Fenpropimorph

A. niger strains used in this study are listed in Table 3. The strains were grown at 30°C (unless otherwise stated) in minimal medium (MM) [61 ] or complete medium (CM), consisting of minimal medium (MM) supplemented with 1% yeast extract and 0.5% casamino acids. Fermentation medium (FM) was composed of 0.75% glucose, 0.45% NH₄Cl, 0.15% KH₂PO₄, 0.05% KCl, 0.05% MgSO₄, 0.1% salt solution [61 ] and 0.003% yeast extract. The pH of FM was adjusted to pH 3. Aureobasidin A was purchased from Takara Bio, FK506 from A.G. Scientific, fenpropimorph from Sigma Aldrich, caspofungin (Cancidas®) from Merck and calcofluor white from BASF.
Table 3

Aspergillus nigerstrains used in this work

Name	Genotype	Reference
N402	cspA1, amdS−	[62 (link)]
MA169.4	kusA::DR-amdS- DR, pyrG−	[63 (link)]
MA78.6	ΔkusA, pyrG+(derivative of MA70.15 containing A. niger pyrG)	[63 (link)]
MA47.1	ΔkusA, pyrG+, ΔrlmA ( derivative of AB4.1)	[12 (link)]
ER2.5	ΔkusA, ΔrhoB::AopyrG	[13 (link)]
ER3.4	ΔkusA, ΔrhoC::AopyrG	[13 (link)]
ER7.6	ΔkusA, ΔrhoD::AopyrG	[13 (link)]
MA84.1	ΔkusA, ΔcftA::hygR	[13 (link)]
MA234.1	pyrG+ (derivative of N402 containing A. niger pyrG)	Unpublished
8.21	ΔcrzA, pyrG+(derivative of MA234.1)	Unpublished
MF3.2	kusA::DR-amdS- DR, ΔrlmA, hph	This study
JH1.1	kusA::DR-amdS-DR, msnA−, hph	This study
MF4.10	kusA::DR-amdS- DR, msnA−, ΔrlmA, hph	This study

Methanol (purity ≥ 99.8) was purchased from Merck (Darmstadt, Germany). Ultrapure water was produced with a Milli-Q system (Millipore, Saint Quentin en Yvelines, France) and was used for pesticide dissolution. The eight high-purity (≥ 98%) pesticides (pestanal^® analytical standards) acetochlor (CAS 34256-82-1, Batch SZB9314XV), bromoxynil (CAS 1689-84-5, Batch SZB8021XV), carbofuran (CAS 1563-66-2, Batch SZB9064XV), chlormequat (CAS 999-81-5, Batch SZB8248XV), ethephon (CAS 16672-87-0, Batch SZBB021XV), fenpropimorph (CAS 67564-91-4, Batch SZB9243XV), glyphosate (CAS 1071-83-6, Batch SZB9320XV), and imidacloprid (CAS 138261-41-3, Batch SZB9112XV), were purchased from Fluka, Sigma Aldrich (Les Ulis, France). They were incorporated into the rat diet, at a nominal dose corresponding to the same proportion as their respective environmental exposure based on French use in 2004 (https://www.airbreizh.asso.fr/), to reach a total dose of 447 μg/kg bw/d, which corresponds to the sum of their respective acceptable daily intake. Theoretical and ingested doses calculated from food consumption during the experiment are shown in Table 1.

Analytical standards of fenpropimorph (96.2%) and fenpropimorph acid (99.7%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and LGC Standards (Teddington, Middlesex, UK), respectively. All organic solvents used in this study were of HPLC grade, purchased from J.T Baker (Phillipsburg, NJ, USA). Other chemicals were of analytical grade, purchased from Junsel Chemical Co. (Chuo-ku, Tokyo, Japan). QuEChERS kits were obtained from Agilent Technologies (Santa Clara, CA, USA).

Phenotypic plate assays were performed on YNB (without amino acids and ammonium sulfate) media supplemented with 2% glucose and 10 mM ammonium sulfate unless stated otherwise. For UV sensitivity, cells were spotted then exposed for 6 sec to UV light at 48 mJ/cm² in a UV Stratalinker (Stratagene, USA). For all other assays, the stressor was added to the media immediately prior to pouring at the following concentrations: 0.001% SDS (Sigma, USA), 5 μg/mL fluconazole (Sigma, USA), 0.25 μg/mL itraconazole (Sigma, USA), 1 μg/mL fenpropimorph (Sigma, USA), 1 M NaCl (Sigma, USA), 1 M KCl (Sigma, USA), 125 mM t-butyl hydroperoxide (Sigma, USA), 1 mM NaNO₂ (Sigma, USA), 1 μg/mL cyclohexamide (Sigma, USA) and 1 mM mercaptopurine (Sigma, USA). Tenfold serial dilutions were prepared immediately prior to testing, with plates being imaged at 24–96 hr. All assays were performed at both 30 and 37 °C.