In this study, we used the fungicide formulation Vibrance^® 500 FS, a commercial flowable concentrate for seed treatment containing 500 g sedaxane^® L^-1, i.e., 43.7% w/w of AI (density 1.17 g mL^-1; pH 6.39). Sedaxane is the ISO common name for a mixture of two cis-isomers, 2′-[(1RS,2RS)-1,1′-bicycloprop-2-yl]-3-(difluoromethyl)-1-methylpyrazole-4-carboxanilideand two trans-isomers 2′-[(1RS,2SR)-1,1′-bicycloprop-2-yl]-3-(difluoromethyl)-1-methylpyrazole-4-carboxanilide (IUPAC). Its minimum purity is 960 g kg^-1, with ranges of 820–890 g kg^-1 for the 2 trans-isomers (SYN508210 – 50:50 mixture of enantiomers), and 100–150 g kg^-1 for the 2 cis-isomers (SYN508211 – 50:50 mixture of enantiomers) (EFSA, 2012).

Plants of the maize hybrid Hydro (Syngenta, Basel, Switzerland) were grown in cylindrical PVC pots (50 cm high, 9 cm diameter, 3.1 L volume) in a greenhouse in the L. Toniolo experimental farm of the University of Padua (Legnaro, NE Italy). The pots were filled with a sterilized mixture (36 h in an oven at 120°C) of silty-loam soil collected from a field on the experimental farm (pH 8.4), and fine sand (1:1 w/w) to facilitate water drainage and root collection, to which was added a standard dose of pre-sowing fertilizer (about 100 kg N ha^-1, 150 kg P₂O₅ ha^-1 and 300 kg K₂O ha^-1). Maize seeds were treated with three increasing doses of sedaxane: 25, 75, and 150 μg AI seed^-1, corresponding to label doses of 2.5, 7.5, and 15 mL of the commercial product Vibrance^® 500 FS (500 g AI L^-1) in 50,000 seeds. Plants grown from treated seeds were compared with untreated controls. The experimental design was completely randomized with 6 replicates.

Three seeds per pot were sown at the end of June, and immediately after emergence plants were thinned to one per pot. At harvest, growth measurements were taken from three pots/plants, and enzymatic activity assays were carried out with a further three.

Water stress was avoided throughout the experiment by regularly watering the plants. Before plant harvest, which took place 20 days after sowing (DAS) at the 3-leaf stage, SPAD (Soil Plant Analysis Development) was measured in the last fully developed leaf with a 502 chlorophyll meter (Konica-Minolta, Hong Kong). Fresh and dry (oven-dried for 24 h at 105°C) weights were measured on three replicate samples of shoots, and roots were collected, gently washed of soil, and stored in a 15% v/v ethanol solution until morphological characterization. Root length, surface area, diameter, and number of tips and forks were measured by analysis of 1-bit 400-DPI images of the roots acquired with a flatbed scanner (Epson Expression 11000XL, Epson, Suwa, Japan) using the WinRhizo software (Regent Instruments, Ville de Québec, QC, Canada).

Three replicates were stored at -80°C until analysis, then shoot and root tissue samples were taken from them for enzymatic activity assays. Each enzymatic assay (n = 9) was carried out in triplicate on each plant.

A further trial was performed following the same procedure and timing of the main experiment, and using the same sand-soil mixture (1:1 w/w), but this time it was not sterilized. We took SPAD readings, and measured fresh and dry weights, and root morphological parameters of plants grown in unsterilized soil, as reported above (Supplementary Table S1).

In order to investigate the biological activity of sedaxane, we measured the reduction in root growth in the model plant watercress (Lepidium sativum L.) to assess auxin-like activity, and the increase in shoot length in lettuce (Lactuca sativa L.) to assess gibberellin-like activity (Audus, 1972).

Watercress and lettuce seeds were surface-sterilized by immersion in 8% hydrogen peroxide for 15 min. After rinsing 5 times with sterile distilled water, 20 seeds were aseptically placed on filter paper in a Petri dish. In the case of watercress, the filter paper was moistened with 1.2 mL of H₂O (controls), or with 1.2 mL of 0.1, 1, 10 and 20 mg L^-1 indoleacetic acid (IAA, natural auxin) (Sigma–Aldrich, St. Louis, MO, United States) to obtain the calibration curve, or with 1.2 mL of a serial dilution of the tested product Vibrance containing 500 g L^-1 of AI sedaxane. The experimental design for lettuce was the same as for watercress, except that the sterile filter paper was moistened with 1.4 mL of the above solutions, while the calibration curve was a progression of 0.0001, 0.001, 0.01, and 0.1 mg L^-1 gibberellic acid (GA) (Sigma–Aldrich, St. Louis, MO, United States).

