In a recent essay, Harker and coauthors stated that considering herbicide resistance as a wicked problem “without clear causes or solutions” ignores what weed scientists know about the biology and management of herbicide-resistant weeds. In this response, we argue that this misrepresents what is meant by “wicked” and that the wicked problem concept is valuable in understanding the multifaceted nature of herbicide resistance as a human-caused phenomenon.

Palmer amaranth is native to the United States, but was discovered in 2015 in Brazil. Palmer amaranth populations in Brazil were very difficult to control using glyphosate, which resulted in many changes to standard weed management practices. A genotyping assay was used to confirm that the population detected in Mato Grosso State, Brazil, was correctly identified as Palmer amaranth and that it was not tall waterhemp. Greenhouse dose–response curves and shikimate accumulation assays showed that the Brazilian population was highly resistant to glyphosate, with an LD50 value (3,982 g glyphosate ha−1) more than twice the typical use rates and very little shikimate accumulation at 1 mM glyphosate concentrations in a leaf-disk assay. The Brazilian population was also resistant to sulfonylurea and imidazolinone acetolactate synthase (ALS) inhibitor herbicides. The resistance mechanisms in the Brazilian population were identified as increased EPSPS gene copy number for glyphosate resistance (between 50- and 179-fold relative EPSPS gene copy number increase) and two different alleles for target-site mutations in the ALS gene (W574L and S653N). These results confirm the introduction of Palmer amaranth to Brazil using a genetic marker for species identification, as well as resistance to glyphosate and ALS inhibitors.

Weed resistance to herbicides occurs when herbicides are overused and can be mitigated by reducing their use. Consensus on herbicide resistance management strategies is problematic given strong industrial profit motive links in the weed science discipline.

Eclipta, widespread in tropical, subtropical, and temperate regions, is one of the main malignant broadleaf weeds and thrives in moist and dryland fields. Field rates of acetolactate synthase (ALS) inhibitors have failed to control eclipta in some farmlands in China. One ALS inhibitor–resistant population (R) collected from Jiangsu province in China was confirmed in the greenhouse in our preliminary work. Whole-plant assays revealed that this R population was highly resistant to four sulfonylureas (pyrazosulfuron-ethyl, 134-fold; bensulfuron-methyl, 172-fold; metsulfuron-methyl, 30-fold; and tribenuron-methyl, 195-fold), two triazolopyrimidines (pyroxsulam, 98-fold; penoxsulam, 30-fold), and one pyrimidinylthio-benzoate (bispyribac-sodium, 166-fold) and was moderately resistant to two imidazolinones (imazethapyr, 10-fold; imazapic, 19-fold). ALS enzyme-activity assays showed insensitivity of the ALS from the R population (resistance index values ranged from 12 to 293) to all of the above ALS inhibitors in vitro. Chromatograms from ALS gene sequence analysis detected a homozygous Pro-197-Ser amino acid substitution in the R population. These results confirmed that the Pro-197-Ser substitution results in broad-spectrum cross-resistance to ALS inhibitors in the eclipta R population. To our knowledge, this study is the first to report broad cross-resistance to ALS inhibitors in eclipta and to obtain the full-length ALS gene sequence.

Resistance to acetolactate synthase (ALS)-inhibitor herbicides due to continuous and repeated selection is widespread in many troublesome weed species, including Palmer amaranth, throughout the United States. The objective of this research was to investigate the physiological and molecular basis of resistance to ALS inhibitors in a chlorsulfuron-resistant Palmer amaranth population (KSR). Our results indicate that the KSR population exhibits a high level of resistance to chlorsulfuron compared with two known susceptible populations, MSS and KSS from Mississippi and Kansas, respectively. MSS is highly susceptible to chlorsulfuron, whereas KSS is moderately sensitive. Dose–response analysis revealed that KSR was more than 275-fold more resistant compared with KSS. Nucleotide sequence analysis of the ALS gene from the plants that survived chlorsulfuron treatment revealed the possibility of evolution of both target site–based and non–target site based resistance to ALS inhibitors in the KSR population. The most common mutation (Pro-197-Ser) in the ALS gene associated with resistance to the sulfonylureas in many weed species was found only in 30% of the KSR population. A preliminary malathion study showed that the remaining 70% of resistant plants might have cytochrome P450–mediated non–target site resistance. This is the first report elucidating the mechanism of resistance to ALS inhibitors in Palmer amaranth from Kansas. Presence of both target site– and non–target site based mechanisms of resistance limits the herbicide options to manage Palmer amaranth in cropping systems.

