Nematicidal toxicity bioassay

Nematodes used in these bioassays (Acrobeloides spp. and Rhabditis spp.) were isolated from the soil samples, and cultured on NGM (nematode growth medium) plates according to Ferris⁵⁴. NGM plates were made as following steps: 3 g NaC1, 2.5 g Bactopeptone (Difco) and 17 g Bacto-agar (Difco) were dissolved in 975 ml distilled water; after autoclaving, 1 ml cholesterol in ethanol (5 mg/ml), 1 ml M CaCl₂, 1 ml M MgSO₄ and 25 ml M potassium phosphate buffer (pH 6.0) are added in order⁵⁵.

Range-finding studies were run to determine the appropriate testing concentrations. A serial dilution of each of the four phenolic acids (five concentrations each) was prepared in distilled H₂O. Nematode suspensions (20 μl) containing 100 juveniles were transferred to vials to which 980 μl of the phenolic acid solution was added. The vials were stored at 25 °C. Dead nematodes were counted daily for 72 h. After the last count, the inactive juveniles were maintained in distilled H₂O for 24 h to test for their revival. Six repetitions for each treatment were performed using distilled H₂O as control. Each entire experiment was conducted three times.