Materials and Methods

Fungal material and substrate
In this study, six ectomycorrhizal fungal species were selected based on their representativeness and availability. The fungal species were supplied by Westerdijk Fungal Biodiversity Institute (Utrecht, the Netherlands) and by the Belgian Coordinated Collections of Microorganisms/Mycothèque de L’Université catholique de Louvain (BCCM/MUCL, Louvain-la-Nueve, Belgium). All the isolates were cultivated in specific agar media (Table 2) for approximately 40 days until the mycelium covered the entire surface of the petri dish. An average of 8 plates for each species was harvested. Upon harvesting, mycelium was divided into three concentric layers, allowing comparing across different ages of the same mycelium. Age was approximated as the average of the growth between the inner edge and the outer edge. The layers were removed from the plate, and the mycelium was then rinsed three times with hot water (85 °C) to wash out the remaining agar without damaging the mycelium. The mycelium was then dried overnight at 70℃. This provided approximately 120 mg of dried mycelium for each layer. The mycelium was put into a nylon mesh bag with 25 µm opening (Van Borselen Filters B.V., Zoetermeer, The Netherlands) and heat-sealed before incubation. Five replicates were prepared for each layer, resulting in 90 bags in total. One of these replicates was used to analyse the concentrations of cell wall components of the initial necromass (Week 0). For Lactarius deliciosus only 65 mg of dry mycelium per bag could be prepared due to the scarcity of biomass. For the decomposition assay, soil from a pine stand located in the Meijendel Dunes area (Wassenaar, The Netherlands, 52°08’57.0”N 4°22’21.6”E) was collected and sieved with a 2 mm mesh sieve. Each bag was incubated in a pot with approximately 400g of soil for a period of six weeks.
Mass loss evaluation
The mesh bags were harvested from the soil after one, two, four and six weeks of decomposition. The bags were rinsed in deionised water to wash off the remaining soil and dried overnight at 65℃. Mass loss was calculated by weighing the dried bags and comparing their weight with the dry weight before incubation. We further calculated the weekly mass loss by dividing the mass difference between two consecutive mass measurements by the number of weeks between the two measurements. The dried mycelium was collected and ground with a pestle for microcentrifuge tubes in a 1.5ml tube for chemical analysis.
Chemical analysis
Chitin was extracted according to the protocol of Fernandez & Koide (2012), with minor adjustments; the concentration in the samples was determined with the spectrophotometer at 653 nm. Known amounts of pure chitin (Merck KGaA, Darmstadt, Germany) were assayed to obtain a standard curve which was used to obtain the concentrations in the samples. Melanin concentrations were measured according to Fernandez et al. (2014). The determination of melanin concentration was made by placing 10 mg of sample in 2 mL of Azure A dye solution and incubating for 24 hours. The solution was filtered through a 0.45 mm nitrocellulose membrane, and the absorbance of the filtrate was measured using the spectrophotometer at 610 nm. A standard curve was constructed using known amounts of melanin previously isolated from the same material used in the decomposition assay. The change in absorbance was used to calculate the melanin content of the sample based on the standard curve.
Glucans were extracted with the Mushroom and yeast beta-glucans assay kit (K-YBGL 08/18, Megazyme). The protocol was followed with a few adjustments; a known amount of glucans was used to calculate the standard curve, which was used to calculate the glucans concentration after measuring the absorbance at 510nm.