2.1 Chemicals

If not stated otherwise, all chemicals were at least of analytical grade and purchased from Roth (Karlsruhe, Germany). The solvents for the isolation and extraction processes were obtained from VWR International (Vienna, Austria). Distillation of acetone occurred prior to its use. Solvents used in HPLC experiments had at least pro analysis (p.a.) quality and were purchased from Merck (Merck KGaA, Darmstadt, Germany). Ultrapure water was obtained via the Sartorius arium® 611 UV purification system (Sartorius AG, Göttingen, Germany).

2.2 Instruments

The fungal agar colonies were ground with the mill Retsch® SM 2000 (RETSCH GmbH, Haan, Germany), which was equipped with a 4.0 mm mesh. The weighing instruments KERN ALS 220-4 (KERN & SOHN GmbH, Balingen-Frommern, Germany) and Sartorius Cubis®-series (Sartorius AG, Göttingen, Germany) were utilized for weighing in of samples. The rotary evaporator Heidolph LABOROTA 4000-efficient (Heidolph Instruments GmbH & CO. KG, Schwabach, Germany) was used together with a vacuum pump and a vacuum controller for the evaporation of solvents under reduced pressure. Moreover, the ultrasonic baths Sonorex RK 106, Sonorex RK 52, and Sonorex TK 52 (BANDELIN electronic GmbH & Co. KG, Berlin, Germany) were used. The samples were mixed with the vortex mixer Vortex-Genie 2 (Scientific Industries, Inc., Bohemia, New York). Volumes were transferred with pipettes and tips from Eppendorf AG (Hamburg, Germany) and STARLAB International GmbH (Hamburg, Germany). Any other specific instruments are mentioned and described in the respective chapters.

2.3 Strains

Pycnoporus cinnabarinus (Jacq.) P. Karst fruiting bodies were collected in the Austrian central alpine area on deciduous trees at altitudes between 650 and 1100 m above sea level (IBF20160231 at c. 650 m, IBF20170021 at c. 1100 m, and IBF20180012 at c. 800 m). Voucher specimens were deposited in the Tiroler Landesmuseum IBF (Index Herbariorum ID 126158, Hall in Tyrol, Tyrol, Austria). Pure culture isolates were obtained for all strains and preserved in the culture collection of the Department of Microbiology, University Innsbruck under the same voucher numbers. The identity was confirmed by morphological hallmarks and barcode sequencing as described previously [22]. The ITS sequences are deposited at the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/, accessed 2023/09/23) under the following GenBank accession numbers: IBF20160321 (OR584232), IBF20170021 (OR584233), and IBF20180012 (OR584234). Cultures were preserved in 10% skim milk and in 40% glycerol at T = –80 °C at the Department of Microbiology in Innsbruck (Tyrol, Austria).

2.4 Media and inocula

Preliminary experiments with malt extract agar (MEA), potato dextrose agar (PDA), and Sabouraud dextrose agar (SDA) supplemented with 2% dextrose revealed MEA as best medium for both growth and pigment production. For the preparation of MEA, malt extract (c = 12 g L−1, bacteriological, Roth, Germany) and agar (c = 19.2 g L−1, Kobe I, Roth, Germany) were autoclaved together at T = 115 °C for t = 15 min. The medium volume (V = 20 mL) per Petri dish was applied with a dispenser. Preparation of the inocula was done by withdrawing mycelial discs from just behind the growing margin of the cultures with a sterile, sharpened corkscrew (inner diameter 6.7 mm) to facilitate a more constant starting point for the lag phase compared to the application of spore suspension as inoculum.

2.5 Cultivation2.5.1 Temperature effect on fungal growth: estimation in terms of radial growth

All isolates were incubated for 7 days at T = 25 °C and a relative humidity of 60% prior to the experiments. For the determination of cardinal temperatures, the three P. cinnabarinus strains were incubated in triplicates under dark conditions (to exclude the factor of light) and at the following ten temperatures: T = 10, 20, 25, 30, 32, 35, 37, 40, 42, and 45 °C. After 5 days of incubation, the cultures were photo-documented (supplementary information, Figure S12–15) and the radial growth, i.e., the culture diameter (cm) was measured.

