**4.** *In situ* **antifungal activity**

112 Fungicides for Plant and Animal Diseases

Potato dextrose agar (PDA) medium normally used by the scientist to maintained fungal isolates and consist of extract of boiled potatoes, 200 mL; dextrose, 20 g; agar, 20 g; deionized

broth (PDB) to a concentration of 107 spores per mL. The spore population needed can be counted using a haemocytometer. Subsequent dilutions can be made from the aforementioned suspension to adjust to the required concentration, which can be used in the antifungal test.

Screening for antifungal effect can be carried out by using the disc diffusion method. The plate containing 25 mL of PDA medium will be seeded with 1 mL of fungal conidial spore suspension containing 105 spores per mL from a 120-h-old culture. Three Whatman filter paper No. 1 discs of 6-mm diameter can be used to screen the antifungal activity. Each sterile disk will be impregnated with 20 mL of the extract corresponding with 100 mg/mL of crude extract, myconazole 30 g/mL, as positive control, or vehicle as negative control. The plates will be refrigerated for 2 h to allow the compounds presents in the extract diffused

measured, and the mean of the three replicates are taken (Bauer et al., 1966). The disc diffusion method is a qualitative test which could provide the information whether the

The minimal inhibitory concentration (MIC) can be determined as the lowest concentration at which no growth occurs and is determined as follows: PDB medium will be prepared and sterilized in universal bottles, each containing 10 mL medium. Different amounts of the tested extract will be added to the broth medium to give the following concentration: 0.3125 to 100 mg/mL. To each flask 0.5 mL of Tween-80 will be added as emulsifying agent. The flasks will be inoculated with 0.5 mL fungal conidial spore suspension containing 105 spores

determined as the lowest concentration of plant extract in the broth medium that inhibited visible growth of the test fungal strains. Each assay should be carried out in triplicate. The

The hyphal growth inhibition test can be used to determine the antifungal activity of the plant extract against fungal strains as previously described Picman et al. (1990). Briefly, dilutions of

concentrations of 100, 10, 1, 0.8, 0.6, 0.4, 0.2, and 0.1 mg/mL of plants extracts. The resultant solution will be thoroughly mixed and approximately 15 mL will be poured onto the petri plate. Plugs of 1 mm of fungal mycelium cut from the edge of actively growing colonies will be inoculated in the center of the agar plate and then incubated in a humid chamber at 25

Control cultures will be received an equivalent amount of vehicle. Three replicates will be used for each concentration. Radial growth is measured when the control colonies almost

the test solutions dissolved in vehicle will be added to sterile melted PDA at 45

C. Spore suspensions can be prepared and diluted in sterile potato dextrose

C for 5 days. Diameter of the inhibition zone will be

C for 5 days. The MIC value is

C to give final

C.

**3.** *In vitro* **antifungal testing** 

**3.2 Screening for the antifungal effect** 

and then will be incubated at 28

crude extract possessed antifungal properties

**3.3 Determination of the minimal inhibitory concentration** 

per mL from a 120-h-old culture and will be incubated at 28

**3.4 Determination of minimum fungicidal concentration** 

MIC test will be quantified the antifungal activity of plant extract.

**3.1 Microbial strain** 

water, 800 mL at 28

Electron microscopy (EM) is one of the many methods available for visual inspection of fungal strains. The effects of potential antifungal extracts from natural sources can also be evaluated by using the EM methods. Hence in this section the microscopical techniques such as Scanning (SEM) and Transmission (TEM) electron microscopy on the *in situ* antifungal activity by plant extract will be discussed.