The management of glyphosate-resistant Palmer amaranth has been a challenge in southern United States cropping systems. Registration of dicamba-resistant crops will provide an alternative management option to control herbicide-resistant Palmer amaranth populations, particularly those having resistance to herbicide Groups 2, 3, 5, 9, 14, and 27. However, repeated use of sublethal doses of dicamba may lead to rapid evolution of herbicide resistance, especially in Palmer amaranth—a species with a strong tendency to evolve resistance. Therefore, selection experiments with dicamba were conducted on Palmer amaranth using sublethal doses. In the greenhouse, a known susceptible Palmer amaranth population was subjected to sublethal dicamba doses for three generations (P1–P3). Susceptibility of the individuals to dicamba was evaluated, and its susceptibility to 2,4-D was characterized. Based on the greenhouse study, following three generations of dicamba selection, the dose required to cause 50% mortality increased from 111 g ae ha−1 for parental individuals (P0) to 309 g ae ha−1 for the P3. Furthermore, reduced susceptibility of the P3 to 2,4-D was also evident. This research presents the first evidence that recurrent use of sublethal dicamba doses can lead to reduced susceptibility of Palmer amaranth to dicamba as well as 2,4-D. Here, we show that selection from sublethal dicamba doses has an important role in rapid evolution of Palmer amaranth with reduced susceptibility to auxin-type herbicides.

Herbicide-resistant weeds pose a considerable threat to agriculture, but their resistance mechanisms are poorly understood. Differential gene expression analysis of a weed subjected to herbicide treatment is a key step toward more mechanistic studies. Such an analysis, often involving quantitative real-time PCR (qPCR), requires suitable reference genes as internal controls. In this study, we identified optimal reference genes in the noxious weed, Japanese foxtail. This weed has evolved resistance to acetyl-coenzyme A carboxylase (ACCase) inhibitors. We analyzed the stability of eight commonly used candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]; ubiquitin [UBQ]; capsine phosphatase [CAP]; beta-tubulin [TUB]; eukaryotic initiation factor 4a [EIF4A]; elongation factor-1 alpha [EF1]; 18S ribosomal RNA [18S]; 25S ribosomal RNA [25S]) from root, stem, and leaf tissue of plants that were either resistant or sensitive to ACCase inhibitors, with or without herbicide stress, using qPCR. The results were further ranked and analyzed using geNorm, NormFinder, and BestKeeper software. These analyses identified EF1 and UBQ in roots, EF1, TUB, CAP, and 18S in stems, and EF1, GAPDH, and 18S in leaves as suitable references for qPCR normalization. We have identified a set of reference genes that can be used to study herbicide resistance mechanisms in Japanese foxtail.

Five rigid ryegrass populations suspected of being resistant to both glyphosate and oxyfluorfen were collected in southern Spain and tested under laboratory-controlled conditions. Four populations (Depuradora, Condado, AlamoRasilla, and Portichuelo) were treated with glyphosate for at least 15 consecutive years, and treatments during the last 5 yr were mixed with oxyfluorfen. The fifth population (4alamos) followed the same glyphosate treatment, although oxyfluorfen was never used to control it. Dose–response assays confirmed glyphosate resistance in all populations, with resistance indexes ranging from 11.7 to 37.5 (GR90). Shikimate accumulation assays consistently supported these data, as the most glyphosate-resistant populations (Depuradora and Condado) displayed the lowest shikimate levels. Surprisingly, four populations (Depuradora, Condado, AlamoRasilla, and Portichuelo) displayed 7.93- to 70.18-fold more resistance (GR90) to oxyfluorfen, despite limited selection pressure, showing a similar resistance pattern as that for glyphosate. The 4alamos population displayed oxyfluorfen GR90 values that were similar to those observed in susceptible plants; however, this population was significantly more resistant in terms of plant survival (LD90). Protoporphyrin IX accumulation assays supported the results of dose–response assays, in that the most oxyfluorfen-resistant populations accumulated less protoporphyrin IX. Although more studies are needed, it seems that these five glyphosate-resistant weed populations display a natural tendency to easily develop resistance to oxyfluorfen, with the populations that have higher resistance to glyphosate also having higher resistance to oxyfluorfen.