2.5.2 Irradiation effect on fungal growth: estimation in terms of dry weight

For the irradiation experiments, cultures were grown at T = 25 °C and a relative humidity of 60% during 7 days prior to the experiments. Fungal inocula were placed on a commercial cellophane membrane that had been cut into discs. The diameter of these discs was 4 mm less than the inner diameter of the Petri dishes (i.e., 82 mm), thus enabling a more convenient applicability under the sterile airstream. Estimation of dry weight: see workflow in Section “Sample preparation and extraction”.

2.6 Irradiation

Irradiation was performed with an irradiation device (LIGHT BOX) and its established handling as described previously [23, 24]. In brief, to achieve highly standardized growth and illumination conditions (i.e., homogeneous illumination at defined wavelengths), this ventilated instrument was used within a climatic chamber. Inoculated Petri dishes were immediately incubated in a LIGHT BOX and kept for 7 days at T = 25 °C and at 60% relative humidity. The cultures were either kept constantly illuminated or in complete darkness (supplementary information, Figure S15).

2.7 Microscopic evaluation and documentation

All pure culture isolates were examined with standard microscopic techniques using water and cotton blue. Microscopic documentation and measurements were made with a Nikon DS Fi1 camera and the corresponding software NIS Elements 4.13.04 (Nikon Europe, Amsterdam, Netherlands). All measurements were made at 1000-fold magnification with a Nikon Plan Fluor 100X/0.5-1.3 oil immersion objective.

2.8 Sample preparation and extraction

The cultures were processed as follows: first, the cellulose hydrate membrane was carefully peeled off from the solidified medium with tweezers. The mycelium was scraped off the membrane, transferred into a glass vial, and stored in a deep freezer (T = –80 °C). After a storage time of approx. 24 h, the frozen sample was subjected to freeze-drying (Benchtop Pro, SP Scientific, Warminster, USA). After the determination of the dry weight, the individual samples were finely ground with mortar and pestle. An aliquot (m = 2–8 mg) of the ground sample was extracted with dimethyl sulfoxide (DMSO, V = 500 µL) by ultra-sonication (t = 5 min), followed by centrifugation (approx. 16 000 rcf, t = 10 min). The supernatant was filtered through cotton wool into a 2 mL volumetric flask. This extraction procedure was repeated twice and all supernatants were collected in the volumetric flask. In a last step, the targeted volume (V = 2 mL) was complemented with DMSO. The obtained solution was directly subjected to HPLC–DAD analysis.

2.9 High-performance liquid chromatography (HPLC)

The HPLC–DAD experiments were conducted using the modular system Shimadzu LC-20AD XR (Shimadzu Europa GmbH, Duisburg, Germany) equipped with a solvent degasser (DGU-20A3), a pump (LC-20AD XR), an auto-sampler (SIL-20AC XR), a column thermostat (CTO-20AC), and a diode-array detector (SPD-M20A). A Synergi Hydro-RP 80 Å column (150 × 4.60 mm, 4 µm) from Phenomenex (Aschaffenburg, Germany) was used as stationary phase. The mobile phase comprised water with 0.1% v/v formic acid (A) and acetonitrile (B). Elution was performed in gradient mode (0 min: 25% B, 11 min: 53.4% B, 12 min: 95% B, 14 min: 95% B, 15 min: 25% B, 17 min: 25% B, followed by 8 min of re-equilibration with 25% B). Flow rate, sample volume, and column temperature were adjusted to Q = 1.2 mL/min, V = 10 µL, and T = 40 °C, respectively. The DAD was set to λdet = 450 nm. The area of the target peak (tr, cinnabarin = 5.21 min) was integrated with Origin 2020 (OriginLab Corporation, North Hampton, United States), see supplementary information, Figure S1 for a representative run. For further data processing, MS Excel 365 (Microsoft Cooperation, Redmond, USA) and R Studio (2020 1.3.1093, R version 4.0.2 (2020-06-22)) were used.

