The presence of glyphosate-resistant smooth pigweed (Amaranthus hybridus L.) biotypes has increased in southern Brazil in the last years, presenting the triple amino acid substitution TAP-IVS in the 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), as previously found in Argentina. Some of these biotypes have morphological characteristics of smooth pigweed and redroot amaranth (Amaranthus retroflexus L.). The present study aimed to identify, through molecular markers, the herbicide-resistant species of Amaranthus from Brazil that have the TAP-IVS substitution and to analyze the occurrence of pollen-mediated gene flow (PMGF) as the source of TAP-IVS substitution in these biotypes. Six biotypes were evaluated by the internal transcribed spacer (ITS) sequences, of which two (AMACHY-S and CAMAQ-R) were molecularly classified as A. hybridus, and four (AMACRET-S, AMACVI-S, ARRGR-R, and SAOJER-R) were unclassified. Interestingly, all the glyphosate-resistant biotypes (ARRGR-R, SAOJER-R, and CAMAQ-R) had the TAP-IVS substitution, and an increase in EPSPS relative copy number; however, only CAMAQ-R was confirmed as A. hybridus. Although the biotypes ARRGR-R and SAOJER-R are closely related to A. hybridus and green pigweed (Amaranthus powellii S. Watson), their species identity could not be resolved. The biotype SAOJER-R also was resistant to ALS-inhibiting herbicides due to a tryptophan to leucine substitution at position 574 in acetolactate synthase (ALS). The evaluation of 119,746 seedlings in an intraspecific hybridization study of A. hybridus indicated an outcrossing frequency of 0.09%. In contrast, an absence of interspecific hybridization (A. hybridus × unclassified biotype – AMACVI-S) was found after screening 111,429 offspring. Unclassified biotypes might be derived from one or more ancient hybridization events and subsequently evolved the triple mutation independently. Alternatively, such biotypes could have evolved from recent hybridization events, which occur at a frequency below the level of detection in our study.

Protoporphyrinogen oxidase (PPO)-inhibiting herbicides remain an important and useful chemistry 60 yr after their first introduction. In this review, based on topics introduced at the Weed Science Society of America 2021 symposium titled “A History, Overview, and Plan of Action on PPO Inhibiting Herbicides,” we discuss the current state of PPO-inhibiting herbicides. Renewed interest in the PPO-inhibiting herbicides in recent years, due to increased use and increased cases of resistance, has led to refinements in knowledge regarding the mechanism of action of PPO inhibitors. Herein we discuss the importance of the two isoforms of PPO in plants, compile a current knowledge of target-site resistance mechanisms, examine non–target site resistance cases, and review crop selectivity mechanisms. Consistent and reproducible greenhouse screening and target-site mutation assays are necessary to effectively study and compare PPO-inhibitor resistance cases. To this end, we cover best practices in screening to accurately identify resistance ratios and properly interpret common screens for point mutations. The future of effective and sustainable PPO-inhibitor use relies on development of new chemistries that maintain activity on resistant biotypes and the promotion of responsible stewardship of PPO inhibitors both new and old. We present the biorational design of the new PPO inhibitor trifludimoxazin to highlight the future of PPO-inhibitor development and discuss the elements of sustainable weed control programs using PPO inhibitors, as well as how responsible stewardship can be incentivized. The sustained use of PPO inhibitors in future agriculture relies on the effective and timely communication from mode of action and resistance research to agronomists, Extension workers, and farmers.

