Methanol acetic acid solution

Cells were irradiated and cultured in 4 μg/mL of Cytochalasin B (Sigma, St Louis, MO, USA) for 22 h [64 (link)]. Harvested cells were suspended in 5 mL of 75 mM KCl solution, centrifuged, and fixed in 3:1 methanol acetic acid solution and formaldehyde (ThermoFisher, Waltham, MA, USA). Cells were dropped onto slides and allowed to air dry at room temperature. Slides were stained in 5% Giemsa solution in GURR solution (Invitrogen, Grand Island, NY, USA) for 5 min. A total of 300 binucleated cells were scored per treatment dosage to obtain micronuclei per binucleated cells.

Soil samples were randomly collected from the top layer (2–10 cm) at two different locations, an oilseed rape field in Weende, Göttingen, Germany (named as field soil) and a campus lawn at the University of Göttingen, Germany (named as grassland soil). Soil was sieved through a 2 mm sieve before use, and one half of fresh soil was sterilized twice at 121°C for 20 min with an overnight interval. The maximal water holding capacity (MWHC) of each soil was determined according to the following equation (Viji and Rajesh, 2012 (link)):
Based on this calculation, soil moisture was adjusted to 6%, 50%, and 90% MWHC by adding the required amount of distilled water. Twelve microsclerotia were spread on the surface of a cellulose acetate membrane (0.4 μm pore size, Sartorius Stedim Biotech GmbH, Germany), covered with another piece of membrane and buried into either sterile or unsterile soil. After 2 days of exposure, the membrane samples containing microsclerotia were carefully removed from the soil and stained with 0.25% Coomassie brilliant blue R250 in a methanol/acetic acid solution (Thermo Fisher Scientific). The germination rate was determined under a stereoscope (Leica Microsystems GmbH, Germany). The experiment was conducted twice with three replicates.