## 2 Materials and Methods
### Antimicrobial Activities  
#### 2.7.1 Antifungal Assays  
The antifungal activity of each extract was tested against _Fusarium oxysporum_ CTM10402, _Aspergillus niger_ CTM 10099, Fusarium graminearum ISPaVe 271, Fusarium culmorum ISPaVe 21w and _Alternaria alternata_ CTM 10230. _Fusarium oxysporum, Aspergillus niger_ and _Alternaria alternata_ were provided by the local culture Collection of Tunisian Microorganisms (CTM) of the Centre of Biotechnology of Sfax (CBS Sfax-Tunisia). _Fusarium graminearum_ ISPaVe 271, _Fusarium culmorum_ ISPaVe 21w were provided by National School of Engineers of Sfax (ENIS Sfax-Tunisia). Fungal strains were maintained on potato dextrose agar (PDA) (Oxoid, Basingstoke, UK) medium and preserved at 4 \({}^{\circ}\)C. All fungal strains were conserved in 50% glycerol (PubChem CID: 753) and 50% potato dextrose broth (_v_/_v_) at -20 \({}^{\circ}\)C.  
For spore preparation, the fungal strains were grown in PDA medium for 7 days at 25 \({}^{\circ}\)C. Sterile distilled water was added to the agar plate surface and spores were liberated from fungal mycelium by scraping with a sterile glass spreader. The liquid mixture was collected and filtered through Miracloth (Calbiochem, Darmstadt, Germany) before a centrifugation at 4000 rpm for 10 min. The spore pelletwas washed twice with sterile distilled water and resuspended in sterile distilled water again to obtain a final spore suspension. Fungal spore concentration was quantified under microscopy. The spore suspension obtained was adjusted to the concentration of 10\({}^{4}\) spores/mL. Spore suspension was directly used after preparation. Antifungal activity was determined by the agar diffusion test [28; 29]. The quantity used for each extract was 50 \(\upmu\)L per disk (at 5 mg/mL), and the plates were incubated at 30 \({}^{\circ}\)C for 72 h [28]. Dimethyl Sulfoxyde (DMSO) was used as a negative control, while Ambhoriccin B (20 \(\upmu\)g/wells) was used as a positive control. Antifungal activity was evaluated by measuring the diameter (mm) of circular inhibition zones around the well. Tests were performed in triplicate. 7.2 Determination of Minimal Inhibitory Concentration (MIC) and the Minimal Fungicidal Concentration (MFC)  
For minimal inhibitory concentration (MIC) determination, each extract was dissolved in a solution of dimethylsulfoxide DMSO (PubChem CID: 679) (Loba Chemie Pvt. Ltd., Mumbai, India)/water (1/9; _v/v_) to a final concentration of 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10 and 20 mg/mL and then filtered through 0.22 \(\upmu\)m pore-size black polycarbonate filters (Millipore). The test was performed in sterile 96-well microplates using 100 \(\upmu\)L as a final volume for each well which contain 10 \(\upmu\)L of cell suspension corresponding to a final concentration of 10\({}^{6}\) CFU/mL [30]. Positive growth control well consisted of fungi in Mueller Hinton broth (Oxoid, Basingstoke, UK) and DMSO/water (1/9) was used as negative control. The plates were then covered and incubated in appropriate temperature for 24 h. The MIC was defined as the lowest concentration in which the microorganism did not demonstrate visible growth. As an indicator of fungal growth, 25 \(\upmu\)L of thiazolly blue tetrazolium bromide (MTT) (PubChem CID: 64965) (Sigma-Aldrich, Taufkirchen, Germany) was added at 0.5 mg/mL to the well microplates and kept for 30 min at 37 \({}^{\circ}\)C. The tetrazolium salt was a colourless electron acceptor molecule which was reduced to a red-coloured formazan product with the growth of the indicator microorganisms. This salt would remain uncoloured if the microbial growth was blocked. MIC values were determined in triplicate. Minimal fungicidal concentrations (MFCs) were determined by serial sub cultivation of 10 \(\upmu\)L in PDA plates and incubated for 72 h at 28 \({}^{\circ}\)C. The lowest concentration with no visible growth was defined as the MFC, indicating \(\geq\)99.9% killing of the original inoculum. The determinations of MIC and MFC values were done in triplicate.