SummaryRapid detoxification of atrazine in naturally tolerant crops such as maize (Zea mays) and grain sorghum (Sorghum bicolor) results from glutathione S‐transferase (GST) activity. In previous research, two atrazine‐resistant waterhemp (Amaranthus tuberculatus) populations from Illinois, U.S.A. (designated ACR and MCR), displayed rapid formation of atrazine‐glutathione (GSH) conjugates, implicating elevated rates of metabolism as the resistance mechanism. Our main objective was to utilize protein purification combined with qualitative proteomics to investigate the hypothesis that enhanced atrazine detoxification, catalysed by distinct GSTs, confers resistance in ACR and MCR. Additionally, candidate AtuGST expression was analysed in an F2 population segregating for atrazine resistance. ACR and MCR showed higher specific activities towards atrazine in partially purified ammonium sulphate and GSH affinity‐purified fractions compared to an atrazine‐sensitive population (WCS). One‐dimensional electrophoresis of these fractions displayed an approximate 26‐kDa band, typical of GST subunits. Several phi‐ and tau‐class GSTs were identified by LC‐MS/MS from each population, based on peptide similarity with GSTs from Arabidopsis. Elevated constitutive expression of one phi‐class GST, named AtuGSTF2, correlated strongly with atrazine resistance in ACR and MCR and segregating F2 population. These results indicate that AtuGSTF2 may be linked to a metabolic mechanism that confers atrazine resistance in ACR and MCR.

To date, the only known mechanism conferring protoporphyrinogen IX oxidase (PPO)-inhibitor resistance in waterhemp ( Amaranthus tuberculatus ) is a glycine deletion in PPO2 (ΔG210), which results in cross-resistance to foliar PPO-inhibiting herbicides. However, a metabolism-based, HPPD-inhibitor resistant waterhemp population from Illinois (named SIR) was suspected of having a non-target site resistance (NTSR) mechanism due to its resistance to carfentrazone-ethyl (CE) but sensitivity to diphenylethers (DPEs). In greenhouse experiments, SIR sustained less injury than two PPO inhibitor-sensitive populations (WCS and SEN) after applying a field-use rate of CE, and after initial rapid necrosis, regrowth of SIR plants was comparable to a known PPO inhibitor-resistant population (ACR) possessing the ΔG210 mutation. Dose-response analysis determined 50% growth reduction rates in CE-resistant (SIR and ACR) and sensitive (SEN) waterhemp populations, which showed SIR was 30-fold resistant compared to SEN and two-fold more resistant than ACR. Deduced amino acid sequences derived from SIR PPX2 partial cDNAs did not contain the ΔG210 mutation found in ACR or other target-site mutations that confer PPO-inhibitor resistance previously reported in Palmer amaranth ( Amaranthus palmeri ). Although several SIR cDNAs contained amino acid substitutions, none were uniform among samples. Additionally, SIR plants treated with malathion and CE showed a significant reduction in biomass accumulation compared to CE alone. These results indicate robust CE resistance in SIR is not mediated by amino acid changes in the PPO2 protein, but instead resistance may be conferred through a NTSR mechanism such as enhanced herbicide metabolism.

Greenhouse experiments were conducted to quantify resistance levels to the 4-hydroxyphenyl-pyruvate dioxygenase (HPPD)-inhibiting herbicides mesotrione (MES) and isoxaflutole (IFT) in NEB (Nebraska HPPD- and atrazine-resistant) and SIR (Stanford, IL, HPPD- and atrazine-resistant) waterhemp [Amaranthus tuberculatus(Moq.) J. D. Sauer] populations. These populations differ in their field-use histories and resistance levels to MES. Foliar growth responses were compared with ACR (HPPD sensitive; metabolic atrazine-resistant) and SEN (sensitive to HPPD and photosystem II [PSII] inhibitors). A greenhouse dose–response study was conducted with each herbicide at two POST timings: early (EPOST) (5 cm; 4 to 5 true leaves) and POST (10 cm; 8 to 9 true leaves). At the EPOST timing, SIR was 10-fold resistant to IFT and 32-fold resistant to MES, while NEB was 4-fold resistant to IFT and 7-fold resistant to MES when compared with ACR. At the POST timing, SIR was 17-fold resistant to IFT and 21-fold resistant to MES, while NEB was 3-fold resistant to IFT and 7-fold resistant to MES when compared with ACR. Results overall indicated greater fold-resistance levels to MES relative to IFT at each timing. However, POST treatments to SIR showed contrasting effects on resistance levels relative to EPOST. To investigate potential management strategies for resistantA. tuberculatuspopulations, a POST interaction study was conducted using combinations of metribuzin and either IFT or MES. A metribuzin rate (191 g ai ha−1) causing an approximately 20% biomass reduction was chosen for interaction studies and combined with varying rates of either IFT or MES. Results indicated 52.5 g ai ha−1of MES combined with metribuzin displayed a synergistic effect on biomass reduction in SIR. However, other combinations of either MES or IFT and metribuzin resulted in additive effects on biomass reduction in both HPPD-resistant populations. These results provide insights into the joint activity between HPPD and PSII inhibitors for controlling metabolism-based, multiple herbicide–resistantA. tuberculatus.

Metabolic resistances to atrazine (atz-R) and mesotrione (meso-R) occur in several waterhemp [Amaranthus tuberculatus (Moq.) Sauer] populations in the United States. Interestingly, although metabolic atz-R but mesotrione-sensitive A. tuberculatus populations have been reported, an Amaranthus population has not been confirmed as meso-R but atrazine-sensitive, implying an association between these traits. Experiments were designed to investigate whether the single gene conferring metabolic atz-R plays a role in meso-R. An F2 population was generated from a multiple herbicide–resistant A. tuberculatus population from McLean County, IL (MCR). A cross was made between a known meso-R male clone (MCR-6) and a herbicide-sensitive female clone from Wayne County, IL (WCS-2) to develop an F1 population. Survival of MCR-6 plants following atrazine POST treatment (14.4 kg ha−1) indicated the male parent was homozygous atz-R. F1 plants were intermated to obtain a segregating pseudo-F2 population. Dose–response and metabolic studies conducted with mesotrione using F1 plants indicated intermediate biomass reductions and metabolic rates compared with MCR-6 and WCS. F2 plants were initially treated with either mesotrione (260 g ha−1) or atrazine (2 kg ha−1) POST, and after 21 d of recovery, vegetative clones from surviving resistant plants were subsequently treated with the other herbicide. When mesotrione was applied first, the meso-R frequency was 8.2%, and when atrazine was applied first, the atz-R frequency was 75%. However, the meso-R frequency increased to 16.5% following preselection for atz-R, and 100% of surviving meso-R plants were atz-R. Our findings indicate that the gene conferring metabolic atz-R is also involved with the meso-R trait within the population tested.