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.