Seeds were germinated in the dark at 25°C. After 48 h, the watercress seedlings were removed from the dishes and the roots measured with a digital gauge; after 72 h, the lettuce seedlings were removed and the shoots measured with a digital gauge.

A linear regression model (y = a + bx) was used to describe the dose-response relationship after logarithmic transformation of IAA, GA and the sedaxane doses, where x was the sedaxane concentration (g L^-1) and y the root or shoot length (mm) (Conselvan et al., 2017).

Fresh leaf and root samples, previously stored at -80°C, were ground to a homogenous powder with liquid N₂. Proteins were extracted by homogenizing 0.5 g of root or shoot materials with 5 mL of 38 mM KH₂PO₄ and 62 mM K₂HPO₄, pH 7, at 4°C. After 2 min, the extract was filtered through three layers of muslin and centrifuged at 15,000 g for 20 min at 4°C. A 50-μL supernatant sample was incubated with 50 μL of Milli-Q water and 2.5 mL of 0.00117 M Bradford reagent. After 15 min, the protein concentration in the extract was determined according to Bradford (1976), using a Jasco V-530 UV/Vis spectrophotometer (Jasco Corporation, Tokyo, Japan) at 595 nm wavelength. The protein concentration was expressed as mg of protein per g of fresh root or shoot.

To extract the enzymes involved in N reduction and assimilation pathways, fresh shoot and root samples were ground to a homogeneous powder with liquid N₂. Each activity assay was carried out in triplicate and with 3 biological repetitions using specific buffers for enzyme extraction.

Glutamine synthetase (GS; EC 6.3.1.2) was extracted by homogenizing 0.6 g of root or shoot material at 4°C with 2.4 mL of a solution of 1 mM Tris(hydroxymethyl)aminomethane HCl (Tris-HCl), 25 mM KH₂PO₄, 10 mM L-cysteine hydrochloride monohydrate and 3% (w/v) bovine serum albumin at pH 7.8 (Baglieri et al., 2014). After 10 min, the extract was filtered through two layers of gauze and centrifuged at 15,000 g for 25 min at 4°C. A 200-μL sample of supernatant was incubated with 200 μL of reaction buffer (50 mM Tris-HCl, 20 mM MgSO₄, 80 mM L-glutamate, 30 mM NH₂OH, 24 mM ATP, pH 7.8) at 37°C for 25 min. Reaction was blocked with a stopping solution (0.5 mL of 370 mM FeCl₂⋅6H₂O and 670 mM HCl). Samples were centrifuged at 15,000 g for 15 min. The amount of γ-glutamyl hydroxamate in the supernatant was determined photometrically (wavelength 540 nm) against an immediately stopped parallel sample (Jezek et al., 2015). A standard curve was made using authentic γ-glutamyl hydroxamate (GHA) proportional to absorbance intensity. Enzyme activity was expressed as μmol of GHA produced per g of fresh root or leaf tissue per minute (Conselvan et al., 2017).

Glutamate synthase (GOGAT; EC 1.4.7.1) was extracted by homogenizing 0.5 g of root or shoot material with 2 mL of a solution of 100 mM Tris-HCl, pH 8.2, 10 mM MgCl₂⋅6H₂O, 2 mM β-mercaptoethanol, 10% (v/v) glycerol and 1 mM Na₂EDTA. After 15 min, the extract was filtered through two layers of gauze and centrifuged at 15,000 g for 30 min at 4°C. The supernatant was centrifuged a second time at 15,000 g for 15 min at 4°C. For the enzyme assay, 100 μL of extract was added to 900 μL of reaction buffer (41.6 mM HEPES, pH 7.5, 1 mM NADH, 10 mM EDTA, 20 mM glutamine) and 300 μL (for leaf extract) or 900 μL (for root extract) of 10 mM α-ketoglutaric acid. The reaction time was 2 min for the shoot extract, and 1.5 min for the root extract at 30°C. GOGAT was assayed spectrophotometrically by monitoring NADH oxidation at wavelength 340 nm according to Avila et al. (1987). GOGAT activity was expressed as nmol NADH reduced per g of fresh root or shoot per minute.