It is generally expected that, in the case of multiple herbicide resistance, different resistance mechanisms within a weed will follow Mendel’s law of independent assortment. Research was conducted to investigate anecdotal observations suggesting that target site–based resistances to inhibitors of acetolactate synthase (ALS) and protoporphyrinogen oxidase (PPO) did not follow independent assortment in common waterhemp. Cosegregation of the two resistances was observed in backcross lines (population sensitive to both herbicides as recurrent parent). Specifically, whereas 52% of backcross plants were resistant to a PPO inhibitor, this percentage increased to 92% when the backcross plants were preselected for resistance to an ALS inhibitor. Molecular marker analysis confirmed that the corresponding genes (ALS and PPX2) were genetically linked. When data from all plants analyzed were pooled, the genetic distance between the two genes was calculated to be 7.5 cM. The two genes were found to be about 195 kb apart in the recently published grain amaranth genome, explaining the observed genetic linkage. There is likely enough recombination that occurs between the linked genes to prevent the linkage from having significant implications in terms of resistance evolution. Nevertheless, documentation of the happenstance linkage between target-site genes for resistance to ALS and PPO inhibitors in waterhemp is a reminder that one should not assume distinct resistance mechanism will independently assort.

Japanese foxtail is a grass weed in eastern China. This weed is controlled by fenoxaprop-P-ethyl, one of the most common acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. Some Japanese foxtail populations have developed resistance to fenoxaprop-P-ethyl, owing to target-site mutations (amino acid substitutions) located within the carboxyl transferase domain of ACCase. In the present study, three mutations were detected in three fenoxaprop-P-ethyl–resistant Japanese foxtail populations: Ile-1781-Leu in JCJT-2, Ile-2041-Asn in JZJR-1, and Asp-2078-Gly in JCWJ-3. Two copies of ACCase (Acc1-1 and Acc1-2) were identified, but mutations were detected only in Acc1-1. The derived cleaved amplified polymorphic sequence (dCAPS) method detected these mutations successfully in Japanese foxtail. The mutation frequencies in JCJT-2, JZJR-1, and JCWJ-3 were approximately 98%, 92%, and 87%, respectively. Different cross-resistance patterns to ACCase inhibitors were found in the three resistant populations. JCJT-2 (Ile-1781-Leu) and JZJR-1 (Ile-2041-Asn) showed cross-resistance to haloxyfop-R-methyl, clodinafop-propargyl, and pinoxaden, but were susceptible to clethodim. JCWJ-3 (Asp-2078-Gly) showed cross-resistance to all tested ACCase-inhibiting herbicides.

