Blackgrass (Alopecurus myosuroides Huds.)

In A. myosuroides, the Trp-2027-Cys amino acid substitution conferred resistance to FOPs and DEN but not DIMs (Li et al. Reference Li, Du, Liu, Yuan and Wang2014; Petit et al. Reference Petit, Bay, Pernin and Délye2010). In contrast, the Ile-2041-Asn mutation confers resistance to fenoxaprop, clodinafop, and haloxyfop, but plants were only moderately resistant to pinoxaden and sensitive to cycloxydim (Délye et al. Reference Délye, Matéjicek and Michel2008; Petit et al. Reference Petit, Bay, Pernin and Délye2010). The Ile-1781-Leu mutation in A. myosuroides conferred resistance to all G1 herbicides, except clethodim at the labeled rate (120 g ha⁻¹) in Europe (Délye et al. Reference Délye, Matéjicek and Michel2008; Scarabel et al. Reference Scarabel, Panozzo, Varotto and Sattin2011). The Gly-2096-Ala mutation endowed resistance to fenoxaprop, clodinafop, haloxyfop, and cycloxydim, while some individuals survived a full rate of clethodim (120 g ha⁻¹), leading the authors to speculate that possible control failures may occur in the field with clethodim if the rate is reduced or grasses are taller than the recommended weed height at application (Délye et al. Reference Délye, Matéjicek and Michel2008). Analysis of a large number of A. myosuroides populations from France has shown NTSR was prevalent and that fenoxaprop was more affected than clodinafop and pinoxaden (Petit et al. Reference Petit, Bay, Pernin and Délye2010).

Lolium spp

Italian Ryegrass [Lolium perenne L. ssp. multiflorum (Lam.) Husnot], Rigid Ryegrass (Lolium rigidum Gaudin), and Perennial Ryegrass (Lolium perenne L.)

In L. perenne ssp. multiflorum, the Trp-2027-Leu amino acid substitution conferred resistance to FOPs but not DIMs or DEN (Kaundun et al. Reference Kaundun, Downes, Jackson, Hutchings and Mcindoe2021). The Ile-1781-Leu mutation in L. perenne ssp. multiflorum conferred resistance to all G1 herbicides, except clethodim at the labeled rate (120 g ha⁻¹) in Europe (Délye et al. Reference Délye, Matéjicek and Michel2008; Scarabel et al. Reference Scarabel, Panozzo, Varotto and Sattin2011). The Trp-1999-Ser mutation in a different L. perenne ssp. multiflorum population conferred high levels of resistance to pinoxaden and FOPs and partial resistance to sethoxydim and cycloxydim while being sensitive to tepraloxydim and clethodim (Kaundun et al. 2013).

In Australia, heterozygous L. rigidum plants carrying the mutant Ile-1781-Leu allele were killed by clethodim (60 g ai ha⁻¹), while most homozygous individuals survived (Yu et al. Reference Yu, Cairns and Powles2007a, Reference Yu, Collavo, Zheng, Owen, Sattin and Powles2007b). In an L. perenne population, metabolic resistance affected the efficacy of pinoxaden but not clethodim (Ghanizadeh et al. Reference Ghanizadeh, Buddenhagen, Harrington, Griffiths and Ngow2022). Metabolic resistance is typically favored under low-dose selection, especially for the less potent G1 herbicides such as diclofop-methyl (Neve and Powles Reference Neve and Powles2005). In cool-season grasses, enhanced metabolism affects predominantly cereal- and dicot-selective G1 herbicides, such as diclofop, fenoxaprop, clodinafop, tralkoxydim, and pinoxaden; in contrast, dicot only–selective cycloxydim, sethoxydim, and clethodim are less affected (Kaundun Reference Kaundun2014). For example, enhanced metabolism identified in the L. rigidum population SLR31 affected most cereal- and dicot-selective FOP and DIM herbicides but not sethoxydim (Vila-Aiub et al. Reference Vila-Aiub, Neve, Steadman and Powles2005). A similar observation was made for NTSR L. perenne ssp. multiflorum populations from the United Kingdom surviving diclofop, clodinafop, and tralkoxydim but remaining sensitive to cycloxydim and pinoxaden (Hull et al. Reference Hull, Tatnell, Cook, Beffa and Moss2014). Furthermore, a L. perenne ssp. multiflorum population with individuals showing high levels of NTSR to FOPs, partial resistance to pinoxaden and cycloxydim, and sensitivity to tepraloxydim and clethodim has been documented (Kaundun et al. Reference Kaundun, Downes, Jackson, Hutchings and Mcindoe2021). In Australia, despite a high frequency of resistance to FOP herbicides, clethodim remains widely used to control L. rigidum (Broster et al. Reference Broster, Boutsalis, Gill and Preston2022).

