Cotton and soybean growers were offered new technologies in 2016, expanding in-crop herbicide options to include dicamba or 2,4-D. Within 3 yr of commercialization, dicamba use in these crops increased 10-fold, and growers began to report Palmer amaranth escapes in dicamba-tolerant production systems in western Tennessee. In 2020, Palmer amaranth seed was collected from eight Tennessee locations where growers witnessed poor control following dicamba. Greenhouse experiments were conducted to evaluate the response of these Palmer amaranth populations to dicamba. In 2021, field experiments were conducted on two tentative dicamba-susceptible populations in Georgia, on three confirmed dicamba-resistant populations in Tennessee, and on a tentative dicamba-susceptible population in Texas to evaluate cotton response following dicamba and to examine if malathion insecticide (a cytochrome P450 inhibitor) would improve weed control and not reduce cotton yield when applied in conjunction with dicamba. Palmer amaranth populations collected in 2020 survived dicamba in the greenhouse at 1, 2, and 4 times the labeled rate. Five Palmer amaranth populations exhibited 15% to 26% survival to the labeled dicamba rate (560 g ha−1) in the greenhouse. These findings were reinforced in the field when research on three of those populations in 2021 showed 55% control with the labeled dicamba rate and 69% control with 2 times the labeled rate. This demonstrates that the dicamba resistance allele or alleles were passed between generations. This result was not consistent in the Macon County, GA, or Worth County, GA, locations, where malathion improved dicamba control of 15- to 38-cm-tall Palmer amaranth. Cotton injury was observed when malathion was applied in combination with dicamba. These results further document the evolution of dicamba-resistant Palmer amaranth in Tennessee. Moreover, the nonreversal of resistance phenotype by malathion may suggest that the resistance mechanism is something other than metabolism.

Since its initial introduction in the late 1950s, chemical control has dominated weed management practices in China. Not surprisingly, the development of herbicide resistance has become the biggest threat to long-term, sustainable weed management in China. Given that China has followed the same laissez-faire approach toward resistance management that has been practiced in developed countries such as the United States, herbicide resistance has evolved rapidly and increased steadily over the years. Previously, we carried out a systematic review to quantitatively assess herbicide-resistance issues in China. In this review, our main objective is to focus on mechanistic studies and management practices to document the (1) history of herbicide application in China; (2) resistance mechanisms governing the eight most resistance-prone herbicide groups, including acetolactate synthase inhibitors, acetyl-CoA carboxylase inhibitors, synthetic auxin herbicides, 5-enolpyruvylshikimate-3-phosphate synthase inhibitors, protoporphyrinogen oxidase inhibitors, photosystem I electron diverters, photosystem II inhibitors, and long-chain fatty-acid inhibitors; and (3) herbicide-resistance management strategies commonly used in China, including chemical, cultural, biological, physical, and integrated approaches. At the end, perspectives and future research are discussed to address the pressing need for the development of integrated herbicide-resistance management in China.

Chinese sprangletop [Leptochloa chinensis (L.) Nees] is one grass weed severely affecting rice (Oryza sativa L.) growth in paddies in China. Cyhalofop-butyl is the main herbicide used to control grass weeds in Chinese paddy fields, especially for controlling L. chinensis; however, L. chinensis has evolved resistance to cyhalofop-butyl due to continuous and extensive application. To investigate cyhalofop-butyl resistance levels and mechanisms in L. chinensis in some of the Chinese rice areas, 66 field populations were collected and treated with cyhalofop-butyl. Of these tested populations, 10 showed a high level of resistance to cyhalofop-butyl; the 50% effective dose ranged within 108.4 to 1,443.5 g ai ha−1 with resistance index values of 9.1 to 121.8 when compared with the susceptible population. Acetyl-coenzyme A carboxylase genes (ACCase) of susceptible and all 10 resistant populations were amplified and sequenced. Among them, Ile-1781-Leu, Trp-2027-Cys, Trp-2027-Ser, and Ile-2041-Asn mutations were found in five resistant populations. No known resistance-related mutations were found in the other five resistant populations, indicating that resistance to cyhalofop-butyl in these populations was likely to be endowed by non–target site resistance mechanisms. Notably, the Ile-1781-Leu and Trp-2027-Cys substitutions have previously been reported, but this is the first report of Trp-2027-Ser and Ile-2041-Asn mutations in L. chinensis. Furthermore, three derived cleaved amplified polymorphic sequence methods were developed to rapidly detect these mutations in L. chinensis.

