Malt extract

Deletion strains were grown on plates with 3% (w/v) malt extract (Merck, Darmstadt, Germany) or Mandels-Andreotti medium¹⁰² containing 1% (w/v) carboxymethylcellulose (CMC) as carbon source, for 2-4 days at 28 °C in constant light (≈1800 Lx) or constant darkness. The growth in constant darkness was analyzed after two days by measuring the colony diameter. The mutant strains were inoculated in the center of the plate and colony diameter reflecting hyphal extension was measured every 24 hours for two to four days. QM6aΔtmus53 was used as control for every set. At least three biological replicates were analyzed for every strain.

The fungal strain JKI-BI-1450 was stored at −80 °C in cryo-tubes (Microbank, Pro-Lab Diagnostics, Richmond Hill, Canada) and routinely cultured on malt peptone agar (MPA) containing 3% (w/v) malt extract (Merck, Darmstadt, Germany), 0.5% (w/v) peptone from soybean (Merck), and 1.8% (w/v) agar-agar (Roth, Karlsruhe, Germany).

The fungal pathogens used in this study are shown in Table 1. The media used in this study were YPD (1% yeast extract [Bioshop, Canada], 2% peptone [Bioshop], 2% dextrose [Bioshop]), PDB (24 g potato dextrose broth [Himedia, India] in 1 L distilled water), RPMI 1640 medium (10.4 g RPMI 1640 powder [Sigma-Aldrich, USA], 34.5 g MOPS [3-(N-morpholino) propanesulfonic acid, Sigma-Aldrich], 2 g dextrose, in 1 L distilled water, with pH adjusted to 7.0 with NaOH), spider medium (10 g nutrient broth [Himedia], 10 g mannitol [Panreac, Spain], 2 g K₂HPO₄, in 1 L distilled water, adjusted to pH 7.2 with H₃PO₄), YNB (0.17% yeast nitrogen base w/o amino acids [Bioshop], 0.5% (NH₄)₂SO_4, 2% dextrose) and modified Dixon medium (36 g malt extract [Merck, Germany], 20 g desiccated oxbile [Sigma-Aldrich], 10 mL Tween 40 [Sigma-Aldrich], 6 g peptone [Bioshop], 2 mL glycerol [Scharlab, Spain], 2 mL oleic acid [Sigma-Aldrich], in 1 L distilled water). All media were solidified by adding 2% agar (Bioshop) if needed, except mDixon medium (1.5% agar).
For synthesis reaction, N, N, N′, N′, N′′-pentamethyldiethylenetriamine [Sigma-Aldrich], alkyl bromides [Sigma-Aldrich], 1, 4-butanesultone [Sigma-Aldrich], and acetonitrile [POCh S. A., Poland] were purchased. All compounds were analytical reagent quality and used without further purification.

Trichoderma reesei wild-type strain QM6a, its derivative QM9414 (ATCC 26921), QM9414Δblr1, Δblr2, and Δenv1 (Castellanos et al., 2010 (link)), and QM6aΔku80 were used throughout this study. Strains were maintained on 3% (w/v) malt extract-agar (malt extract: Merck, Darmstadt, Germany; agar-agar: Roth, Karlsruhe, Germany). For quantitative reverse transcription-PCR (qRT-PCR) analysis, biomass determination, SDS-PAGE, and secreted cellulase activity, T. reesei was grown in liquid culture in 100 ml Mandels-Andreotti minimal medium (Mandels and Andreotti, 1978 ) supplemented with 0.1% (w/v) peptone (Roth, Karlsruhe, Germany) and with 1% (w/v) microcrystalline cellulose (Alfa Aesar, Karlsruhe, Germany) as a carbon source for 72 h at 28°C on a rotary shaker (200 rpm). Strains were grown either in the presence of constant illumination (Osram L 18W/835; day light simulating wave length distribution) with different light intensities ranging from 700 to 5,000 lux or in constant darkness. In the latter case, cultures were harvested under red safety light (Fischer Photolamp 230V 15W^*5F, Diez, Germany) using Miracloth filtration material (Calbiochem/Merck, Darmstadt, Germany) to separate the culture from the supernatant.

Upon collection of the ascocarps, several analyses were performed. First, material for genetic identification was collected directly upon sterilization of the tissue surface. Second, in those individuals with an intact fruiting body (16 individuals), fungal isolation was attempted either by germinating spores or by direct culturing pieces of hymenia. Isolation was performed on potato dextrose agar (PDA, Potato infusion powder, Sigma-Aldrich, 4 g/L + D(+) - glucose monohydrate, Roth, 20 g/L + Agar-Agar, Merck, 15 g/L). Pure isolates were obtained by successive plating in the same medium. In addition, two Chinese cultivars (NEU142 and NEU143) and one morel specimen from our collection (M84) were grown in PDA to be included in the analysis. Sclerotia were obtained by culturing on malt agar (Malt extract, Fluka, 12 g/L + Agar-Agar, Merck, 15 g/L; MA). All media were autoclaved at 121°C before pouring into Petri dishes.