Wild Oat (Avena fatua L.)

The Ile-2041-Asn mutation in A. fatua from Mexico conferred resistance to FOPs and DIMs but not pinoxaden (Cruz-Hipolito et al. Reference Cruz-Hipolito, Osuna, Dominguez-Valenzuela, Espinoza and De Prado2011). In contrast, the Asp-2078-Gly mutation in A. fatua from Chile conferred cross-resistance to all three ACCase families. Lower resistance indices for DIMs and DEN in A. fatua could be explained by a ploidy and dilution effect (i.e., due to homoeologous ACCase genes) of the TSR mechanisms in this species (Yu et al. Reference Yu, Ahmad-Hamdani, Han, Christoffers and Powles2013). In A. fatua, ACCase genes from all three genomes are expressed and, for example, a single Ile-2041-Asn mutation may not be sufficient to endow resistance to DIMs and pinoxaden.

Large Crabgrass [Digitaria sanguinalis (L.) Scop.]

In greenhouse and field trials conducted in Ontario, Canada, D. sanguinalis was resistant to all G1 herbicides (FOPs, DIMs, and DEN) with the exception that clethodim (DIM) was still effective (Figure 1). Group 1 resistance in this D. sanguinalis population is thus active ingredient-specific within the DIM herbicides; preliminary unpublished research indicates resistance is due to gene overexpression (M Laforest, personal communication).

Figure 1. Control of Digitaria sanguinalis with quizalofop-p-ethyl (left) and clethodim (right) in a field in Ontario, Canada.

Other Examples of G1 Resistance

In Japanese foxtail (Alopecurus japonicus Steud.), the Trp-2027-Cys amino acid substitution conferred resistance to fenoxaprop, clodinafop-propargyl, fluazifop-P-butyl, quizalofop-P-ethyl, haloxyfop-R-methyl, cyhalofop-butyl, metamifop, and pinoxaden but not clethodim and sethoxydim (Xu et al. Reference Xu, Zhu, Wang, Li and Dong2013). In slough grass [Beckmannia syzigachne (Steud.) Fernald], the Trp-2027-Cys amino acid substitution conferred resistance to the FOPs and DEN but not pinoxaden (Li et al. Reference Li, Du, Liu, Yuan and Wang2014; Petit et al. Reference Petit, Bay, Pernin and Délye2010). The Trp-1999-Cys mutation affected the efficacy of fenoxaprop in sterile oat (Avena sterilis L.), but plants were sensitive to clodinafop-propargyl and sethoxydim (Liu et al. Reference Liu, Harrison, Chalupska, Gornicki, O’Donnell, Adkins, Haselkorn and Williams2007). Resistance to cyhalofop-butyl has been detected at a low frequency (3% or 12%, depending on the survey) in Echinochloa spp. in Arkansas rice fields (Rouse et al. Reference Rouse, Roma-Burgos, Norsworthy, Tseng, Starkey and Scott2018). In a barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] population from China, a TSR mechanism via amino acid substitution (Asp-2078-Glu) conferred resistance to FOP and DEN herbicides but not metamifop (Fang et al. Reference Fang, He, Liu, Li and Dong2020). In two E. crus-galli populations collected from the U.S. Midsouth, an NTSR mechanism conferred resistance to FOPs (Hwang et al. Reference Hwang, Norsworthy, González-Torralva, Piveta, Barber and Butts2022).