Late watergrass [Echinochloa phyllopogon (Stapf) Koso-Pol.] is one of the most persistent weeds in rice fields and shows resistance to some acetolactate synthase (ALS)-inhibiting herbicides, such as penoxsulam. Previous studies of E. phyllopogon’s herbicide resistance have focused on non–target site resistance mechanisms. In this study, E. phyllopogon populations from Heilong Jiang Province, China, that were possibly resistant to penoxsulam were used to identify the target site–based mechanisms of resistance. Population HSRH-520 showed a 25.4-fold higher resistance to penoxsulam than the sensitive population, HSRH-538. HSRH-520 was resistant to other ALS inhibitors, with resistance indexes ranging from 17.1 to 166. Target-gene sequence analysis revealed two different ALS genes in E. phyllopogon; a Pro-197-Ser substitution occurred in the ALS-2 gene of HSRH-520. In vitro activity assays revealed that the penoxsulam concentrations required to inhibit 50% of the ALS activity were 13.7 times higher in HSRH-520 than in HSRH-538. Molecular-docking tests showed that the Pro-197-Ser mutation reduced the binding affinity between ALS and ALS inhibitors belonging to the triazolopyrimidine, sulfonylaminocarbonyltriazolinone, and sulfonylurea families, and there were almost no effects on binding affinity when the ALS inhibitors were of the pyrimidinylthiobenzoate and imidazolinone families. Overall, the results indicated and verified that the Pro-197-Ser mutation leads to increased ALS activity by reducing the binding affinity of the inhibitor and ALS. This is the first report on the Pro-197-Ser mutation in the complete ALS gene of E. phyllopogon and will aid future research of target site–based resistance mechanisms of E. phyllopogon to ALS inhibitors.

Monensin (Mon) is an anticoccidial polyether ionophore widely used to control coccidiosis. The extensive use of polyether ionophores on poultry farms resulted in widespread resistance, but the underlying resistance mechanisms are unknown in detail. For analysing the mode of action by which resistance against polyether ionophores is obtained, we induced in vitro Mon resistance in Toxoplasma gondii-RH strain (MonR-RH) and compared it with the sensitive parental strain (Sen-RH). The proteome assessment of MonR-RH and Sen-RH strains was obtained after isotopic labelling using stable isotope labelling by amino acid in cell culture. Relative proteomic quantification between resistant and sensitive strains was performed using liquid chromatography-mass spectrometry/mass spectrometry. Overall, 1024 proteins were quantified and 52 proteins of them were regulated. The bioinformatic analysis revealed regulation of cytoskeletal and transmembrane proteins being involved in transport mechanisms, metal ion-binding and invasion. During invasion, actin and microneme protein 8 (MIC8) are seem to be important for conoid extrusion and forming moving junction with host cells, respectively. Actin was significantly upregulated, while MIC8 was downregulated, which indicate an invasion reduction in the resistant strain. Resistance against Mon is not a simple process but it involves reduced invasion and egress activity of T. gondii tachyzoites while intracellular replication is enhanced.

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.