Comparing fitness of herbicide-resistant and herbicide-susceptible weed biotypes is important for managing herbicide resistance. Previous research suggests there is little to no fitness penalty from amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (a mechanism of glyphosate resistance) in Palmer amaranth (Amaranthus palmeri S. Watson) in controlled studies in the greenhouse or growth chamber. A field study was conducted in North Carolina at three locations naturally infested with A. palmeri to determine vegetative, reproductive, and germination fitness of plants with and without EPSPS amplification grown season-long with cotton (Gossypium hirsutum L.). Seed number was not correlated with EPSPS copy number. However, when plants were binned into two groups, those having an EPSPS copy number ≥2 (relative to reference genes) and those having an EPSPS copy number <2, plant fresh weight and seed number were 1.4 and 1.6 times greater, respectively, for plants with fewer than 2 EPSPS copies. Amaranthus palmeri height and seed germination, and yield of cotton, did not differ when comparing the two binned groups. These data suggest that A. palmeri plants with EPSPS amplification are relatively less fit in the absence of glyphosate, but this reduced fitness does not translate into differences in interference with cotton.

Owing to the lack of effective POST herbicide options, producers typically rely on nicosulfuron as the main POST grass herbicide in sweet corn production systems. In 2019, a Wisconsin sweet corn producer reported fall panicum control escapes after spraying nicosulfuron. Seeds from mature plants were collected to (1) measure fall panicum response to acetolactate synthase (ALS)-inhibiting herbicides, (2) elucidate the resistance mechanism, and (3) evaluate its response to alternative POST herbicides. Greenhouse and laboratory investigations were conducted to assess fall panicum response to ALS-inhibiting herbicides and elucidate the resistance mechanism. Dose–response results showed that fall panicum was highly resistant to nicosulfuron with a resistance ratio of >12.9-fold (survived rates >254 g ai ha−1, or 8× the field label rate). Molecular and genetic studies indicated that there are multiple ALS gene copies in fall panicum and that resistance was due to a mutation in one copy, resulting in an Asp-376-Glu amino acid substitution. Additional greenhouse experiments indicate that clethodim (105 g ai ha−1), quizalofop-p-ethyl (70 g ae ha−1), glyphosate (864 g ae ha−1), and glufosinate (650 g ai ha−1) are effective POST options to manage the ALS-resistant fall panicum (>90.0% control and 96.8% biomass reduction) in rotational years. The 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides isoxaflutole (105 g ai ha−1), mesotrione (105 g ai ha−1), tembotrione (92 g ai ha−1), and tolpyralate (39 g ai ha−1) did not provide effective POST fall panicum control. Because these herbicides are commonly used for POST weed control in sweet corn, more investigations are required to evaluate combinations of HPPD-inhibiting herbicides with herbicides from other sites of action for POST fall panicum control. Herein we confirm the first case of herbicide resistance in fall panicum in the United States.

Waterhemp [Amaranthus tuberculatus (Moq.) Sauer] is one of the most troublesome agronomic weeds in the midwestern United States. The rapid evolution and selection of herbicide-resistance traits in A. tuberculatus is a major challenge in managing this species. An A. tuberculatus population, designated CHR, was identified in 2012 in Champaign County, IL, and previously characterized as resistant to herbicides from six site-of-action groups: 2,4-D (Group 4), acetolactate synthase inhibitors (Group 2), protoporphyrinogen oxidase inhibitors (Group 14), 4-hydroxyphenylpyruvate dioxygenase inhibitors (Group 27), photosystem II inhibitors (Group 5), and very-long-chain fatty-acid synthesis inhibitors (Group 15). Recently, ineffective control of CHR was observed in the field after dicamba application. Therefore, this research was initiated to confirm dicamba resistance, quantify the resistance level, and investigate its inheritance in CHR. Multiple field trials were conducted at the CHR location to confirm poor control with dicamba and compare dicamba treatments with other herbicides. Greenhouse trials were conducted to quantify the resistance level in CHR and confirm genetic inheritance of the resistance. In field trials, dicamba did not provide more than 65% control, while glyphosate and glufosinate provided at least 90% control. Multiple accessions were generated from controlled crosses and evaluated in greenhouse trials. Greenhouse dicamba dose–response experiments indicated a resistance level of 5- to 10-fold relative to a sensitive parental line. Dose–response experiments using F1 lines indicated that dicamba resistance was an incompletely dominant trait. Segregation analysis with F2 and backcross populations indicated that dicamba resistance had moderate heritability and was potentially a multigenic trait. Although dicamba resistance was predominantly inherited as a nuclear trait, minor maternal inheritance was not completely ruled out. To our knowledge, CHR is one of the first cases of dicamba resistance in A. tuberculatus. Further studies will focus on elucidating the genes involved in dicamba resistance.

