Herbicide Metabolism Inhibition by NBD-Cl

To verify the contribution of GSTs toward S-metolachlor resistance in A. palmeri, three populations (14CRI-G, 15CRI-A and SS) were tested in an agar-based plate assay with a GST inhibitor. The growth medium was prepared by dissolving 2.2 g of Murashige and Skoog basal salt mix (PhytoTech Labs Inc., Lenexa, KS) and 4 g of agar (Himedia Labs, West Chester, PA) in 500 mL deionized water. The pH was adjusted to 6.3 and the mixture was autoclaved. Analytical grade S-metolachlor (Sigma-Aldrich Inc., St. Louis, MO) and the GST inhibitor 4-chloro-7-nitrobenzofurazan (NBD-Cl; Sigma-Aldrich) were dissolved and diluted in chloroform. Work solutions were prepared through serial dilutions to achieve S-metolachlor concentrations of 0, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, and 8 μM, and NBD-Cl concentrations of 0 and 25 μM in the growth medium. Herbicide and inhibitor rates were chosen based on preliminary experiments (data not shown). A final chloroform concentration of 0.14% was kept constant in all plates by adding 24 μL of each work solution to 30 mL of growth medium to each plate. The plates were sterile, square, gridded Petri dishes (100 × 100 × 15 mm, 13-mm grids, Simport Scientific Inc., Beloeil, QB, Canada). Each plate had 30 seeds. The plates were sealed with Parafilm tape (Bemis Company Inc, Neenah, WI) and kept in a growth chamber at 30/28°C day/night temperature, and 16-h daylight. At 14 DAT, the plates were photographed and root lengths were measured using ImageJ (Schneider et al., 2012). The square grids were used as scale for unit conversion. From each plate, 10–15 representative roots were measured, which were considered biological replicates. The experiment was repeated and data from both runs were pooled as results across runs did not differ statistically. Root length data were fitted to a four-parameter, log logistic model using the package drc in R 4.0.3, as defined previously in Eq. 1. To determine the sole effect of NBD-Cl in the absence of herbicide, a subset of the data was submitted to ANOVA and means were compared using a Tukey's HSD test in the multcomp package in R.