Skimmed milk agar

A. alternata was tested for extracellular enzyme activities in the chromogenic media described by Kwon et al. (2007) [24 (link)] with some modifications. Preculturing of A. alternata was done on PDA at 28 °C for 7 days. To detect fungal extracellular enzyme activity, the precultures were transferred onto a medium containing Skimmed Milk Agar (SMA) for the protease activity [25 (link)], starch from potato (Sigma Aldrich, St. Louis, MO, USA) for amylase activity [26 ], 0.5% xylan (Megazyme) for xylanase activity [27 (link)], and 0.5% carboxymethylcellulose (CMC) (Sigma-Aldrich, USA) for cellulase activity [28 ,29 (link)]. Congo red dye or iodine solution was used for the chromogenic reaction to detect the enzymatic activities. After 5 days of incubation at 28 °C, a halo around the fungal plug was observed and measured, indicative of the hydrolytic activities. Each test was conducted in triplicate and repeated three times. The halo measurement was expressed as the mean of the obtained values.

Overnight cultures were grown to 0.7 (OD₆₀₀) in TSB and were spotted onto 10% skimmed milk agar (Sigma-Aldrich, St. Louis, MO, United States). Plates were incubated at 37°C for 24–48 h and lysis halos were measured from the border of the spotted bacterial growth until the border of the halo. The strains were categorized into three percentiles (high, medium, and low) depending on the size of the halos produced.