Photosystem II (PS II)-inhibitor herbicide resistance in Ontario waterhemp [Amaranthus tuberculatus (Moq.) Sauer] populations is conferred via target-site resistance (TSR) and non–target site resistance (NTSR) mechanisms. Metribuzin-resistant (MR) A. tuberculatus is due to TSR. Conversely, in other populations of PS II–inhibitor resistant A. tuberculatus, plants are resistant to atrazine but metribuzin sensitive (MS). The objective of this study was to determine the biologically effective dose of metribuzin applied preemergence and postemergence for the control of MS and MR A. tuberculatus. Ten field experiments were conducted in 2019 and 2020 to determine the effective doses of metribuzin for 50%, 80%, and 95% control of MS and MR A. tuberculatus. Metribuzin applied preemergence at the calculated doses of 133, 350, and 1,070 g ai ha−1 controlled MS A. tuberculatus 50%, 80%, and 95%, respectively, whereas the calculated doses of 7,868 and 17,533 g ai ha−1 controlled MR A. tuberculatus 50% and 80%, respectively, at 12 wk after application (WAA). Metribuzin applied postemergence at the calculated doses of 245 and 1,480 g ai ha−1 controlled MS A. tuberculatus 50% and 80%, respectively; the calculated dose for 50% MR A. tuberculatus control was greater than the highest dose (17,920 g ai ha−1) included in this study. Metribuzin at 560 and 1,120 g ha−1 and pyroxasulfone/flumioxazin (240 g ai ha−1) applied preemergence controlled MS A. tuberculatus 88%, 95%, and 98%, respectively, at 12 WAA, whereas the same treatments only controlled MR A. tuberculatus 0%, 4%, and 93%, respectively, at 12 WAA. Metribuzin at 560 and 1,120 g ha−1 and fomesafen (240 g ai ha−1) applied postemergence controlled MS A. tuberculatus 65%, 70%, and 78%, and MR A. tuberculatus 0%, 1%, and 49%, respectively, at 12 WAA. Based on these results, PS II–inhibitor resistant A. tuberculatus with NTSR (enhanced metabolism) is controlled with metribuzin applied preemergence and postemergence; in contrast, PS II–inhibitor resistant A. tuberculatus with TSR (glycine-264-serine altered target site) is not controlled with metribuzin.

During the 2017 to 2019 growing seasons, samples of waterhemp and Palmer amaranth that had reportedly survived field-rate applications of protoporphyrinogen oxidase (PPO)–inhibiting herbicides were collected from the American Midwest and tested for target-site mutations known at the time to confer resistance. Target-site resistance was identified in nearly all (135 of 145) tested common waterhemp populations but in only 8 of 13 Palmer amaranth populations. Follow-up research on one population of Palmer amaranth (W-8), which tested negative for all such mutations, confirmed it was resistant to lactofen, with a magnitude of resistance comparable to that conferred by the ΔG210 PPO2 mutation. Gene sequences from both isoforms of PPO (PPO1 and PPO2) were compared between W-8 and known PPO inhibitor–sensitive sequence. A glycine-to-alanine substitution at the 399th amino acid position (G399A) of PPO2, recently identified to reduce target-site herbicide sensitivity, was observed in a subset of resistant W-8 plants. Because no missense mutation completely delimited resistant and sensitive sequences, we initially suspected the presence of a secondary, non-target-site resistance mechanism in this population. To isolate G399A, a segregating F2 population was produced and screened with a delimiting rate of lactofen. χ2 goodness-of-fit analysis of dead/alive ratings indicated single-locus inheritance of resistance in the F2 population, and molecular markers for the W-8 parental PPO2 coding region co-segregated tightly, but not perfectly, with resistance. More research is needed to fully characterize Palmer amaranth PPO inhibitor–resistance mechanisms, which appear to be more diverse than those found in common waterhemp.