Understanding the mechanism of herbicide resistance is fundamental for designing sustainable weed control strategies and exploiting herbicides rationally. Shortawn foxtail is a problem grass weed infesting several important crops in China. The repeated use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has resulted in herbicide resistance in this weed. The ACCase gene of resistant individuals of a shortawn foxtail population (JSLS-1) has an Ile-2041-Thr mutation. F2 generation seeds, originated from the same heterozygous plant, were harvested, and two homozygous mutant (JSLS-1RR) and wild (JSLS-1SS) populations for the Ile-2041-Thr mutation were obtained. In whole plants, the JSLS-1RR population conferred high resistance to fenoxaprop and clodinafop, moderate resistance to haloxyfop, low resistance to pinoxaden, and no obvious resistance to clethodim and sethoxydim, compared with JSLS-1SS and a proven susceptible population (HNXY-1). A derived cleaved amplified polymorphic sequence (dCAPS) marker was developed to rapidly detect the rare Ile-2041-Thr mutation in the shortawn foxtail population. This is the first report of the cross-resistance pattern of Ile-2041-Thr mutation, and the robust dCAPS marker could quickly detect this mutation in shortawn foxtail.

Few studies on herbicide resistance report data to establish unambiguously the correlation between genotype and phenotype. Here we report on the importance of the EPSPS prolyl106 point mutation to serine (P106S) in conferring resistance to glyphosate in a goosegrass population from Davao, Mindanao Island, the Philippines (Davao). Initial rate-response studies showed clear survivors within the Davao population at glyphosate rates that completely controlled the standard sensitive goosegrass population (STD1). Assessment of potential resistance mechanisms identified the presence of P106S mutant individuals in the Davao population. Polymerase chain reaction (PCR) amplification of specific alleles (PASA) analysis established that the mixed-resistant Davao population was comprised of 39.1% homozygous proline wild-type (PP106), 3.3% heterozygous serine mutant (PS106), and 57.6% homozygous serine mutant (SS106) genotypes. Further rate-response studies on plants with a predetermined genotype estimated the Davao SS106 individuals to be approximately 2-fold more resistant to glyphosate compared to Davao PP106 individuals. Extensive analysis at different goosegrass growth stages and glyphosate rates established strong correlation (P < 0.001) between presence of P106S in EPSPS and the resistant phenotype. Importantly, no differences in the pattern of absorbed or translocated 14C–glyphosate were observed between PP106 and SS106 Davao genotypes or Davao and STD1 individuals, suggesting that glyphosate resistance in the Davao population was attributable to an altered target site mechanism. This study demonstrates that whilst P106S in EPSPS confers a moderate resistance level to glyphosate, the mechanism is sufficient to endow glyphosate failure at the recommended field rates.

In 2006 and 2007, farmers from two counties in Illinois reported failure to control waterhemp with glyphosate. Subsequent onsite field experiments revealed that the populations might be resistant to multiple herbicides. Greenhouse experiments therefore were conducted to confirm glyphosate resistance, and to test for multiple resistance to other herbicides, including atrazine, acifluorfen, lactofen, and imazamox. In glyphosate dose-response experiments, both populations responded similarly to a previously characterized glyphosate-resistant population (MO1). Both Illinois populations also demonstrated high frequencies of resistance to the acetolactate synthase (ALS) inhibitor, imazamox. Additionally, one of the populations demonstrated high frequencies of resistance to both atrazine and the protoporphyrinogen oxidase (PPO) inhibitor, lactofen. Furthermore, using combinations of sequential and tank-mix herbicide applications, individual plants resistant to herbicides spanning all four site-of-action groups were identified from one population. Molecular experiments were performed to provide an initial characterization of the resistance mechanisms and to provide confirmation of the presence of multiple resistance traits within the two populations. Both populations contained the W574L ALS mutation and the ΔG210 PPO mutation, previously shown to confer resistance to ALS and PPO inhibitors, respectively. Atrazine resistance in both populations is suspected to be metabolism-based, because a triazine target-site mutation was not identified. A P106S EPSPS mutation, previously reported to confer glyphosate resistance, was identified in one population. This mutation was identified in both resistant and sensitive plants from the population; however, and so more research is needed to determine the glyphosate-resistance mechanism(s). This is the first known case of a weed population in the United States possessing multiple resistance to herbicides from four site-of-action groups.

