*4.5. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of Nitric-Extract*

The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) are dilution end points of a substance which completely inhibits the growth or kills the fungi tested; both are widely used in routine tests of substances with antimicrobial activity [9,81]. The minimum inhibitory concentration (MIC), defined as the lowest concentration of the test substance that inhibits visible growth, was determined with a microdilution method. For each pathogen, in a 2.0 mL tube, 400 μL of "Nitric-extract" at specific concentrations were added to 400 μL of sterile PDB and to 200 μL of spores suspension (concentration 104 spores/mL) to obtain 10 serial dilutions (1 mL each) of the

substance tested (final concentrations 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0%). Then, the tubes were incubated at 25 ◦C for 3 days.

After the incubation period, the MIC was the lowest concentration where no cloudiness was visible in the tubes, which means that no pathogen growth was observed. The determination of MFC was an additional step of the MIC test. The MFC is defined as the lowest concentration of a substance required to kill a fungal pathogen corresponding to no visible subculture growth on an unamended culture medium in environmental conditions favorable to the growth. In the present study, the evaluation of the MFC was carried out by transferring 10 μL from each of the wells where solution cloudiness was not observed into PDA medium. The inoculated plates were incubated at 25 ◦C for 3 days. The MFC for each pathogen was represented by the plated concentration that did not lead to any mycelial growth after the incubation period.
