Materials and reagents

Fungal and bacterial strains

1. Fungal strain to test [Serpula lacrymans, origin: Innsbruck, no. 1SLIBK2018 (Embacher et al., 2021)]

2. Bacterial strains to test [e.g., Microbacterium spp. (Embacher et al., 2021 and 2022)]

   
   

Materials

1. Autoclaved wood blocks (50 mm × 25 mm × 15 mm) (e.g., Picea abies; all blocks for one experiment should originate from the same batch timber)

2. Vessels (88 mm height, Ø 75 mm, volume 300 mL, e.g., ROTILABO^®, catalog number: EP28.1) filled with 25 mL malt extract agar

3. Petri dishes 94 mm × 16 mm, without vents (Greiner Bio-One, catalog number: 632180)

4. Pre-cultured bacterial isolates to be tested

5. Pre-cultured fungal strain(s) to be tested (e.g., the wood rotting fungus S. lacrymans)

6. Fisherbrand^TM Easy Reader^TM conic centrifuge tubes 15 mL, PP (Fisher Scientific, catalog number: 11819650)

7. Sterile toothpicks

8. 1.5 mL microcentrifuge tubes (Greiner Bio-One, catalog number: 616201)

   
   

Reagents

1. Casein peptone (Roth, catalog number: 8952.2)

2. Soy peptone (Roth, catalog number: 2365.2)

3. NaCl (Roth, catalog number: 0601.1)

4. Agar (Roth, catalog number: 5210.2)

5. Malt extract (Roth, catalog number: X976.2)

6. dH₂O

7. D(+)-Glucose monohydrate (Roth, catalog number: 6780.1)

8. (NH₄)₂HPO₄ (Roth, catalog number: P736.1)

9. KH₂PO₄ (Roth, catalog number: 3904.1)

10. MgSO₄·7H₂O (Roth, catalog number: T888.1)

11. CaCl₂·2H₂O (Roth, catalog number: T885.1)

12. FeCl₃ (Roth, catalog number: 5192.1)

13. Thiamine-HCl (Roth, catalog number: T911.1)

14. EDTA disodium salt (Roth, catalog number: 8043.1)

15. ZnSO₄·7H₂O (Roth, catalog number: K301.2)

16. H₃BO₃ (Roth, catalog number: 5935.1)

17. MnCl₂·4H₂O (Roth, catalog number: 0276.1)

18. CoCl₂·6H₂O (Roth, catalog number: 7095.1)

19. CuSO₄·5H₂O (Roth, catalog number: 8175.6)

20. (NH₄)₆Mo₇O₂₄·4H₂O (Roth, catalog number: 7311.1)

21. FeSO₄·7H₂O (Roth, catalog number: P015.1)

22. KOH (Roth, catalog number: 6751.1)

23. NaOH (9356.1)

24. PCR reagents:

    1. Red Taq 2× DNA Polymerase Master Mix (VWR, Radnor, USA)

    2. Primers 27F (5′-AGA GTT TGA TCA TGG CTC A-3′) and 1492R (5′-TAC GGT TAC CTT GTT ACG ACT T-3′) (both 10 μM)

    3. Distilled water

    4. Bovine serum albumin (BSA) 2% [(Roth, catalog number: 3854.2); see as well (Embacher et al., 2021)]

25. 1% agarose gel for gel electrophoresis system [e.g., agarose powder (Sigma, catalog number: A9539)]

26. Polyethylene glycol 6000 (PEG) 20% (Roth, catalog number: 0158.1)

27. 80% EtOH (-20 °C)

28. Nuclease-free water

29. Tryptone soya agar (TSA) plates (see Recipes)

30. TSA soft agar (freshly prepared on harvesting day, store at ~50 °C until usage) (see Recipes)

31. Liquid Tryptone soya (TS) medium (see Recipes)

32. Malt extract agar (MEA) (see Recipes)

33. Modified Melin-Norkrans (MMN) (see Recipes)

34. Hutner’s Trace metals (see Recipes)

35. 0.85% NaCl solution (see Recipes)

