Potato dextrose agar (pda)

For the antifungal activity analysis, dual culture assays were carried out. For the strain’s activation, CMRP 4490 was cultured in LBA (Luria Bertani Agar, Neogen Corporation, United States) at 28°C for 24 h. Then, CMRP 4490 was inoculated, using an inoculation loop, at four points at the edges of plates containing PDA medium (Potato Dextrose Agar, Neogen Corporation, United States). On the center of the Petri dish, a 6-mm mycelial plug taken from the edge of actively growing colonies of three phytopathogenic fungi (Sclerotinia sclerotiorum, Macrophomina phaseolina, Botrytis cinerea, and Rhizoctonia solani) was placed. The experiment was incubated at 25°C with a 12-h/12-h photoperiod for 7 days. For comparison purposes, positive control was performed with only the mycelial disc at the center of the Petri dish incubated under the same conditions. Growth was calculated as the average of the two orthogonal diameters (mm), and growth inhibition was determined using the following formula:
where cd = diameter in control, td = diameter in treatment, and MGI = mycelial growth inhibition.

Isolates of Sclerotinia sclerotiorum, Macrophomina phaseolina, Fusarium oxysporum, and B. cinerea were provided by Dra. Maria Isabel Balbi-Peña of the Plant Pathology Laboratory of the Department of Agronomy of the State University of Londrina (UEL). Dr. Artur Soares (Simbiose-Agro, Cruz Alta, RS, Brazil) provided an isolate of Colletotrichum truncatum. The isolates were grown on potato dextrose agar (PDA) (Neogen Corporation, Lansing, MI, USA) in 90 mm × 10 mm polystyrene plates and incubated for 7 days at 25 ± 1 °C, with 12 h of fluorescent light and 12 h of darkness. All isolates were deposited in the microbial culture collection of the Laboratory of Microbial Biotechnology—LABIM, UEL, Londrina, Brazil.

The bacterial strains Enterococcus faecalis, Bacillus subtillus, Bacillus cereus, Staphylococcus aureus, Vibrio fluvialis, Vibrio damsela, Pseudomonas aeruginosa, and Salmonella typhimurium; and the fungal strains Aspergillus fumigatus, Aspergillus terreus, Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, and Penicillium oxalicum used in this work were provided by the Department of Microbiology, National Institute of Oceanography and Fishers, Red Sea branch, Egypt. These strains were isolated from marine sources and identified by Dr. Moaz M. Hamed. The strains were kept at 2 °C on nutrient agar slants for bacteria and Potato Dextrose Agar (PDA) (Neogen Corporation, Lansing, MI, USA) for fungi slants. The slants were folded with 25% glycerol to ensure long-term preservation.

C. albicans SC5314 and C. albicans SC5314 carrying the green fluorescence protein (GFP) reporter gene (C. albicans–GFP) [28 (link)] kindly provided by J. Berman (Tel Aviv University, Israel), were grown for 24–48 h at 30 °C on Potato Dextrose Agar (PDA) (Neogen, Lansing, MI) plates. The yeast cells were resuspended at an OD₆₀₀ = 0.05 in RPMI medium (Sigma-Aldrich, St. Louis, MO, USA) and used for biofilm assays by incubation at 37 °C. Control samples were treated with corresponding ethanol concentrations. Blank samples were CBD in RPMI in the absence of fungi.