Failure to control Palmer amaranth with glyphosate and protoporphyrinogen IX oxidase (PPO)-inhibitor herbicides was reported across southwestern Nebraska in 2017. The objectives of this study were to 1) confirm and 2) validate glyphosate and PPO-inhibitor (fomesafen and lactofen) resistance in 51 Palmer amaranth accessions from southwestern Nebraska using genotypic and whole-plant phenotypic assay correlations and cluster analysis, and 3) determine which agronomic practices might be influencing glyphosate resistance in Palmer amaranth accessions in that location. Based on genotypic assay, 88% of 51 accessions contained at least one individual with amplification (>2 copies) of the 5-enolypyruvyl-shikimate-3-phosphate synthase (EPSPS) gene, which confers glyphosate resistance; and/or a mutation in the PPX2 gene, either ΔG210 or R128G, which endows PPO-inhibitor resistance in Palmer amaranth. Cluster analysis and high correlation (0.83) between genotypic and phenotypic assays demonstrated that EPSPS gene amplification is the main glyphosate resistance mechanism in Palmer amaranth accessions from southwestern Nebraska. In contrast, there was poor association between genotypic and phenotypic responses for PPO-inhibitor resistance, which was attributed to segregation for PPO-inhibitor resistance within these accessions and/or the methodology that was adopted herein. Genotypic assays can expedite the process of confirming known glyphosate and PPO-inhibitor resistance mechanisms in Palmer amaranth from southwestern Nebraska and other locations. Phenotypic assays are also a robust method for confirming glyphosate resistance but not necessarily PPO-inhibitor resistance in Palmer amaranth. Moreover, random forest analysis of glyphosate resistance in Palmer amaranth indicated that EPSPS gene amplification, county, and current and previous crops are the main factors influencing glyphosate resistance within that geographic area. Most glyphosate-susceptible Palmer amaranth accessions were found in a few counties in areas with high crop diversity. Results presented here confirm the spread of glyphosate resistance and PPO-inhibitor resistance in Palmer amaranth accessions from southwestern Nebraska and demonstrate that less diverse cropping systems are an important driver of herbicide resistance evolution in Palmer amaranth.

Resistance to protoporphyrinogen oxidase (PPO) inhibitors was first observed in waterhemp in 2001 and was conferred by the deletion of a glycine residue at the 210th position (ΔGly-210) of the PPO enzyme. PPO-inhibitor resistance in Palmer amaranth was first observed in 2011, 10 years later. The objectives of this study were to directly compare PPO inhibitor responses in plants of both species with or without the ΔGly-210 mutation. Using greenhouse experiments, early (EPOST) and late (LPOST) postemergence dose responses using lactofen and fomesafen, and preemergence (PRE) dose responses using fomesafen and flumioxazin, were obtained for a sensitive and resistant population each of waterhemp and Palmer amaranth. An additional spray study confirmed each sensitive population used in the dose responses was representative of its respective species, with regards to PPO-inhibitor sensitivity. When treated at either POST timing, Palmer amaranth was more tolerant than waterhemp, and the ΔGly-210 mutation provided greater resistance in Palmer amaranth (48-fold to >3,440-fold, depending on timing and herbicide) than in waterhemp (31-fold to 123-fold). The level of tolerance in Palmer amaranth was striking; the sensitive Palmer amaranth population treated LPOST survived as well or better than the resistant waterhemp population treated EPOST. With PRE applications, response differences both between species and between resistant and sensitive populations generally were less pronounced, relative to POST applications. Collectively, this research indicates Palmer amaranth tolerance to POST-applied PPO inhibitors could have initially slowed (relative to waterhemp) evolution of resistance to these herbicides, and resistant and sensitive populations of both species are more likely to be effectively controlled with PRE rather than POST applications.

