## 4 Materials and Methods
### Antimicrobial Activities
#### 4.3.2 Agar Disc Diffusion Assay  
The disc diffusion method measured the antibacterial activity of methanolic extracts [56]. First, 100 \(\upmu\)L of bacterial suspension containing 1.5 \(\times\) 10\({}^{8}\) CFU/mL or yeast suspension containing 1.5 \(\times\) 10\({}^{6}\) CFU/mL was spread on Mueller Hinton agar (MHA, Sigma-Aldrich, St. Louis, MO, USA) or Sabouraud dextrose agar medium (SDA, BTL, Lodz, Poland), respectively. The turbidity of the tested strains was standardized to 0.5 McFarland (spectrophotometer VIS-723G, Rayleigh, Beijing). Then, paper discs (Whatman no. 1, England, 6 mm diameter) were placed on the agar surface and impregnated with 30 \(\upmu\)L of stock solutions of 100 \(\upmu\)g/mL. For a negative control and a solvent, 10% DMSO was used. In addition, filter discs impregnated with 30 \(\upmu\)g/mL tetracycline (Sigma-Aldrich) or amphoteric B (Gibco, Life Technologies, USA) were used as a positive reference control for bacteria and fungi, respectively. The plates, after remaining at 4 \({}^{\circ}\)C for 2 h, were incubated at 37 \({}^{\circ}\)C (24 h) for bacterial strains and at 30 \({}^{\circ}\)C (48 h) for yeasts. Antimicrobial activity was evaluated by measuring the diameters of inhibition zones in millimeters. The experiment was done in triplicate, and mean values are presented (Table 2).  
3.3 Determination of Minimum Inhibitory Concentration, Minimum Bactericidal Concentration and Half Inhibitory Concentration  
The minimum inhibitory concentrations (MICs) were determined by a serial dilution method [57] in 96-well plates (Tissue Culture Plates VWR European). The bacterial species were cultured in Mueller-Hinton agar (MHA, Sigma-Aldrich, St. Louis, MO, USA) at 36 \({}^{\circ}\)C (_Pectobacterium_ spp. at 28 \({}^{\circ}\)C) for 20 h. After cultivation, bacterial suspensions were made in Mueller-Hinton broth (MHB, Sigma-Aldrich, St. Louis, MO, USA), and their turbidity was standardized to 0.5 McFarland (spectrophotometer VIS-723G, Rayleigh, Beijing). The bacterial inoculum's final optical density (OD) was -5 \(\times\) 10\({}^{5}\) CFU/mL. The dry methanolic extracts were dissolved in 10% DMSO (dimethylsulfoxide) and were prepared in a 500 to 62.5 \(\upmu\)g/mL concentration. As a positive control, a solvent (10% DMSO) was used as negative control along with tetracycline (30 \(\upmu\)g/mL, Sigma-Aldrich). The plates were then incubated at 36 \({}^{\circ}\)C (_Pectobacterium_ spp. at 28 \({}^{\circ}\)C), and the bacterial growth was observed after 20 h by measuring the optical density (OD) with a microplate photometer (Thermo Scientific, Waltham, MA, USA, MultiscanGO; \(\uplambda\) = 570 nm). The experiments were done in three series at four repetitions. The minimum inhibitory concentration (MIC\({}_{90}\)) was the lowest concentration of assayed extracts that caused 90% growth inhibition as compared to the positive control. It was determined by calculating the percentage inhibition of bacterial growth by the following formula [(Equation (1))]:  
\[\%\ Inhibition=\frac{\mathrm{ODpc-ODs}}{\mathrm{ODpc}}\times 100 \tag{1}\]  
where:  
ODpc is the mean value of the measured optical density of the positive control.  
ODs is the mean value of the measured optical density of the extracts of the test sample.  
Minimum bactericidal concentration (MBC) was defined as the lowest extract concentration that killed 99% of microorganisms. To determine MBC, cultures were taken from each well without visible growth and inoculated on MHA.