4.6.2. Determination of ZnO-NPs’ Nematicidal Activity by Mortality Assay of Meloidogyne incognita

Meloidogyne incognita pure culture was originated from the single egg mass of an identified M. incognita female. The soil surrounding the root of a tomato infected for 45 days was used as a source of root-knot nematodes (RKNs). The infective second-stage juveniles (J₂s) of M. incognita were isolated using the sieving and decanting procedure [100]. The nematicidal activity of ZnO-NP nanofluid against M. incognita J₂s was tested in vitro using a mortality assay after 24, 48 and 72 h of exposure. In sterile test tubes, a stock solution of ZnO-NP nanofluid was diluted with sterile distilled water to reach a final concentration of 100 µg/mL of ZnO-NPs and a final volume of 5 mL, with 100 ± 5 J₂s of M. incognita. In addition, a treatment that contained the same volume of base nanofluid without the ZnO-NPs and an autogenic control that contained only sterile distilled water, both including 100 ± 5 J₂s of M. incognita, were also tested. Treatments and controls were performed in ten replicates and all tubes were maintained in an incubator at 30 °C. The count of live and mortal juveniles after each time interval was estimated using a one mL counting slide. The mobility of nematodes or their winding shape indicated their vitality, while immobility indicated mortality. The mortality percentage was estimated as [(number of live nematode larvae in control treatment) − (number of live nematode larvae counted in other treatments)/(number of live nematode larvae in control treatment)] × 100.