A glyphosate-resistant Palmer amaranth biotype was confirmed in central Georgia. In the field, glyphosate applied to 5- to 13-cm-tall Palmer amaranth at three times the normal use rate of 0.84 kg ae ha−1 controlled this biotype only 17%. The biotype was controlled 82% by glyphosate at 12 times the normal use rate. In the greenhouse, I 50 values (rate necessary for 50% inhibition) for visual control and shoot fresh weight, expressed as percentage of the nontreated, were 8 and 6.2 times greater, respectively, with the resistant biotype compared with a known glyphosate-susceptible biotype. Glyphosate absorption and translocation and the number of chromosomes did not differ between biotypes. Shikimate was detected in leaf tissue of the susceptible biotype treated with glyphosate but not in the resistant biotype.

The mechanism of glyphosate resistance in horseweed was investigated. Eleven biotypes of putative sensitive (S) and resistant (R) horseweed were obtained from regions across the United States and examined for foliar retention, absorption, translocation, and metabolism of glyphosate. Initial studies used spray application of 14C-glyphosate to simulate field application. When S and R biotypes were compared in the absence of toxicity at a sublethal dose, we observed comparable retention and absorption but reduced root translocation in the R biotypes. S and R biotypes from Delaware were further examined at field use rates and results confirmed similar retention and absorption but reduced root translocation in the R biotypes. Application of 14C-glyphosate to a single leaf demonstrated reduced export out of the treated leaf and lower glyphosate import into other leaves, the roots, and the crown in R relative to S biotypes. Examination of the treated leaf by autoradiography showed that glyphosate loading into the apoplast and phloem was delayed and reduced in the R biotype. Our results consistently showed a strong correlation between impaired glyphosate translocation and resistance. Tissues from both S and R biotypes showed elevated levels of shikimate suggesting that 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) remained sensitive to glyphosate. Analysis of tissue shikimate levels demonstrated reduced efficiency in EPSPS inhibition in the R biotypes. Our results suggest that resistance is likely due to altered cellular distribution that impaired phloem loading and plastidic import of glyphosate resulting in reduced overall translocation as well as inhibition of EPSPS.

Rigid ryegrass, an important annual weed species in cropping regions of southern Australia, has evolved resistance to 11 major groups of herbicides. Dose–response studies were conducted to determine response of three clethodim-resistant populations and one clethodim-susceptible population of rigid ryegrass to three different frost treatments (−2 C). Clethodim-resistant and -susceptible plants were exposed to frost in a frost chamber from 4:00 P.M. to 8:00 A.M. for three nights before or after clethodim application and were compared with plants not exposed to frost. A reduction in the level of clethodim efficacy was observed in resistant populations when plants were exposed to frost for three nights before or after clethodim application. In the highly resistant populations, the survival percentage and LD50 were higher when plants were exposed to frost before clethodim application compared with frost after clethodim application. However, frost treatment did not influence clethodim efficacy of the susceptible population. Sequencing of the acetyl coenzyme A carboxylase (ACCase) gene of the three resistant populations identified three known mutations at positions 1781, 2041, and 2078. However, most individuals in the highly resistant populations did not contain any known mutation in ACCase, suggesting the resistance mechanism was a nontarget site. The effect of frost on clethodim efficacy in resistant plants may be an outcome of the interaction between frost and the clethodim resistance mechanism(s) present.