Summary and Implications for Future Weed Management

Group 1 herbicides are far from being a homogeneous group with respect to herbicide resistance. Phenotypes of plants displaying TSR to G1 herbicides frequently depend on herbicide family, active ingredient, target-site amino acid substitution, ploidy level, and number of mutant ACCase alleles. Metabolic resistance is often unpredictable in conferring cross-resistance in weeds (Jugulam and Shyam Reference Jugulam and Shyam2019) and can be broad, class specific, or even active ingredient specific, depending on the species, resistance genes/enzymes involved and occasionally environmental conditions (Matzrafi et al. Reference Matzrafi, Seiwert, Reemtsma, Rubin and Peleg2016; Refatti et al. Reference Refatti, de Avila, Camargo, Ziska, Oliveira, Salas-Perez, Rouse and Roma-Burgos2019). The unpredictable nature of metabolic resistance in weeds may also be due to the mostly unknown substrate specificity, or lack of specificity (Atkins Reference Atkins2020; Brazier-Hicks et al. Reference Brazier-Hicks, Franco-Ortega, Watson, Rougemont, Cohn, Dale, Hawkes, Goldberg-Cavalleri, Onkokesung and Edwards2022), of Phase I or II herbicide-metabolizing enzymes or Phase III parent herbicide/herbicide–metabolite transporters (Jugulam and Shyam Reference Jugulam and Shyam2019; Kreuz et al. Reference Kreuz, Tommasini and Martinoia1996; Yu and Powles Reference Yu and Powles2014); these areas clearly warrant additional physiological research.

Barnyardgrass (Echinochloa crus-galli)

Echinochloa crus-galli populations have evolved resistance to G2 herbicides via an altered target site, either an Ala-122-Val or Ala-122-Thr substitution (Riar et al. Reference Riar, Norsworthy, Srivastava, Nandula, Bond and Scott2013). One population (Ala-122-Val) was resistant to IMIs but sensitive to PTBs and TPs. Conversely, the other E. crus-galli population (Ala-122-Thr) was resistant to IMIs and TPs but sensitive to PTBs. A separate study documented the Ala-122-Asn amino acid substitution in the ALS gene, which conferred significant cross-resistance to each G2 herbicide (IMI, PTB, TP, and SU) (Panozzo et al. Reference Panozzo, Scarabel, Rosan and Sattin2017).

Common Ragweed (Ambrosia artemisiifolia L.)

A study conducted in Illinois reported imazamox (IMI) resistance was greater than cloransulam-methyl (TP) resistance in A. artemisiifolia populations (Zheng et al. Reference Zheng, Patzoldt and Tranel2005). The study revealed that only 54% of the imazamox-resistant plants contained one or more Leu-574 ALS alleles, and the presence of a Leu-574 allele contributing to imazamox resistance depended on the population. Leu-574 alleles were not identified in plants from populations resistant to imazamox but sensitive to cloransulam-methyl, indicating these populations had a different mechanism of G2 resistance. In contrast, the Leu-574 ALS allele was determined to be the primary factor for cloransulam-methyl resistance in A. artemisiifolia.

Downy Brome (Bromus tectorum L.)

A population of B. tectorum from Montana exhibited 5-fold resistance (low level) to pyroxsulam (TP), 14-fold resistance (moderate) to propoxycarbazone-sodium (SCT), and 110-fold resistance (high) to imazamox (IMI) (Kumar and Jha Reference Kumar and Jha2017). Sequence analysis revealed a single target-site mutation, Ser-653-Asn, in resistant plants. Interestingly, this population remained sensitive to the SU herbicide, sulfosulfuron. In a study conducted in Oregon, a B. tectorum population exhibited high resistance to the SU herbicides, primisulfuron-methyl and sulfosulfuron, and the SCT herbicide, propoxycarbazone-sodium, but was sensitive to the IMI herbicide, imazamox. Resistance was attributed to a Pro-197-Ser mutation (Park and Mallory-Smith Reference Park and Mallory-Smith2004).

Eastern Black Nightshade (Solanum ptycanthum Dunal)

Solanum ptycanthum populations from Illinois, Indiana, and Wisconsin displayed high levels of resistance (>100-fold) to IMI herbicides in the greenhouse, but either lacked cross-resistance to other G2 herbicides (Milliman et al. Reference Milliman, Riechers, Wax and Simmons2003) or displayed low-level resistance to the sulfonylurea herbicide, primisulfuron-methyl (5.9-fold; Volenberg et al. Reference Volenberg, Stoltenberg and Boerboom2000). In the populations from Illinois and Indiana, a single base pair mutation in the ALS gene led to an Ala-122-Thr substitution (Milliman et al. Reference Milliman, Riechers, Wax and Simmons2003), which is known to confer IMI-specific resistance (Powles and Yu Reference Powles and Yu2010).