For the phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) assay, 1 g of shoot material was homogenized with 0.1 g of poly(vinylpolypyrrolidone) (PVPP) and 5 mL of 100 mM potassium phosphate buffer (pH 8.0) containing 1.4 mM β-mercaptoethanol. After 10 min, the extract was filtered through two layers of gauze and centrifuged at 15,000 g for 20 min at 4 °C. A 60 μL sample of supernatant was incubated with 400 μL of 100 mM Tris-HCl buffer (pH 8.8), 140 μL of 100 mM phosphate buffer and 200 μL of 40 mM phenylalanine, at 37°C for 30 min. Reaction was stopped with 200 μL 6N HCl (El-Shora, 2002). After centrifuging at 10,000 g for 15 min, the absorbance of the supernatant was measured at 280 nm against an immediately stopped parallel sample. A standard curve was made using authentic cinnamic acid at increasing dilutions. PAL activity was expressed as nmol cinnamic acid produced per mg of protein in the sample per minute.

Soluble phenolic acids were extracted by homogenizing 200 mg of leaf material with 600 mL of pure methanol. The extract was kept on ice for 30 min then centrifuged at 15,000 g for 30 min at 4°C. Total phenols were measured according to the procedure described by Arnaldos et al. (2001). In brief, 1 mL of 2% Na₂CO₃ and 75 μL of Folin-Ciocalteau reagent (Sigma–Aldrich, St. Louis, MO, United States) were added to 50 μL of the phenolic extract. After incubation in the dark for 15 min at 25°C, absorbance was measured at 725 nm. A standard curve was made using authentic gallic acid. The soluble phenol content was expressed as mg of gallic acid equivalent (GAE) per g of fresh shoot material.

Free phenolic acid concentrations were revealed on 0.1 g shoot samples treated with 5 mL 80% (v/v) acetonitrile (ACN) in 10-mL tubes for 5 min at room temperature with agitation (70 rpm). After centrifugation (5 min, 10,000 RCF), clear supernatant was filtered at 0.2 μm (Acrodisc syringe filters with GHP membranes) and kept in clean tubes at -20°C until processing. HPLC analysis was carried out according to the method described by Adom et al. (2003) with modifications. Samples were manually shaken, then 200 μL was extracted and placed in vials for HPLC autosampling. The mobile phase was 0.25% (v/v) trifluoroacetic acid (TFA, solvent A) and pure ACN (solvent B). The HPLC gradient was linear: after 2 μL sample injection, solvent B was kept at 4% for 1.16 min, then increased gradually to 12% over 1.16 min, to 23% over 4.63 min, to 95% over 1.85 min, and to 100% over 1.16 min, with final rate maintained for a further 2.78 min. The duration of the analysis was 11.58 min at a solvent flow rate of 1.1 mL min^-1. The HPLC equipment (Shimadzu, Kyoto, Japan) had a UV diode array detector (SPD-M20A) at wavelength 282 nm, and an Ultra Tech sphere C18 analytical column (33 mm × 4.6 mm i.d., 1.5 μm particle size; Cil Cluzeau, Sainte-Foy-La-Grande, France) kept at 36°C. Control sample solutions of shoots containing known phenolic acid concentrations were analyzed at the beginning of each new batch analysis, and measurement accuracy was verified by checking expected concentrations.

Each peak was identified by analyzing the retention time and absorbance spectrum of each pure compound (i.e., p-Coumaric, caffeic, syringic, vanillic and t-ferulic acids; Supplementary Figure S1). The coefficients of determination of all calibration curves were >99%.

Shoots (5 g) were homogenized in methanol (20 mL) with an Ultra Turrax T25 at 13,500 rpm for 30 s until they attained uniform consistency. Samples were filtered once through filter paper (589 Schleicher), and a second time through cellulose acetate syringe filters (0.45 μm). The extract was then ready for HPLC analysis, for which we used a Jasco X-LC liquid chromatography system (Jasco Inc., Easton, MD, United States) consisting of a PU-2080 pump, an MD-2015 multiwavelength detector, an AS-2055 autosampler, and a CO-2060 column oven interfaced to a PC using the ChromNAV chromatography data system software (Jasco Inc., Easton, MD, United States).

