Fungal Hyphae Fragmentation Protocol

We followed the FF method as described previously (13 (link)). Mutagenized hyphae cultured in CB medium (2 mL) for a few weeks were transferred to a 15-mL centrifuge tube (Greiner, Tokyo, Japan) and fragmented using the SoniMix ultrasonic homogenizer UX-050 (Mitsui Electric, Chiba, Japan) with an output power setting of 50% for 10 s. Five hundred microliters of homogenized hyphae were transferred to Ultrafree centrifugal filter units (5-μm pore; Millipore, Billerica, MA, USA) and centrifuged at 12,000×g for 1 min. The filtrate was spread onto solid CB medium and incubated at 28°C for one month to generate colonies.