Detecting the effect of ACCase-targeting herbicides on ACCase activity utilizing a malachite green colorimetric functional assay

Abstract

Research was conducted to evaluate acetyl-Coenzyme A carboxylase (ACCase) enzyme activity using a functional malachite green colorimetric assay previously identified as resistant to sethoxydim, and select aryloxyphenoxypropionate (FOPs) herbicides, fenoxaprop, and fluazifop. Two resistant southern crabgrass [Digitaria ciliaris (Retz.) Koeler] biotypes, R1 and R2, containing an Ile-1781-Leu amino acid substitution and previously identified as resistant to sethoxydim, pinoxaden, and fluazifop but not clethodim was utilized as the resistant chloroplastic ACCase source compared to known susceptible (S) ACCase. Dose-response studies with sethoxydim, clethodim, fluazifop-p-butyl, and pinoxaden (0.6 to 40 µM) were conducted to compare the ACCase enzyme-herbicides interaction of R1, R2, and S using the malachite green functional assay. Assay results indicated that R biotypes required more ACCase-targeting herbicides to inhibit ACCase activity compared to S. IC50 values of all four herbicides for R biotypes were consistently an order of magnitude greater than S. No sequencing differences in the carboxyltransferase domain was observed for R1 and R2, however, R2 IC50 values were greater across all herbicides. These results indicate the malachite green functional assay is effective in evaluating ACCase enzyme activity of R and S biotypes in the presence of ACCase-targeting herbicides, which can be used as a replacement for the 14C-based radiometric functional assay.

Discrimination of ACCase-inhibiting herbicides-resistant Digitaria ciliaris populations with three diagnostic bioassays

“…Clethodim was 17 and 4 times more leaching electrolyte to S than R2 and R1, respectively. Previous research also reported the R1 and R2 biotypes had differential resistance to clethodim when foliar was applied (Yu et al, 2017) and comparatively lower ACCase enzyme activity to clethodim than the other inhibitors sethoxydim, pinoxaden, and fluazifop (Basak et al, 2019;Basak et al, 2021). Using the electrical conductivity assay, the viability of seeds from a variety of crops, including Brassica spp.…”

“…Experiments were conducted in the Department of Crop, Soil and Environmental Sciences, Auburn, AL, USA. Two resistant biotypes (R1 and R2) of D. ciliaris with confirmed resistance to select ACCase herbicides (Yu et al, 2017;Basak et al, 2019;Basak et al, 2021), and one susceptible biotype (S), a total of 360 plantlets from each biotype was included in this study. Seeds of the test plant D. ciliaris were grown in separate plastic flats containing commercial potting soil and peat moss (2:1 v/v) under greenhouse conditions.…”