Sugars were separated in a HyperRez XP Carbohydrate Pbþþ analytical column (7.7 mm × 300 mm; ThermoFisher Scientific, Waltham, MA, United States), operating at 80°C. Isocratic elution was carried out with water at a flow rate of 0.6 mL min^-1. D-(β)-glucose and D-(β)- fructose were quantified by a calibration method. Standards were dissolved in water and the calibration curves were generated with concentrations ranging from 100 mg L^-1 to 1,000 mg L^-1 (Nicoletto et al., 2013).

The data are the means of measurements from three different pots per treatment. The analysis of variance (ANOVA) was performed in the SPSS 23 (IBM Corp) software, and was followed by pairwise post hoc analyses (Student–Newman–Keuls test) to determine significant differences among means at P ≤ 0.05.

Ahead of the analysis, the Audus test was used to determine the biostimulant properties of the active ingredient sedaxane. As with the natural auxin IAA, which reduces root elongation in the model plant watercress and is dose-proportional, increasing concentrations of sedaxane led to a progressive reduction in root length, suggesting an auxin-like effect (Figure 1). We also found sedaxane to exhibit gibberellin-like activity, as it enhanced the shoot growth of lettuce and had a similar dose-proportionality to exogenous gibberellic acid (Figure 2). Both regression curves were significant (P < 0.02 for root responses, P ≤ 0.05 for shoot responses), revealing the hormone-like activity of this fungicide.

Under sterile conditions, fungicide treatment did not significantly enhance plant growth, although the medium dose of sedaxane (75 μg seed^-1) appreciably increased shoot (+21%) and root (+10%) biomasses as compared to untreated controls (Table 1). The effects of the seed treatments were more evident on other root features: Root length increased by 60% and root area by 45% at the intermediate fungicide dose. While root diameter was slightly smaller (P > 0.05), the number of root tips and forks increased, most noticeably with the intermediate (tips +27%, forks +51%) and maximum doses (tips +17%, forks +48%), although only the root branching increase was significant. These results show that root stimulation by sedaxane may be dose-dependent up to saturation.

Leaf greenness, measured in terms of SPAD values, was very stable across treatments at the end of the trial (Table 2), while protein content was significantly influenced by sedaxane (P < 0.001), with an increase of 14% at the intermediate and highest AI doses (Table 2). A similar effect was found in the roots, with protein content increasing significantly at the highest AI dose (+20% vs. untreated controls).

Fungicide treatment did not affect the shoot and root glucose content, the former having an average concentration of 3374 μg g^-1 FW, the latter 3766 μg g^-1 FW. The only variation found with regard to fructose was that it was significantly reduced in the shoot at the lowest and highest sedaxane doses (-21% and -15%, respectively, vs. untreated controls) (Table 2).

Glutamine synthetase (GS) activity and glutamate synthase (GOGAT) activity were, respectively, 3.8 and 2.1 times higher, on average, in the shoots than in the roots. Seed treatment with sedaxane significantly increased GS activity in the shoots (P < 0.01) at the lowest (+145% vs. controls) and intermediate AI doses (+45%), and in the roots (P < 0.001), particularly at the intermediate and highest AI doses (both +66%, P ≤ 0.05) (Figure 3).

Sedaxane treatments did not affect GOGAT activity in the shoots, while slight, but insignificant, reductions were observed in the roots (Figure 3).

A significant increase in soluble phenolic acids in the shoots was observed at the lowest sedaxane dose (+14% vs. untreated controls), while values similar to controls were detected at greater AI doses (P ≤ 0.05) (Table 3). However, when individual compounds were analyzed, large differences among treatments were detected for caffeic acid, and, to a lesser extent, for syringic and p-coumaric acids (P ≤ 0.05). Significantly higher concentrations of caffeic acid were found in all treated plants compared with untreated controls (P ≤ 0.05). Sedaxane increased caffeic acid by 41–58%, depending on the dose, and p-coumaric acid, the most abundant phenolic compound, by 23% at the lowest and 19% at the intermediate dose. There were only slight differences in the vanillic and t-ferulic acid contents in treated plants as compared with controls (P > 0.05).

The ANOVA revealed a significant increase (P ≤ 0.05) in PAL enzyme activity in the shoots with the lowest and highest fungicide doses (+29% and +43%, respectively) as compared with untreated controls (Table 3).