Quantifying Fungal Diversity in Soil

To count the number of growing fungal colonies, soil samples were processed within 15 days from collection, using the Dilution Plate Technique [33 ], partially following the protocol of Landínez-Torres et al. [34 (link)]. Soil dilutions at 10⁻³ were prepared for four replicates of each sample, and 100 µL of these dilutions were spread on malt extract agar (MEA; Merck KGaA, Darmstadt, Germany) plates. Moreover, in order to estimate the number of fungal strains with strong abilities of degrading recalcitrant materials, fungal counts were also performed on humic acid agar (1 g of commercial humic acids, 3.26 g of Bushnell-Haas broth, 15 g of agar; Merck KGaA, Darmstadt, Germany) and lignocellulose agar (4 g of lignocellulose, 3.26 of Bushnell-Haas broth, 15 g of agar) plates [35 (link)]. Inoculated plates were incubated at 25 °C in the dark and observed every day for 2 weeks. The number of developed fungal colonies was expressed as CFU (Colony-Forming Units) per gram of soil dry weight.