2.10 Annotation process

UHPLC-HRMS/MS: The UHPLC analysis of a Pycnoporus cinnabarinus acetone extract (c = 2.5 mg/mL, solvent for dissolution = DMSO) was performed on a Vanquish system (Thermo Scientific, Waltham, MA, USA) consisting of quaternary pump, auto-sampler, column oven, and variable wavelength detector connected to a Thermo Scientific Exploris 120 Orbitrap HRMS unit. Separation was carried out on a Waters Acquity BEH C18 column (100 mm × 2.1 mm; particle size 1.7 µm) protected by a SecurityGuard ULTRA guard C18 pre-column. The mobile phase comprised water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The applied gradient was as follows: from 1 to 99% B in 9 min, followed by an isocratic step at 99% B for 1 min. Finally, the column was re-equilibrated with the initial solvent composition (i.e., 0% B) for 6 min. The flow rate, column temperature, auto-sampler temperature, and injection volume were adjusted to 0.5 mL/min, 40 °C, 20 °C, and 1 µL, respectively. The detection wavelengths were set to 468 and 519 nm.

The instrument was controlled by Thermo Scientific Xcalibur 4.4 software. Calibration of the mass analyzer was done via the Thermo Scientific proprietary calibration mix and the respective automatic calibration function. The mass spectrometric parameters were as follows: heated-ESI ionization source, positive polarity with static spray voltage (3000 V), sheath gas (N2): 37 arbitrary units, auxiliary gas (N2): 10 arbitrary units, sweep gas (N2): 0 arbitrary units. Temperature of the ion transfer tube and vaporizer was adjusted to 370 and 420 °C, respectively. MS data (range 100–1000 m/z) were recorded from 0 to 16 min with a resolution of 60 000 FWHM for MS1. Data-dependent experiments were conducted with stepped collision energies (15, 30, and 45 eV) at a resolution of 15 000 FWHM. The number of dependent scans was set to 3. The following selection of filters was employed: intensity threshold filter (5.0E5), dynamic exclusion (parent ions were placed in the exclusion list for 2 s after detection), isotope exclusion, charge state, and apex filter. In addition, a specific exclusion list was created for the measurement using DMSO as a background extract with an IODA Mass Spec notebook [25].

Data processing with MZmine3: The data obtained in positive mode (.raw format, Thermo Scientific) were imported into MZmine3 software [26]. For mass detection at the MS1 level, the noise level was set to 1.0E6. For MS2 detection, the noise level was set to 0.00. The ADAP chromatogram builder parameters were set as follows: minimum group size in number of scans, 4; group intensity threshold, 1.0E6; minimum highest intensity, 1.0E6 and scan to scan accuracy (m/z) of 0.0030 or 10.0 ppm. The ADAP feature resolver algorithm was used for chromatogram deconvolution with the following parameters: S/N threshold, 30; minimum feature height, 1.0E6; coefficient/area threshold, 110; peak duration range, 0.01–1.0 min; retention time (RT) wavelet range, 0.01–0.10 min. Isotopes were detected using the 13C isotope filter (formerly: isotope grouper) with a m/z tolerance of 0.0030 or 10.0 ppm, a retention time tolerance of 0.05 min (absolute), the maximum charge set to 1, and the representative isotope used was the most intense. Alignment was done with the Join aligner (m/z tolerance, 0.0030 or 10.0 ppm; RT tolerance, 0.05 min; Weight for RT, 70) and the aligned list was filtered using the Duplicate peak filter (m/z tolerance, 0.0030.0 or 10.0 ppm; RT tolerance, 0.10 min). The files were filtered with the Feature list rows filter (RT, 0.00–10.00 min) and only the ions with an associated MS2 spectrum were kept.

SIRIUS: After data processing, the feature corresponding to cinnabarin (RT 3.26 min—UHPLC analysis) was exported to SIRIUS (https://bio.informatik.uni-jena.de/software/sirius/ [27]) using the dedicated function in Mzmine3 with the Merge MS/MS option activated and the m/z tolerance set to 0.0030 m/z or 10.0 ppm. Following parameters were used for the SIRIUS molecular formula calculation: possible ionizations: [M + H]+, [M + K]+, [M + Na]+; instrument: Orbitrap, MS2 mass accuracy (ppm): 5 ppm, candidates stored: 10; min candidates per ion stored: 1; use DB formulas only: all. ZODIAC was used with default settings. The CSI:FingerID fingerprint prediction [28] was carried out with subsequent parameters: fallback adducts: [M + H]+, [M]+, [M + K]+, [M + Na]+; general: score threshold activated, search DBs: all. Lastly, the CANOPUS compound class prediction was performed [29,30,31].