36. Sodium borate buffer (20× Stock solution, pH 8) (for agarose gel electrophoresis, see Recipes)

    
    

Recipes

1. Tryptone soya agar (TSA) plates

   Casein peptone 1.5% (w/v), 15 g

   Soy peptone 0.5% (w/v), 5 g

   NaCl 0.5% (w/v), 5 g

   Agar 1.8% (w/v), 18 g

   dH₂O, add up to 1 L

   (pH = 7.3 ± 0.2)

   Prepare TSA medium, autoclave (120 °C), let it cool down, pour into Petri dishes, and let cool to room temperature (RT). Store at 4 °C.

   
   

2. TSA soft agar

   Casein peptone 1.5% (w/v), 15 g

   Soy peptone 0.5% (w/v), 5 g

   NaCl 0.5% (w/v), 5 g

   Agar 0.7% (w/v), 7 g

   dH₂O, add up to 1 L

   (pH = 7.3 ± 0.2)

   Prepare TSA medium and autoclave (120 °C). Store at ~50 °C until usage for plate casting after Koch.

   
   

3. Liquid Tryptone soya TS medium

   Casein peptone 1.5% (w/v), 15 g

   Soy peptone 0.5% (w/v), 5 g

   NaCl 0.5% (w/v), 5 g

   dH₂O, add up to 1 L

   (pH = 7.3 ± 0.2)

   Prepare TS liquid medium, autoclave (120 °C), and let it cool down. Store at 4 °C.

   
   

4. Malt extract agar (MEA)

   Malt extract 3% (w/v), 30 g

   Soy peptone 0.3% (w/v), 3 g

   Agar 1.8% (w/v), 18 g

   dH₂O, add up to 1 L

   (pH = 3.5 ± 0.2)

   Prepare MEA medium, autoclave (120 °C), and cool it down to 50 °C. Pour into Petri dishes and let cool to RT. Store at 4 °C.

   
   

5. Modified Melin-Norkrans (MMN)[after Tauber et al. (2016)]

   Glucose, 5 g

   (NH₄)₂HPO₄, 0.25 g

   KH₂PO₄, 0.5 g

   MgSO₄·7H₂O, 0.15 g

   CaCl₂·2H₂O, 0.067 g

   NaCl, 0.025 g

   FeCl₃ (1%), 1.2 mL

   Thiamine-HCl (10%), 1 μL

   Hutner’s trace metals (0.01×), 100 μL

   Agar, 20 g

   dH₂O, add up to 1 L

   (pH = 5.6)

   Prepare FeCl₃, Thiamine-HCl (10%), and Hutner’s trace metals (0.01×) (Hutner et al., 1950) separately. Add them to the media while mixing on the rotary shaker. Measure the pH (and adjust if necessary) and adjust the media to 1,000 mL. Autoclave at 120 °C, let it cool down, pour into Petri dishes, and let cool to RT. Store at 4 °C.

   
   

6. Hutner’s trace metals

   EDTA disodium salt, 50 g in 250 mL dH₂O

   ZnSO₄·7H₂O, 22 g in 100 mL

   H₃BO₃, 11.4 g in 200 mL

   MnCl₂·4H₂O, 5.06 g in 50 mL

   CoCl₂·6H₂O, 1.61 g in 50 mL

   CuSO₄·5H₂O, 1.57 g in 50 mL

   (NH₄)₆Mo₇O₂₄·4H₂O, 1.10 g in 50 mL

   FeSO₄·7H₂O, 4.99 g in 50 mL

   20% KOH solution (w/v)

   For 1 L final mix, dissolve each component in the volume of water indicated. The EDTA should be dissolved in boiling water, and the FeSO₄·7H₂O should be prepared last to avoid oxidation.