The first case of evolved protoporphyrinogen oxidase (PPO)-inhibitor resistance was observed in 2001 in common waterhemp [Amaranthus tuberculatus (Moq.) Sauer var. rudis (Sauer) Costea and Tardif]. This resistance in A. tuberculatus is most commonly conferred by deletion of the amino acid glycine at the 210th position (ΔGly-210) of the PPO enzyme (PPO2) encoded by PPX2. In a field in Kentucky in 2015, inadequate control of Amaranthus plants was observed following application of a PPO inhibitor. Morphological observations indicated that survivors included both A. tuberculatus and Palmer amaranth (Amaranthus palmeri S. Watson). Research was conducted to confirm species identities and resistance and then to determine whether resistance evolved independently in the two species or via hybridization. Results from a quantitative PCR assay based on the ribosomal internal transcribed spacer confirmed that both A. tuberculatus and A. palmeri coexisted in the field. The mutation conferring ΔGly-210 in PPO2 was identified in both species; phylogenetic analysis of a region of PPX2, however, indicated that the mutation evolved independently in the two species. Genotyping of greenhouse-grown plants that survived lactofen indicated that all A. tuberculatus survivors, but only a third of A. palmeri survivors, contained the ΔGly-210 mutation. Consequently, A. palmeri plants were evaluated for the presence of an arginine to glycine or methionine substitution at position 128 of PPO2 (Arg-128-Gly and Arg-128-Met). The Arg-128-Gly substitution was found to account for resistance that was not accounted for by the ΔGly-210 mutation in plants from the A. palmeri population. Results from this study provide a modern-day example of both parallel and convergent evolution occurring within a single field.

Waterhemp [Amaranthus tuberculatus (Moq.) J. D. Sauer] and Palmer amaranth (Amaranthus palmeri S. Watson) are troublesome weeds of row-crop production in the United States. Their dioecious reproductive systems ensure outcrossing, facilitating rapid evolution and distribution of resistances to multiple herbicides. Little is known, however, about the genetic basis of dioecy in Amaranthus species. In this work, we use restriction site–associated DNA sequencing (RAD-Seq) to investigate the genetic basis of sex determination in A. tuberculatus and A. palmeri. For each species, approximately 200 plants of each sex were sampled and used to create RAD-Seq libraries. The resulting libraries were separately bar-coded and then pooled for sequencing with the Illumina platform, yielding millions of 64-bp reads. These reads were analyzed to identify sex-specific and sex-biased sequences. We identified 345 male-specific sequences from the A. palmeri data set and 2,754 male-specific sequences in A. tuberculatus. An unexpected 723 female-specific sequences were identified in a subset of the A. tuberculatus females; subsequent research, however, indicated female specificity of these markers was limited to the population from which they were identified. Primer sets designed to specifically amplify male-specific sequences were tested for accuracy on multiple, geographically distinct populations of A. tuberculatus and A. palmeri, as well as other Amaranthus species. Two primer sets for A. palmeri and four primer sets for A. tuberculatus were each able to distinguish between male and female plants with at least 95% accuracy. In the near term, sex-specific markers will be useful to the A. tuberculatus and A. palmeri research communities (e.g., to predict sex for crossing experiments). In the long-term, this research will provide the foundational tools for detailed investigations into the molecular biology and evolution of dioecy in weedy Amaranthus species.

