4.8. Fungal Growth Inhibition Bioassays

The purified and refolded PcOSM was assayed for its ability to inhibit mycelial growth of Phytophthora capsici oomycete by in vivo leaf infiltration method. In total, 100 µL of PcOSM in 20 mM Tris Cl at concentrations 100 and 200 µg/mL were infiltrated into the basal side of Piper nigrum leaves. Leaves were previously surface sterilized using 0.1% HgCl₂ followed by rinsing in sterile water. Infiltration was carried out using needless syringe [48].

After 1 h of PcOSM infiltration, infection was initiated by placing a P. capsici plug isolated from a fresh plate of P. capsici maintained in sterile potato dextrose agar (PDA) medium, following established protocol [6]. The plugs were placed on the abaxial surface of the leaves and leaves were placed in a moist chamber to obtain high relative humidity. After 24 h of incubation, leaf discs were separated for P. capsici staining.

For Trypan blue staining of fungal hyphae, leaf discs were placed in a 12-well plate containing solution A (Acetic acid: ethanol (1:3 v/v) and incubated at room temperature in a rocker overnight. After incubation, solution A was removed and solution B (acetic acid:ethanol:glycerol (1:5:1) was added and incubated for 3 h to remove chlorophyll followed by overnight staining in 0.1% Trypan blue. After destaining with 60% glycerol for 3–6 h, the leaf discs were mounted and visualized in a Nikon Eclipse Ni microscope, Japan.