The failure of PRE and POST applications of atrazine to control Palmer amaranth in recent field studies prompted further investigation to determine whether this population had evolved resistance to multiple herbicide sites of action, including glyphosate (Group 9), thifensulfuron (Group 2), and atrazine (Group 5). Greenhouse and laboratory experiments were conducted to: (1) confirm the presence of resistance to glyphosate, an ALS inhibitor (thifensulfuron), and atrazine in a single Palmer amaranth population; and (2) establish the molecular basis for resistance to these herbicide sites of action. In the greenhouse, glyphosate+thifensulfuron+atrazine at 1.26 kg ae ha−1+0.0044 kg ai ha−1+1.12 kg ai ha−1 provided 55% control of the suspected multiply resistant (MR) Palmer amaranth population and 93% control of the known susceptible population (S). The decreased sensitivity of the MR population compared with the S population at labeled use rates of these herbicides indicated that this population was likely resistant to three different herbicide site of action groups. The RF values for POST applications of glyphosate, thifensulfuron, and atrazine were 12.2, 42.9, and 9.3 times, respectively, for the MR Palmer amaranth population relative to the S population. The RF value for atrazine PRE for the MR population was 112.2 times. Laboratory experiments confirmed that the mechanisms for resistance to ALS-inhibiting herbicides and glyphosate in the MR Palmer amaranth population were target-site based, via amino acid substitution and amplified EPSPS copy number, respectively. There was a Pro to Leu substitution at site 197 in the ALS inhibitor–resistant plants, and there was a greater than 50-fold increase in EPSPS copy number in the glyphosate-resistant plants. There were no nucleotide changes in the psbA gene; therefore, atrazine resistance in this population was not target-site mediated. The evolution of this multiple herbicide-resistant Palmer amaranth population poses significant management challenges to Michigan farmers.

Keng stiffgrass is a serious farmland grass weed distributed globally in winter wheat fields and rice–wheat double-cropping areas. The intensive use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has led to the evolution of resistance in a growing number of grass weeds. In this study, whole-plant pot bioassay experiments were conducted to establish that a Keng stiffgrass population from eastern China, JYJD-2, has evolved high-level resistance to fenoxaprop-P-ethyl and moderate resistance to quizalofop-P-ethyl and pinoxaden. Using the derived cleaved amplified polymorphic sequence method, a tryptophan-to-cysteine mutation at codon position 1999 (W1999C) was detected in the ACCase gene of the resistant population JYJD-2. Of the 100 JYJD-2 plants tested, we found 47 heterozygous resistant and 53 homozygous sensitive individuals. In vitro ACCase assays revealed that the IC50 value of the ACCase activity of the resistant population JYJD-2 was 6.48-fold higher than that of the susceptible population JYJD-1. To the best of our knowledge, this is the first report of the occurrence of W1999C mutation in the ACCase gene of fenoxaprop-P-ethyl–resistant Keng stiffgrass. This study confirmed the resistance of Keng stiffgrass to the ACCase inhibitor fenoxaprop-P-ethyl, cross-resistance to other ACCase inhibitors, and the resistance being conferred by specific ACCase point mutations at amino acid position 1999.

A biotype of annual bluegrass with suspected resistance to pronamide was collected from a golf course in Georgia. The objectives of this research were to determine the level of resistance to pronamide and the mechanisms associated with resistance. From POST applications, the pronamide rate that reduced shoot biomass 50% from the nontreated bluegrass measured>10 times higher for the resistant (R) biotype as compared with susceptible (S) biotypes. The R biotype was not controlled by PRE applications of dithiopyr or prodiamine, but was controlled >92% by PRE applications of pronamide at 0.56 and 1.68 kg ha−1. Mature plants (3- to 5-tiller) of the R biotype absorbed 32% less [14C]pronamide than the S biotype after 72 h in hydroponic culture and accumulated 39% less radioactivity per gram basis of dry shoot mass. The R biotype metabolized [14C]pronamide similar to the S biotype, averaging 16% of the extracted radioactivity. The resistance to POST pronamide applications in the R biotype is associated with reduced absorption and translocation compared with the S biotype.

Knowledge of Palmer amaranth biology and physiology is essential for the development of effective weed management systems. The aim of this study was to investigate the response of Palmer amaranth gender to nutrient deficiency and light stress. Differential gender responses were observed for all the growth, phenology, and photochemistry parameters measured. Female plants, for example, invested more in height, stem, and total dry weight, whereas male plants invested more in leaf area and leaf dry weight. The growth rate of females was higher than that of male Palmer amaranth plants, although both followed similar declining trends as the experimental period progressed. Initiation of flowering of female plants occurred 6 to 8 d earlier compared with male plants. Nitrogen and to a certain extent phosphorous were the most influential nutrients that affected measured parameters in both Palmer amaranth genders, particularly under high light intensity. Electron transport rate and chlorophyll content of female Palmer amaranth plants compared with male plants was lower at high light intensity in combination with nitrogen and phosphorous deficiencies. There is a potential to manipulate Palmer amaranth population structure by altering microenvironments at the field level.

