Salicylhydroxamic acid sham

The following commercial fungicide formulations were supplied by their representative manufacturers and used in all tests: trifloxystrobin as Flint 50 WG (Bayer AG, Leverkusen, Germany); cyprodinil as Chorus 50 WG (Syngenta Crop Protection AG, Basel, Switzerland); dodine as Syllit 544 SC (Arysta LifeScience Benelux Sprl, Ougrée, Belgium); difenoconazole as Score 25 EC (Syngenta Crop Protection AG, Basel, Switzerland); boscalid as Cantus 50 WG (BASF SE, Ludwigshafen, Germany); fludioxonil as Geoxe 50 WG (Syngenta Crop Protection AG, Basel, Switzerland); dithianon as Delan 70 WG (BASF Agro BV, Arnhem, The Netherlands); and captan as Merpan 80 WG (Adama Makhteshim Ltd, Beer Sheva, Israel). The fungicides were dissolved in sterilized distilled water and stock solutions of 1000 mg L⁻¹ active ingredient (a.i.) were prepared. To obtain the desired concentrations of each fungicide (mg L⁻¹ of a.i. in growth medium), 10-fold serial dilutions were made from the stock solutions. To inhibit the alternative respiration function, the QoI fungicide trifloxystrobin was tested with the addition of 100 mg L⁻¹ salicylhydroxamic acid (SHAM; Sigma-Aldrich, Darmstadt, Germany) to the medium. The final concentration of the solvent in the growth medium did not exceed 1% (v/v).

The molecular probe 2',7'-dichlorofluorescein diacetate (H₂DCF- DA; Sigma-Aldrich, St Louis, MO, USA) and the NO fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA; Invitrogen, Eugene, OR, USA) were dissolved in DMSO to produce a stock solution, which was aliquoted. Catalase (CAT, bovine liver), d-glucose, diphenyleneiodonium chloride (DPI), MES, EGTA, lanthanum chloride (LaCl₃), sodium azide (NaN₃), salicylhydroxamic acid (SHAM), and tungstate were obtained from Sigma-Aldrich. The remaining chemicals of the highest analytical grade were bought from Chinese companies.

Optical rotations were measured on a Jasco P2000 digital polarimeter (Jasco, Cremella, Italy). UV spectra were acquired on a Jasco V-650 spectrophotometer, NMR spectra were recorded on a Bruker Avance DRX 600 (Bruker, Milan, Italy) equipped with a cryoprobe operating at 600 MHz for protons. Chemical shift values are reported in ppm (δ) and referenced to internal signals of residual protons (CD₃OD ¹H δ3.34, ¹³C 49.0 ppm). High-resolution mass spectra were acquired on a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Milan, Italy); HPLC analyses have been performed on a Jasco system (PU-2089 Plus-quaternary gradient pump equipped with a Jasco MD-2018 Plus photodiode array detector). [1-¹³C]-acetate, [2-¹³C]-acetate and [1,2-¹³C₂]-acetate, [1-¹³C]-glycolate (all sodium salts), and salicylhydroxamic acid (SHAM) were obtained from Sigma Aldrich (Milan, Italy). Solvents were purchased from VWR (Milan, Italy) and were HPLC-grade.