*2.12. Antibacterial Activity of Mycosynthesized ZnO NPs against Xanthomonas oryzae*

A disc diffusion method was carried out to assess the presence of antibacterial activity of ZnO NPs. A bacterial culture of 0.5 McFarland standard was used to lawn Muller Hinton agar plates evenly using a sterile swab. The discs loaded with ZnO NPs 1 mg/mL (PGT4, PGT5, PGT13 and PGTA) were placed on the Mueller Hinton agar plates. Each test plate had a positive control, a negative control and four treated ZnO NP (PGT4, PGT5, PGT13 and PGTA) discs. The standard tetracycline (0.5 mg/mL) was used as a positive control, and the negative control used was distilled water. The plates were incubated at 28 ± 2 ◦C for 24 h. After the incubation, the plates were examined and results were recorded in mm [47].

The synthesized ZnO NPs were subjected for antibacterial activity by minimum inhibitory concentration (MIC), which was determined using a broth microdilution method with minor modifications [30]. The desired different concentrations in 100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL and 6.25 µg/mL was obtained by diluting the ZnO NP stock solution (1 mg/mL) with dilution in Mueller Hinton Broth (MHB) medium, which was added to a 96-well sterile microtiter plate. The bacterial suspension measuring 10 µL was added to each well, which were then incubated for 24 h at 28 ◦C. MHB served as a negative control, the positive control being the tetracycline at the concentration of 100 µg/mL; all these tests were conducted in triplicates. After 24 h of incubation, the addition of 20 µL of iodonitrotetrazolium chloride dye (INT) (0.5 µg/mL) to each well determined the MIC values of the synthesized ZnO NPs. The microtiter plates were incubated for 60 min at 28 ◦C. MICs determine the lowest concentration of the drug that prevents the change of color from being colorless to red, where colorless tetrazolium salt acts as an electron acceptor and gets reduced to a red-colored formazan product by biologically active organisms [30].