In a predictable natural selection process, herbicides select for adaptive alleles that allow weed populations to survive. These resistance alleles may be available immediately from the standing genetic variation within the population or may arise from immigration via pollen or seeds from other populations. Moreover, because all populations are constantly generating new mutant genotypes by de novo mutations, resistant mutants may arise spontaneously in any herbicide-sensitive weed population. Recognizing that the relative contribution of each of these three sources of resistance alleles influences what strategies should be applied to counteract herbicide-resistance evolution, we aimed to add experimental information to the resistance evolutionary framework. Specifically, the objectives of this experiment were to determine the de novo mutation rate conferring herbicide resistance in a natural plant population and to test the hypothesis that the mutation rate increases when plants are stressed by sublethal herbicide exposure. We used grain amaranth (Amaranthus hypochondriacus L.) and resistance to acetolactate synthase (ALS)-inhibiting herbicides as a model system to discover spontaneous herbicide-resistant mutants. After screening 70.8 million plants, however, we detected no spontaneous resistant genotypes, indicating the probability of finding a spontaneous ALS-resistant mutant in a given sensitive population is lower than 1.4 × 10−8. This empirically determined upper limit is lower than expected from theoretical calculations based on previous studies. We found no evidence that herbicide stress increased the mutation rate, but were not able to robustly test this hypothesis. The results found in this study indicate that de novo mutations conferring herbicide resistance might occur at lower frequencies than previously expected.

Experiments were initiated to characterize a waterhemp population (CHR) discovered in a central Illinois corn field after it was not controlled by the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor topramezone. Field experiments conducted during 2014–2015 indicated that acetolactate synthase (ALS)-, protoporphyrinogen oxidase (PPO)-, photosystem II (PSII)-, and HPPD-inhibiting herbicides and the synthetic auxin 2,4-D did not control the CHR population. Laboratory experiments confirmed target site–based resistance mechanisms to ALS- and PPO-inhibiting herbicides. Herbicide doses required to reduce dry biomass 50% (GR50) were determined in greenhouse dose–response experiments, and indicated 16-fold resistance to the HPPD inhibitor mesotrione, 9.5-fold resistance to the synthetic auxin 2,4-D, and 252-fold resistance to the PSII inhibitor atrazine. Complementary results from field, laboratory, and greenhouse investigations indicate that the CHR population has evolved resistance to herbicides from five sites of action (SOAs): ALS-, PPO-, PSII-, and HPPD-inhibiting herbicides and 2,4-D. Herbicide use history for the field in which CHR was discovered indicates no previous use of 2,4-D.

Herbicide resistance within key driver weeds, such as common waterhemp [Amaranthus tuberculatus (Moq.) Sauer var. rudis (Sauer) Costea and Tardif ], constrains available management options for crop production. Routine surveillance for herbicide resistance provides a mechanism to monitor the development and spread of resistant populations over time. Furthermore, the identification and quantification of resistance mechanisms at the population level can provide information that helps growers develop effective management plans. Populations of Amaranthus spp., including A. tuberculatus, redroot pigweed (Amaranthus retroflexus L.), and Palmer amaranth (Amaranthus palmeri S. Watson), were collected from 51 fields in Ohio during the 2016 growing season. Twenty-four A. tuberculatus populations were screened for resistance to the herbicides lactofen, atrazine, and glyphosate. Phenotypically resistant plants were further investigated to determine the frequency of known resistance mechanisms. Resistance to lactofen was infrequently observed throughout the populations, with 8 of 22 populations exhibiting resistant plants. Within those eight resistant populations, the ΔG210 resistance mechanism was observed in 17 of 30 phenotypically resistant plants, and the remainder lacked all known resistance mechanisms. Resistance to atrazine was observed in 12 of 15 populations; however, a target-site resistance mechanism was not observed in these populations. Resistance to glyphosate was observed in all populations. Gene amplification was the predominant glyphosate-resistance mechanism (147 of 322 plants) in the evaluated populations. The Pro-106-Ser mutation was identified in 24 plants, half of which also possessed gene amplification. In this study, molecular screening generally underestimated the phenotypically observed resistance. Continued mechanism discovery and marker development is required for improved detection of herbicide resistance through molecular assays.

