detected with good sensitivity.