The physiological basis of glufosinate resistance for two resistant (R) rice mutants, lines ‘R11-2’ and ‘R11-3’, was studied. Seven days after the application of 0.54 mM glufosinate, two susceptible (S) lines, i.e., variety (var.) ‘FSK’ and its inbred line ‘FSK-3’, and a reference var. Tainung 67 (TNG 67) suffered severe injury, whereas the two R lines exhibited resistance. Dose–response analysis and survival rate 14 d after treatment with 1.5 mM glufosinate also supported this observation. A 14C-glufosinate experiment showed that more labeled herbicide was absorbed by leaves of R11-2 than S lines 48 h after treatment (HAT), but the partitioning of absorbed glufosinate to each part of the shoot did not differ between R and S lines. Although a higher degradation of glufosinate in R line R11-2 was found as compared with the two S lines, i.e., 46% vs. 38 to 40%, the actual concentration of glufosinate in R line was still higher than that in S lines. Foliar application of glufosinate resulted in less inhibition of in vivo activity of glutamine synthetase (GS; EC 6.3.1.2) as well as a lower accumulation of ammonium 24 HAT in R line than in S lines. Further kinetic study of GS showed that cytosolic GS in line R11-2, with a higher enzyme-inhibition constant (Ki) value to glufosinate, was less sensitive to the toxic action of this herbicide. Therefore, a higher metabolism of, and more important, a lower susceptibility of, the target protein GS to this herbicide are suggested to contribute significantly to glufosinate resistance in these rice lines.

Landoltia was collected and cultured from a canal in Lake County, Florida, where diquat was used repeatedly during the past 20–30 yr for duckweed control. Recent applications of diquat failed to provide adequate control of duckweed, and a new commercial formulation of diquat was suspected. The new formulation was not the cause of reduced efficacy. Static exposures (48 h) to various concentrations of diquat were used to compare the susceptibility of the Lake County landoltia accession to one never exposed to diquat. These static tests indicated that landoltia, from a population with no prior history of herbicide treatment, was extremely susceptible to diquat. The accession from Lake County, FL had developed resistance to diquat, and was also cross resistant to paraquat. The resistance factor was 50 for diquat and 29 for paraquat. The Lake County accession also exhibited reduced ion leakage after diquat exposure under light and dark conditions. This suggests the resistance mechanism to the bipyridylium herbicides in landoltia is independent of photosynthetic electron transport. This research documents the first aquatic plant species that has developed resistance to the bipyridylium herbicides.

Greenhouse studies were established in Fayetteville, AR, to investigate glyphosate resistance in Arkansas common ragweed populations. Common ragweed seed were collected from plants in Pope and Jackson counties in Arkansas. Plants grown from seed were sprayed with one of seven glyphosate rates. Populations in Pope and Jackson counties were 21-fold and 10-fold more tolerant to glyphosate, respectively, than a known susceptible population. Based on 14C-glyphosate absorption and translocation studies, reduced glyphosate absorption or translocation was not the resistance mechanism in Arkansas glyphosate-resistant common ragweed. Shikimate accumulation did not differ among the known susceptible and the two resistant populations at 3 d after treatment (DAT). However, by 5 DAT, shikimate accumulation in the two resistant populations was lower than the known susceptible population. Data indicate that glyphosate-resistant common ragweed is present in at least two locations in Arkansas, and the resistance mechanism is not an insensitive target site or reduced glyphosate absorption or translocation.

A suspected glyphosate-resistant Italian ryegrass biotype was collected from a filbert orchard near Portland, OR, where glyphosate was applied multiple times per year for about 15 yr. Greenhouse studies were conducted to determine if this biotype was glyphosate resistant. The plants were sprayed with glyphosate (0.01 to 3.37 kg ae ha−1) 14 d after planting and shoot biomass was determined 3 wk after herbicide treatment. Based on the dose–response experiments conducted in the greenhouse, the suspected Italian ryegrass biotype was approximately fivefold more resistant to glyphosate than the susceptible biotype. Plants from both susceptible and resistant biotypes were treated with glyphosate (0.42 and 0.84 kg ha−1) and shikimic acid was extracted 12, 24, 48, and 96 h after treatment. The susceptible biotype accumulated between three and five times more shikimic acid than did the resistant biotype. Leaf segments from both susceptible and resistant biotypes were incubated with different glyphosate concentrations (0.5 to 3000 μM) for 14 h under continuous light. Shikimic acid was extracted from each leaf segment and quantified. At a concentration up to 100 μM, leaf segments from the susceptible biotype accumulated more shikimic acid than leaf segments from the resistant biotype. The epsps gene was amplified and sequenced in both susceptible and resistant biotypes; however, no amino acid change was found in the resistant biotype. The level of resistance in this biotype is similar to that reported for a glyphosate-resistant Italian ryegrass biotype from Chile.

