Treatment Application

The liquid cultures of the single fungal isolates received gradual and abrupt copper treatments. The treatments were designed according to a dose-day approach and, thus, the copper addition for the gradual treatment had to be started earlier than for the abrupt one. This way, we kept the duration of incubation the same for both treatments to assure that experimental units received the same overall dose (note that dose-days are calculated as the area under the curve). The final concentration of copper was always the same between abrupt and gradual treatments (see Supplementary Figure S1 in Supplementary Material). The final copper concentration in the growth medium in both treatments was 1 mM of copper sulfate pentahydrate. This concentration caused a significant decrease in biomass production, but was not lethal, according to our preliminary tests.

Fungal cultures were pre-incubated for 5 days without copper addition and then, during the treatment phase (copper addition), incubated for 9 days. In the gradual treatment, copper concentration was increased every 24 h by adding 0.1 ml of copper-nutrient solution. In the abrupt treatment, copper concentration was increased on the fifth day directly to the maximal concentration; on all other days, 0.1 ml of nutrient solution (Czapek-Dox broth) was added. For the control samples, 0.1 ml of medium was added daily to prevent a difference in nutrient supply among treatments.