Protoporphyrinogen IX oxidase (PPO)–inhibiting herbicides (WSSA Group 14) have been used in agronomic row crops for over 50 yr. Broadleaf weeds, including glyphosate-resistant Palmer amaranth, have been controlled by this herbicide site of action PRE and POST. Recently, Palmer amaranth populations were reported resistant to PPO inhibitors in 2011 in Arkansas, in 2015 in Tennessee, and in 2016 in Illinois. Historically, the mechanism for this resistance involves the deletion of a glycine at position 210 (ΔG210) in a PPO enzyme encoded by the PPX2 gene; however, the ΔG210 deletion did not explain all PPO inhibitor–resistant Palmer amaranth in Tennessee populations. Recently, two new mutations within PPX2 (R128G, R128M) that confer resistance to PPO inhibitors were identified in Palmer amaranth. Therefore, research is needed to document the presence and distribution of the three known mutations that confer PPO inhibitor resistance in Tennessee. In 2017, a survey was conducted in 18 fields with Palmer amaranth to determine whether resistance existed and the prevalence of each known mutation in each field. Fomesafen was applied at 265 g ai ha–1 to Palmer amaranth infestations within each field to select for resistant weeds for later analysis. Where resistance was described (70% of surviving plants), the ΔG210 mutation was detected in 47% of resistant plants. The R128G mutation accounted for 42% of resistance, similar to the frequency of the ΔG210 mutation. The R128M mutation was less frequent than the other two mutations, accounting for only 10% of the resistance. All mutations detected in this study were heterozygous. Additionally, no more than one of the three PPX2 mutations were detected in an individual surviving plant. Similar to previous research, about 70% of PPO resistance was accounted for by these three known mutations, leaving about 30% of resistance not characterized in Tennessee populations. Survivors not showing the three known PPO mutations suggest that other resistance mechanisms are present.

Recently, several incidents of glyphosate failure on junglerice [Echinochloa colona (L.) Link] have been reported in the midsouthern United States, specifically in Mississippi and Tennessee. Research was conducted to measure the magnitude of glyphosate resistance and to determine the mechanism(s) of resistance to glyphosate in E. colona populations from Mississippi and Tennessee. ED50 (dose required to reduce plant growth by 50%) values for a resistant MSGR4 biotype, a resistant TNGR population, and a known susceptible MSGS population were 0.8, 1.62, and 0.23 kg ae ha−1 of glyphosate, respectively. The resistance index calculated from the these ED50 values indicated that the MSGR4 biotype and TNGR population were 4- and 7-fold, respectively, resistant to glyphosate relative to the MSGS population. The absorption patterns of [14C]glyphosate in the TNGR and MSGS populations were similar. However, the MSGS population translocated 13% more [14C]glyphosate out of the treated leaf compared with the TNGR population at 48 h after treatment. EPSPS gene sequence analyses of TNGR E. colona indicated no evidence of any point mutations, but several resistant biotypes, including MSGR4, possessed a single-nucleotide substitution of T for C at codon 106 position, resulting in a proline-to-serine substitution (CCA to TCA). Results from quantitative polymerase chain reaction analyses suggested that there was no amplification of the EPSPS gene in the resistant populations and biotypes. Thus, the mechanism of resistance in the MSGR population (and associated biotypes) is, in part, due to a target-site mutation at the 106 loci of the EPSPS gene, while reduced translocation of glyphosate was found to confer glyphosate resistance in the TNGR population.