Clethodim resistance was identified in 12 rigid ryegrass populations from winter cropping regions in four different states of Australia. Clethodim had failed to provide effective control of these populations in the field and resistance was suspected. Dose–response experiments confirmed resistance to clethodim and butroxydim in all populations. During 2012, the LD50 of resistant populations ranged from 10.2 to 89.3 g ha−1, making them 3 to 34–fold more resistant to clethodim than the susceptible population. Similarly, GR50 of resistant population varied from 8 to 37.1 g ha−1, which is 3 to 13.9–fold higher than the susceptible population. In 2013, clethodim-resistant populations were 7.8 to 35.3–fold more resistant to clethodim than the susceptible population. The higher resistance factor in 2013, especially in moderately resistant populations, could have been associated with lower ambient temperatures during the winter of 2013. These resistant populations had also evolved cross-resistance to butroxydim. The resistant populations required 1.3 to 6.6–fold higher butroxydim dose to achieve 50% mortality and 3 to 27–fold more butroxydim for 50% biomass reduction compared to the standard susceptible population. Sequencing of the target-site ACCase gene identified five known ACCase substitutions (isoleucine-1781-leucine, isoleucine-2041-asparagine, aspartate-2078-glycine, and cysteine-2088-arginine, and glycine-2096-alanine) in these populations. In nine populations, multiple ACCase mutations were present in different individuals. Furthermore, two alleles with different mutations were present in a single plant of rigid ryegrass in two populations.

A population of common ragweed from Delaware was not controlled in the field by herbicides that inhibit acetolactate synthase (ALS) or protoporphyrinogen oxidase (PPO). Research was conducted to ascertain if this population was resistant to these herbicidal modes of action and, if so, to determine the resistance mechanism(s). Resistance was confirmed by dose-response studies on greenhouse-grown plants with multiple ALS- and PPO-inhibiting herbicides. DNA sequence data revealed that resistance to ALS-inhibiting herbicides was due to the previously reported W574L ALS mutation. To assist in determining the mechanism of resistance to PPO-inhibiting herbicides, an F2 population was derived from a cross between the resistant biotype (Del-R) and a sensitive biotype (DV1-S). This population segregated in the ratio of three resistant : one sensitive when treated with fomesafen, indicating that resistance to PPO-inhibiting herbicides was conferred by a single, (incompletely) dominant, nuclear gene. Sequences of the target-site genes, PPX1 and PPX2, for PPO-inhibiting herbicides were obtained through the screening of a common ragweed cDNA library and subsequent cDNA extension (5′-RACE). Molecular marker analysis with the F2 population revealed that the PPX2 gene cosegregated with resistance to PPO-inhibiting herbicides. A mutation substituting an arginine codon for a leucine codon at a conserved location (R98L) of the PPX2 gene was suspected of being responsible for resistance. By using a transgenic Escherichia coli system, it was demonstrated that the R98L mutation was sufficient to confer resistance to PPO-inhibiting herbicides. The level of resistance to acifluorfen conferred by the R98L mutation in the E. coli system was about 31-fold, similar to the level of resistance seen in the whole-plant dose-response study. Last, a DNA-based assay was developed to identify the presence or absence of the common ragweed PPX2 R98L mutation. The R98L PPX2 mutation is the second mechanism identified for evolved resistance to PPO-inhibiting herbicides.