Agar diffusion method for antifungal activity evaluation

The antifungal effects of the prepared extracts were tested against A. alternata, B. cinerea, F. oxysporum, A. niger, C. gloeosporioides, C. cladosporioides and G. candidum. Potato dextrose agar (PDA) (Difco-USA) plates of 20 mg/ml of aqueous or ethanolic extracts of P. juliflora leaves or fruits were prepared by adding the appropriate volume of stock solutions previously prepared to 50 °C PDA media²⁶, both ethanolic extract stock solutions dissolved in sterile distilled water and in 4% DMSO were tested. Once the media solidified, a plug of 6 mm of agar was aseptically removed from the center of each Petri dish. Plugs of four-day-old fungi culture of the same size were transferred aseptically to the experimental plate. The experiment was run in four replications. Plates with 1 ml of Clotrimazole per plates (10 mg/ml) were used as a positive control. Pure PDA plates and PDA plates with 4% DMSO were also inoculated and used as a negative control. All plates were incubated at 25 °C for 5 days. Percent inhibition of mycelial growth (PIMG) was then calculated using the formula below. Mean diameter mycelial growth was determined by measuring the diameter of the infected area in two perpendicular directions³³.

dc and dt are the average mycelium diameter in the negative control plate and in the treated plate, respectively⁵.