The Enlist™ traits provide 2,4-D resistance in several crops. Though corn is naturally tolerant to 2,4-D, the engineered trait conferred by the aryloxyalkanoate dioxygenase-1 (AAD-1) enzyme provides enhanced 2,4-D tolerance and confers resistance to the graminicide herbicide family, the aryloxyphenoxypropionates. The objectives of this research were 2-fold: (1) measure and compare uptake, translocation, and metabolism of 2,4-D in Enlist™ (E, +AAD1) and non–AAD-1 transformed (NT, −AAD1) isogenic corn hybrids; and (2) and investigate the effect of glyphosate and/or the Enlist™ adjuvant system (ADJ) on these factors and corn injury. Uptake of radiolabeled 2,4-D acid applied alone in corn was not altered by the addition of ADJ when tank mixed at 24 h after application (HAA). By contrast, uptake of radiolabeled 2,4-D was significantly lower (69%) compared with 2,4-D plus ADJ (89%) at 24 HAA with a premixed formulation of 2,4-D choline plus glyphosate-dimethylamine (Enlist Duo™ herbicide [EDH]). Translocation of 2,4-D between the two corn hybrids was not different. E corn metabolized more 2,4-D (100% of absorbed) than NT corn (84%), and glyphosate did not alter 2,4-D metabolism. Furthermore, the metabolism of 2,4-D to nonphytotoxic dichlorophenol (DCP) and subsequent DCP-derived metabolites formed in E corn was examined. Injury to E corn is not typically observed in the field; however, injury symptoms were clearly evident in E corn (within 24 HAA) when formulated acetochlor was tank mixed with EDH, which correlated with an increase in 2,4-D uptake during this time period. In summary, the lack of injury in E corn following EDH applied alone may be attributed to a relatively low amount of 2,4-D uptake and the combination of natural and engineered 2,4-D metabolic pathways.

Palmer amaranth’s ability to evolve resistance to different herbicides has been studied extensively, but there is little information about how this weed species might be evolving other life-history traits that could potentially make it more aggressive and difficult to control. We characterized growth and morphological variation among 10 Palmer amaranth populations collected in Florida and Georgia from fields with different cropping histories, ranging from continuous short-statured crops (vegetables and peanut) to tall crops (corn and cotton) and from intensive herbicide use history to organic production. Palmer amaranth populations differed in multiple traits such as fresh and dry weight, days to flowering, plant height, and leaf and canopy shape. Differences between populations for these traits ranged from 36% up to 87%. Although glyphosate-resistant (GR) populations collected from cropping systems including GR crops exhibited higher values of the aforementioned variables than glyphosate-susceptible (GS) populations, variation in traits was not explained by glyphosate resistance or distance between populations. Cropping system components such as crop rotation and crop canopy structure better explained the differences among populations. The higher growth of GR populations compared with GS populations was likely the result of multiple selection forces present in the cropping systems in which they grow rather than a pleiotropic effect of the glyphosate resistance trait. Results suggest that Palmer amaranth can evolve life-history traits increasing its growth and reproduction potential in cropping systems, which explains its rapid spread throughout the United States. Furthermore, our findings highlight the need to consider the evolutionary consequences of crop rotation structure and the use of more competitive crops, which might promote the selection of more aggressive biotypes in weed species with high genetic variability.