Glyphosate-resistant (GR) common waterhemp (CW) is a localized weed in Ontario and one of the most problematic weeds in the US Corn Belt. First confirmed in Ontario in 2014, GR CW has now been confirmed in forty fields in three counties in Ontario as of 2015. Historically, the primary POST herbicides used for the control of CW in soybean were glyphosate, acifluorfen and fomesafen, but resistance to all three has been confirmed in many US states. Research was conducted in 2015 and 2016 to determine the control of GR CW with some of the new herbicide-resistant soybean technologies including glufosinate (LibertyLink), 2,4-D and glyphosate (Enlist), and isoxaflutole, mesotrione, and glufosinate (HPPD-resistant). Glyphosate-resistant CW was controlled (≥90%) all season with a two-pass weed control system across all herbicide-resistant soybean technologies evaluated. The two-pass weed control system in this research is defined as a PRE herbicide followed by a POST herbicide. At 12 WAA, the two-pass programs in LibertyLink, Enlist, and HPPD-resistant systems controlled GR CW up to 98, 98, and 92%, respectively, and reduced GR CW densities to 0 to 2% of the weedy control at 4 WAA. The two-pass programs provided greater GR CW control than PRE or POST herbicides alone. This study found that the use of two-pass weed control programs in glufosinate-resistant, glyphosate DMA/2,4-D choline-resistant and HPPD-resistant soybean can provide excellent control of GR CW, and can be valuable tools to reduce the selection intensity for herbicide-resistant weeds. Through the rotational use of different technologies, growers may be able to better manage their weed populations in reducing the risk of resistance when compared to the use of one herbicide repeatedly.

Glyphosate-resistant (GR) common waterhemp is the fifth GR weed species confirmed in Canada, and the fourth in Ontario. As of 2017, GR common waterhemp has been confirmed in Lambton, Essex, and Chatham-Kent counties in Ontario. Greenhouse and field dose–response experiments revealed that GR common waterhemp in Ontario had a resistance level of 4.5 and 28, respectively, when compared with known glyphosate-susceptible populations. At 12 wk after application, pyroxasulfone/flumioxazin (240 g ai ha−1), pyroxasulfone/sulfentrazone (300 g ai ha−1), and S-metolachlor/metribuzin (1,943 g ai ha−1) controlled GR common waterhemp 97%, 92%, and 87%, respectively. Pyroxasulfone/sulfentrazone or S-metolachlor/metribuzin applied PRE followed by acifluorfen (600 g ai ha−1) or fomesafen (240 g ai ha−1) applied POST controlled GR common waterhemp 98% and performed better than PRE or POST alone. This research is the first to determine the resistance factor of GR common waterhemp in Ontario and identifies control strategies in soybean to mitigate the impact of common waterhemp interference in soybean crop production.

A 2,4-D-resistant tall waterhemp population (FS) from Nebraska was evaluated for resistance to other TIR1 auxin receptor herbicides and to herbicides having alternative mechanisms of action using greenhouse bioassays and genetic markers. Atrazine, imazethapyr, lactofen, mesotrione, glufosinate, and glyphosate were applied in a single-dose bioassay, and tissue was collected from marked plants for genetic analysis. The FS population was not injured by atrazine or by imazethapyr. Approximately 50% of the plants survived lactofen and were actively growing 28 d after treatment. The population was susceptible to mesotrione, glufosinate, and glyphosate. Ametryn, chlorimuron-ethyl, 2,4-D, aminocyclopyraclor, aminopyralid, and picloram were applied in dose–response studies. The FS population was sensitive to ametryn, and the Ser-264-Gly substitution in the D1 protein was not detected, suggesting the lack of response to atrazine is not due to a target-site mutation. The FS population exhibited less than 50% injury to chlorimuron-ethyl at application rates 20 times the labeled use rate. The Ser-653-Asn acetolactate synthase (ALS) substitution, which confers resistance to imidazolinone herbicides, was present in the FS population. However, this does not explain the lack of response to the sulfonylurea herbicide, chlorimuron-ethyl. Sequencing of a portion of the PPX2L gene did not show the ΔG210 mutation that confers resistance to protoporphyrinogen oxidase–inhibiting herbicides, suggesting that other factors were responsible for waterhemp survival after lactofen application. The FS population was confirmed to be at least 30-fold resistant to 2,4-D relative to the susceptible populations. In addition, it was at least 3-fold less sensitive to aminopyralid and picloram, two other TIR1 auxin receptor herbicides, than the 2,4-D-susceptible populations were. These data indicated that the FS population contains both target and non–target site mechanisms conferring resistance to herbicides spanning at least three mechanisms of action: TIR1 auxin receptors, ALS inhibitors, and photosystem II inhibitors.