2.11 Biomaterial and extraction

Large quantities of P. cinnabarinus fruiting bodies were collected in Innsbruck (Hungerburg/Hoch-Innsbruck: 47.29° N, 11.40° E, Tyrol (Austria)) in August 2019. The freshly harvested fruiting bodies were thoroughly cleaned with a brush, frozen over night at T = − 80 °C, and freeze-dried afterward. The dried fruiting bodies were stored in paper bags at room temperature in the dark until further use.

The dried biomaterial was ground using a laboratory mill (mesh size = 4.0 mm). The highly voluminous powder (m = 47.53 g) was successively extracted with petroleum ether (V = 1.4 L, n = 3), dichloromethane (V = 1.3 L, n = 3), and acetone (V = 1.5 L, n = 3) by ultra-sonication (t = 10 min each). Extracts were vacuum filtrated (filter: MN615, retention capacity 4–12 µm, Macherey-Nagel GmbH & Co. KG, Düren, Germany) and solvents were removed by rotary vacuum evaporation at T = 40 °C. Extract yields were for petroleum ether η = 323.4 mg (0.68% DW), for dichloromethane η = 444.4 mg (0.93% DW), and for acetone η = 355.9 mg (0.75% DW).

2.12 Isolation and characterization of cinnabarin

Based on a previous protocol [32], an optimized isolation process was developed: an aliquot (m \(\sim\) 30–40 mg) of the acetone extract was covered with 10 mL of acetone. After the sample was partially dissolved using an ultrasonic bath (t = 10 min), it was centrifuged (2000 rcf, t = 5 min). The supernatant was recovered and transferred into a round-bottom flask. These steps were repeated until the extract was completely dissolved. The solution in the round-bottom flask was diluted with ultrapure water (q.s., approx. V = 100 mL) and acetone was evaporated under reduced pressure. As the acetone evaporated, an orange–red precipitate was formed. The acetone-free aqueous solution was vacuum filtered (used filter paper: retention capacity 4–12 µm) and the filter cake washed with hot ethanol (n = 4, V = 10 mL, T ~ 60 °C) and hot dioxane (n = 4, V = 5 mL, T ~ 60 °C). Finally, the precipitate was dried by lyophilization (η ~ 2–3 mg (5–10%)).

Cinnabarin (CAS 146-90-70) was obtained as a red solid from the acetone extract of P. cinnabarinus. Experimental data were in line with [19]. Furthermore, UHPLC high-resolution MS/MS-based analyses were performed as additional annotation tool confirming the chemical formula C14H10N2O5 via i.a. the molecular ion peak [M + H]+ at m/z = 287.0649. All relevant spectra for the classic chemical characterization (Fig S1–S6) and the annotation workflow are displayed in the SI part (Fig S7–11 and Table S1). 1H-NMR (400 MHz, DMSO-d6, 25 °C): δ = 9.60 (brs, 1H, NHα), 8.74 (brs, 1H, NHβ), 7.55 (dd, 1H, J = 7.6, 7.5 Hz, CarH-7), 7.53 (dd, 1H, J = 7.7, 2.1 Hz, CarH-8), 7.49 (dd, 1H, J = 7.6, 2.0 Hz, CarH-6), 6.63 (s, 1H, CarH-4), 5.50 (t, 1H, J = 5.0 Hz, OH), 4.89 (d, 2H, J = 4.6 Hz, CH-12). UV/Vis (MeOH): λmax = 202, 233, 430, and 446 nm. IR (cm−1) = 3267w, 2916s, 2849s, 1672w, 1655m, 1591s, 1580s, 1466m, 1302w, 1187w, 1081m, 974m, 912w, 857w, 787m, 689m, 608w, 562w, 506w, 450w,434w.

2.13 Calibration curve

Two separately weighed stock solutions in DMSO (c = 100 µg/mL) were used to prepare 14 calibration levels (i.e., 7 per stock solution) by dilution in DMSO. The resulting solutions were immediately analyzed by HPLC–DAD as described above. Integration of the target peak was done with Origin 2020 and the calibration curve was calculated using a linear regression in MS Excel 365 (Microsoft Cooperation, Redmond, USA).

The following calibration curve parameters were determined for cinnabarin based on the ICH guideline [33]: (i) regression equation y = 391,079.03 x – 32.30, (ii) R2 = 0.9997, (iii) linear range = 2.28 E−4 - 0.11 mg mL−1, (iv) LOD = 4.96 E−4 mg mL−1, and (v) LOQ = 1.50 E−3 mg mL−1.