   Mix all solutions except EDTA. Bring to boil, then add the EDTA solution. The colour of the mixture turns to green. When everything is dissolved, let cool to 70 °C. While keeping the temperature at 70 °C, add 85 mL of hot KOH (20%). Cool to RT and fill up to 1 L final volume. Close the flask with a cotton plug (allows air exchange) and swirl it once a day while incubating for 1–2 weeks. Usually, the solution will initially be clear green but turns dark red or purple over the next few days, leaving a rust-brown precipitate. If no precipitate forms or the solution remains green, check the pH (should be at approximately 6.7; if there is a big deviation, try adding either KOH or HCl to adjust it).

   Filter through two layers of Whatman #1 filter paper and repeat, if necessary, until the solution is clear. Store refrigerated or frozen in convenient aliquots.

   
   

7. 0.85% NaCl Solution

   Sodium chloride 0.85% (w/v), 8.5 g

   dH₂O, add up to 1 L

   Prepare solution, autoclave (120 °C), and let cool down. Store at RT.

   
   

8. Sodium borate buffer (20× stock solution, pH = 8)

   Boric acid, 48 g (concentration 1 M)

   NaOH pellets, 8 g

   dH₂O, add up to 1 L

   Prepare 900 mL of dH₂O in a suitable container and add the boric acid and the sodium hydroxide to the solution. Stir until all solids are dissolved. Adjust pH to 8 by adding boric acid or NaOH. Fill to 1 L with dH₂O.

   Dilute the 20× stock solution to 1× before usage for gel electrophoresis (can be used for preparation for agarose gels and in the gel tank).

Equipment

1. Eppendorf centrifuge 5810 equipped with A-4-81 rotor (Eppendorf SE, Hamburg, Germany)

2. Heraeus Fresco^TM 17 microcentrifuge equipped with 24 × 1.5/2.0 mL rotor with ClickSeal^TM biocontainment lid (Thermo Scientific, Waltham, Massachusetts, USA)

3. Counting chamber Thoma (depth: 0.01 mm, 0.0025 mm², Assistent, Glaswarenfabrik Karl Hecht, Sondheim vor der Rhön, Germany)

4. Autoclave (HMC Europe, HICLAVE, catalog number: HGS-133)

5. Incubator (temperature range: 10–30 °C, e.g., Heraeus Vötsch, Vienna, Austria)

6. Benchtop orbital incubator shaker (New Brunswick Scientific, Edison, model: Innova^® 40/40R)

7. Chemically resistant diaphragm vacuum pump model N810 FT.18 (KNF LABOPORT, Hamburg, Germany)

8. Vacuum filter manifold and sterile filtration funnels (Millipore Sigma^TM, Microfi^l®, Merck Millipore Billerica, USA)

9. Nitrocellulose filter (0.45 μm; diam. 47 mm, part no: 1215230, GVS North America, Sanford, USA)

10. Overhead shaker model REAX2 (Heidolph Instruments, type: 541-21001-00)

11. Heating oven, 3.8 cu ft, 230 VAC (Memmert, UNB500/230 Basic Oven)

12. Balance (PM4600 DeltaRange^®, Mettler-Toledo, Vienna, Austria)

13. Microbiological safety cabinet (Platinum SF, Kojair Tech Oy, Mänttä-Vilppula, Finland)

14. Carton package (to guarantee incubation in darkness)

15. Sterile glass Petri dishes (100 mm × 20 mm Rotilabo^®, Carl Roth, Karlsruhe, Germany)

16. Sterile plier

17. Sterilizable punch (that gives e.g., plugs with ~0.8 cm × 0.7 cm × 1.1 cm)

18. Tweezers

19. PCR cycler (Primus 96 advanced, Peqlab Biotechnologie, Erlangen, Germany)

20. Horizontal gel electrophoresis system (RunOne^TM Electrophoresis Cell, EmBi Tech, San Diego, USA)

21. Gel Doc^TM EZ Imager 30016395-0000 171800 (Bio-Rad Laboratories, Hercules, California, USA)

22. Thermomixer^® comfort 5355 (Eppendorf SE, Hamburg, Germany)

Software

1. Microsoft Excel version 2209

2. RStudio version 4.0.3 (2020-10-10)

Procedure