2.4.1. Agar Well Diffusion Assay

The anti-fungal activity of the ZnNPs was evaluated through agar well diffusion assay [33] involving estimation of the mycelial growth-inhibiting potential of the synthesized ZnNPs on the three test maize pathogenic fungal cultures [31]. The PDA media was poured in sterilized petri dishes (90 × 14 mm, Tarsons triple vent radiation sterile polystyrene, Code: 460091, Tarsons, Kolkata, India) and allowed to gel. The wells in the solidified media were prepared using a sterile cork borer (diameter 8.0 ± 0.2 mm, CAS No. LA737, Himedia Laboratories, Mumbai, India). The ZnNPs stock solutions were prepared in the HPLC grade water and these suspensions were bath sonicated for 30 min at room temperature. The stock solutions were then utilized for the preparation of the working concentrations (0, 5, 10, 20, and 40 mg L−<sup>1</sup> ). These suspensions were then given a quick bath sonication treatment for another five minutes and 20 µL of the suspensions were loaded in the agar wells. The respective fungal growth on PDA media served as the negative control. The fungal disc (8.0 mm diameter) of the freshly grown confluent growth (one-week old culture plate) was placed at the center of each plate and the inoculated petri plates were incubated in a BOD incubator at 27 ± 2 ◦C for seven days. The diameter of the zone of inhibition of the mycelial growth at or near the well containing the ZnNPs was taken as indicator of the decreased mycelial growth.
