2.1. Preparation of Fungal Cultures

Czapek–Dox broth (35 mL) supplemented with different nitrogen sources, in other words, 50 mM ammonium sulphate, ammonium nitrate, sodium nitrate and potassium nitrate, respectively, was inoculated with eight mycelial discs of Ganoderma boninense PER71 in each 125 mL-conical flask. Untreated G. boninense PER71 in Czapek–Dox broth was used as control for nitrogen treatments. Three replicates were prepared for each treatment and control. The flasks were kept in the dark as static cultures at 30 °C. The mycelia of G. boninense were harvested at 6 days post-inoculation (dpi).

For phytohormone and H₂O₂ treatments, 35 mL potato dextrose broth (PDB) was prepared in each 125 mL-conical flask and inoculated with eight mycelial discs. The medium was supplemented with 1 mM salicylic acid (SA) (Sigma-Aldrich, St. Louis, MO, USA), or 1 mM jasmonic acid (JA) (Sigma-Aldrich, USA) or 1 mM H₂O₂. Untreated G. boninense in PDB was used as control. Three replicates were prepared for each treatment and control. All treatments were kept in the dark and incubated as static cultures at 30 °C for 4 days. The mycelia of G. boninense were harvested at 4 dpi. The amount of phytohormones was determined based on [24,25,26] while the amount of H₂O₂ was determined based on [28].

The mycelia were collected by filtration using Whatman filter paper No. 1, rinsed with distilled water to remove the remaining medium and dried by pressing on filter paper. The mycelia collected were frozen in liquid nitrogen and stored at −80 °C for RNA extraction.