# Antimicrobial Efficacy of Edible Mushroom Extracts: Assessment of Fungal Resistance
## 2 Materials and Methods
### Antifungal Test: Agar Plate Assay  
Zone of inhibition assays were initially performed to assess the antifungal activity of the mushroom extracts against _C. albicans_ ATCC 10231 and _A. fumigatus_ AF293 as described previously (2017). Methanol or water extracts from mushroom samples were each tested at 0.625, 1.25, 2.5, 5.0, or 10.0% (\(w/v\)) on PDA Petri plates (100 mm \(\times\) 15 mm; Corning Inc.-Life Sciences, Tewksbury, MA, USA). Mushroom extracts were dissolved in a vehicle (DMSO:water (50%:50%)) before spotting (5 \(\upmu\)L) each concentration onto the fungus-streaked (lawn) PDA. Control contained the DMSO vehicle only (5 \(\upmu\)L) at levels equivalent to that of cohorts receiving the mushroom extracts, within the same set of experiments. The level of zone of inhibition was monitored for three to five days at 35 \({}^{\circ}\)C.  
The level of antifungal activity of _T. camphoratus_ methanol extract (TcM) was investigated further by monitoring the radial growth of seven fungal pathogens/contaminants (_N. fischeri, A. flavus, A. parasiticus, P. expansum_ W1, W2, FR2, FR3) on PDA containing 0.0%, 0.8%, or 1.6% (\(w/v\)) of TcM in triplicate wells of 6-well plates (6 \(\times\) 16.8 mL; Corning Inc.-Life Sciences, Tewksbury, MA, USA) according to the modified methods described previously (2017). This assay was also performed with juice agar (JA) containing apple or  
\begin{table}
\begin{tabular}{c c c} \hline
**Fungi** & **Strain Characteristics** & **Source** \\ \hline _P. expansum_ FR3 & FLU resistant; derived from W2 & WSU, (2017) \\ _Neosartorqa fischeri_ 96468 & Food spoilage fungus; heat resistant & ATCC \\ _Candidida albicans_ 10231 & Human pathogen (candidiasis) & ATCC \\ \hline
**Bacteria** & & \\ _Staphylococcus aureus_ 6538 & Food pathogen & ATCC \\ _Listeria monocytogenes_ RM2194 & Food pathogen & USDA \\ _Lactobacillus acidophilus_ 43560 & Commensal & ATCC \\ _Lactobacillus rhamnosus_ 53103 & Commensal & ATCC \\ _Lactobacillus reuteri_ 23272 & Commensal & ATCC \\ _Escherichia coli_ K-12 MG 1655 & Laboratory strain (Stand-in for _E. coli_ pathogens) & USDA \\ _Salmonella enterica_ PGFP & Food pathogen & USDA \\ \hline \end{tabular}  
* FLU, Fludioxonil; MAPK, Mitogen-Activated Protein Kinase; ATCC, American Type Culture Collection, Manassas, VA, USA; NRRL, National Center for Agricultural Utilization and Research, USDA-ARS, Peoria, IL, USA; USDA, United States Department of Agriculture, Albany, CA, USA; UT, The University of Texas, MD Andersen Cancer Center, Houston, TX, USA; WSU, Washington State University, Wenatchee, WA, USA.  
\end{table}
Table 1: Cont.  
-grape juice (70%, final concentration) w/1.5% select agar base. Organically produced white grape (pH 3.08) and honey crisp apple (pH 3.48) juices were purchased from local grocery stores (Berkeley, CA, USA). Fungal inoculum (1 \(\times\) 10\({}^{3}\) CFU in 20 \(\upmu\)L) was applied to a blank BD Taxo disc (6 mm; BD Life Sciences Franklin Lakes, NJ, USA) at the center of each agar well. Fungal growth (% radial growth) was monitored for five to seven days at 35 \({}^{\circ}\)C, except for _P. expansum_ (28 \({}^{\circ}\)C).