Giant ragweed is a highly competitive weed that continually threatens crop production systems due to evolved resistance to acetolactate synthase–inhibiting herbicides (ALS-R) and glyphosate (GR). Two biotypes of GR giant ragweed exist and are differentiated by their response to glyphosate, termed here as rapid response (RR) and non–rapid response (NRR). A comparison of data from surveys of Indiana crop fields done in 2006 and 2014 showed that GR giant ragweed has spread from 15% to 39% of Indiana counties and the NRR biotype is the most prevalent. A TaqMan® single-nucleotide polymorphism genotyping assay was developed to identify ALS-R populations and revealed 47% of GR populations to be ALS-R as well. The magnitude of glyphosate resistance for NRR populations was 4.6 and 5.9 based on GR50 and LD50 estimates, respectively. For RR populations, these values were 7.8 to 9.2 for GR50 estimates and 19.3 to 22.3 for LD50 estimates. A novel use of the Imaging-PAM fluorometer was developed to discriminate RR plants by assessing photosystem II quantum yield across the entire leaf surface. H2O2 generation in leaves of glyphosate-treated plants was also measured by 3,3′-diaminobenzidine staining and quantified using imagery analysis software. Results show photo-oxidative stress of mature leaves is far greater and occurs more rapidly following glyphosate treatment in RR plants compared with NRR and glyphosate-susceptible plants and is positively associated with glyphosate dose. These results suggest that under continued glyphosate selection pressure, the RR biotype may surpass the NRR biotype as the predominant form of GR giant ragweed in Indiana due to a higher level of glyphosate resistance. Moreover, the differential photo-oxidative stress patterns in response to glyphosate provide evidence of different mechanisms of resistance present in RR and NRR biotypes.

Fluazifop-P-butyl, a selective graminicide, has been widely used to control annual grass weeds for more than three decades in Taiwan, and a resistant (R) biotype of goosegrass has consequently appeared. In this study, liquid chromatography/tandem mass spectrometry (LC/MS/MS) was applied to analyze metabolites of fluazifop-P-butyl in a R biotype of goosegrass. Six signals, including mass-to-charge ratios (m/z) 512, 432, 423, 415, 314, and 160, in positive scanning mode, and four signals, including m/z 788, 623, 593, and 162, in negative scanning mode, were found in the metabolites of the R and S biotypes, respectively. All of the signals of these metabolites in the R biotype showed higher intensity than those of the S biotype, except for m/z 162. Based on the molecular weights of the fragments (MS2 signal) from the molecules (MS1 signal) and comparison with the Metabolite Link Metabolomics database, MassBank database, and related references, one reduced form of fluazifop acid, i.e., 2-[4-(5- trifluoromethyl-2-pyridyloxy) phenoxy] propanol (MW 313); two types of intermediates of MW 163, i.e., 5-trifluoromethyl-2-pyridone and 5-trifluoromethyl-2-hydroxypyridine (or 2-hydroxy-5-trifluoromethyl pyridine); and five possible conjugated compounds containing a common core fragment (MW 255) from fluazifop acid were identified. In addition, another compound, likely degraded from one of the five conjugated compounds, was also detected. Accordingly, the metabolic pathway of fluazifop-P-butyl in goosegrass is described in this study. An enzyme kinetic study on glutathione S-transferase showed that the R biotype has higher affinities to the substrates reduced glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene, with S/R Km ratios of 3.0 and 2.4, respectively. No difference in Vmax was found, revealing that the S biotype has a strong ability to bind GSH and herbicide or target molecules and showed susceptibility to fluazifop.

Italian ryegrass has invaded wheat fields in China and is becoming a predominant, troublesome weed. Fenoxaprop-P-ethyl has been widely used for weed control on Chinese farms since the 1990s. However, overuse has led to fenoxaprop-P-ethyl resistance in Italian ryegrass in Chinese wheat fields. In this study, we identified a putative fenoxaprop-P-ethyl–resistant population of Italian ryegrass, HZYC-6, from Henan province, China. Mutations involving Asp-2078-Gly and Ile-1781-Leu substitutions were identified in the carboxyl-transferase domain of acetyl-coenzyme A carboxylase in this population, and these mutations are the likely cause of the target site–based resistance to fenoxaprop-P-ethyl. In addition, we identified cytochrome P450–mediated metabolism of herbicides (non–target site based resistance) in the HZYC-6 population, indicating that multiple mechanisms of resistance may be segregating in this population. Furthermore, HZYC-6 was also highly resistant to haloxyfop-R-methyl and quizalofop-P-ethyl, moderately resistant to clodinafop-propargyl and sethoxydim, and had low resistance to clethodim and pinoxaden.