The isolates are available at the Department of Diagnostics and Plant Pathophysiology, University of Warmia and Mazury in Olsztyn. Isolates are stored as mycelium/spore I-BET-762 supplier suspensions in 15% glycerol at − 25 °C. YES agar medium (yeast extract 20 g L−1, sucrose 150 g L−1, MgSO4.7H2O 0.5 g L−1, agar 20 g L−1) recommended for secondary metabolite analysis was used. Propiconazole and tebuconazole (Sigma-Aldrich, Germany) were dissolved

in 0.65 mL of acetone and then added to autoclaved YES medium to obtain the final concentrations: 0.25 mg L−1, 0.5 mg L−1, 2.5 mg L−1, and 5 mg L−1. Recommended field doses of both azoles completely inhibited fungal growth on the media. The control sample was supplemented with an identical volume of acetone. Experiments were performed on Petri plates (Ø 80 mm). Petri plates containing 10 mL of YES medium were inoculated with fungal hyphae with a sterile tip and incubated at 25 °C in darkness. For each condition, plates (in triplicate) were incubated at 25 °C for 4 days. The total

RNA was extracted from 4-day-old cultures from three F. graminearum field isolates grown on YES medium with or without supplementation VE-821 clinical trial of the tested azole. Two biological replications were prepared for each condition independently in time. Mycelium (350 mg) was ground in liquid nitrogen with mortar and pestle. Total RNA was extracted using a Quick-RNA™ MiniPrep kit (Zymo Research) following the manufacturer recommendations. Total RNA was reverse-transcribed using the SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen). Reverse transcription was performed immediately after RNA extraction with a Mastercycler ep gradient (Eppendorf AG, Germany) with the thermal cycling conditions recommended by the manufacturer (Invitrogen). cDNA samples were stored at − 25 °C for RT-qPCR analysis. To design primer/probe sets for RT-qPCR analyses, the F. graminearum sequence data of ef1α, tri4, tri5, and tri11 published in the NCBI database were aligned with geneious pro 4.0.0 (Drummond et al., 2011). To prevent amplification

of genomic DNA, at least one primer and/or probe from each set of primers/probes was designed on exon–intron boundaries using primer express 3.0 (Applied Biosystems, Foster City; Table 1). ef11 ef12 ef1α probe Cell press TCGACAAGCGAACCATCGA CCCAGGCGTACTTGAAGGAA VIC-CGAGAAGGAAGCCGC-MGB tri41 tri42 tri4probe TGCATGAAATAGGTGGACTGAGA AACTTGAAGTACAAGGAGCATGTCA FAM-ATGGGAGTTCCTTTAGGG-MGB tri51 tri52 tri5probe AACGAGCACTTTCCCAACGT ATCCAACATCCCTCAAAAAAGTC FAM-TCATTGAACCTTATCCGTAGCA-MGB tri111 tri112 tri11probe CCAGCATCATGCGCATCTC AATCGGACCACGGAATTGTATT FAM-CGTAGGCAAGGTTCATA-MGB Probes, conjugated with an MGB group, were labeled at the 5′-end with FAM, while the ef1α probe was labeled at the 5′-end with VIC. All primers were synthesized by Genomed (Warsaw, Poland), while MGB probes were ordered from ABI PRISM Primers and TaqMan Probe Synthesis Service.