Mayweed Chamomile (Anthemis cotula L.)

Studies were conducted to characterize resistance of six A. cotula populations collected across the U.S. Pacific Northwest to three classes of G2 herbicides: SUs, IMIs, and TPs (Intanon et al. Reference Intanon, Perez-Jones, Hulting and Mallory-Smith2011). The findings revealed cross-resistance to thifensulfuron + tribenuron/chlorsulfuron (SUs), imazethapyr (IMI), and cloransulam-methyl (TP), albeit with varying degrees of resistance within each herbicide class and population. Resistance was solely attributed to mutations in ALS1, as mutations were not found in ALS2. The amino acid substitutions at Pro-197 differed among the resistant populations, and multiple mutations were detected within a single resistant population. This study highlights genotypic variation associated with cross-resistance to G2 herbicides both within and among populations.

Amaranthus spp

Rigid ryegrass (Lolium rigidum)

The first occurrence of G2-resistant L. rigidum was documented in Australia in 1982 (Heap Reference Heap2024). In Western Australia, researchers identified six ALS mutations in L. rigidum that contribute to resistance: Pro-197-Ala, Pro-197-Arg, Pro-197-Gln, Pro-197-Leu, Pro-197-Ser, and Trp-574-Leu (Yu et al. Reference Yu, Han and Powles2008). Each Pro-197 mutation was associated with sulfometuron (SU) resistance, while the Trp-574-Leu mutation conferred resistance to sulfometuron (SU) and imazapyr (IMI).

Wild Radish (Raphanus raphanistrum L.)

In Western Australia, researchers identified five G2 resistance–conferring mutations (Pro-197-Ala, Pro-197-Thr, Pro-197-Ser, Asp-376-Glu, and Trp-574-Leu) in R. raphanistrum (Yu et al. Reference Yu, Han, Li, Purba, Walsh and Powles2012). Plants carrying homozygous ALS mutations at Pro-197 exhibited cross-resistance to SU and TP herbicides. Similarly, plants homozygous for the Trp-574-Leu mutation demonstrated resistance to SU, TP, and IMI herbicide families. Raphanus raphanistrum plants homozygous for Asp-376-Glu displayed high resistance to chlorsulfuron (SU; R/S = 172) and metosulam (TP; R/S >110). In contrast, these plants had moderate resistance to imazamox (IMI; R/S = 3) and imazethapyr (IMI; R/S = 8), while remaining sensitive to imazapyr (IMI; R/S = 0.76).

Other Examples of G2 Resistance

Amino acid substitutions at position Pro-197 in ALS1 conferred G2 resistance in common groundsel (Senecio vulgaris L.) from France (Délye et al. Reference Délye, Causse and Michel2016). A Pro-197-Leu substitution conferred resistance to all SU herbicides tested, as well as imazamox and thiencarbazone, but not florasulam. However, a Pro-197-Ser substitution conferred resistance to all SU herbicides tested as well as florasulam and thiencarbazone, but not imazamox. A Pro-197-Gln substitution in common chickweed [Stellaria media (L.) Vill.] from the United Kingdom was associated with resistance to metsulfuron (SU), but not florasulam (TP), whereas a Trp-574-Leu substitution conferred resistance to both herbicides (Marshall et al. Reference Marshall, Hull and Moss2010).

Negative Cross-Resistance to Other G2 Herbicides

Negative cross-resistance occurs when a weed population resistant to a herbicide becomes more sensitive to other herbicides; these herbicides may have the same or different sites of action (Poston et al. Reference Poston, Hirata and Wilson2002). An understanding of negative cross-resistance is crucial for weed management, because it reveals alternative herbicides that could still control resistant weeds. It also highlights the need to diversify herbicide use and employ integrated weed management strategies to reduce herbicide resistance.

G2 Resistance and Negative Cross-Resistance to G14 and G27 Herbicides

Summary and Implications for Future Weed Management

Resistance to G2 herbicides can evolve in weeds after relatively few applications. Several TSR (ALS mutations) and NTSR-based mechanisms confer resistance to the G2 herbicides. The level of resistance and cross-resistance to other G2 herbicides can vary among the resistance mechanisms present in weed populations. This variation underscores the complexity of managing G2-resistant weeds and the critical information that is often lost due to generalization across the six ALS-inhibiting herbicide families and 58 active ingredients. Additionally, occasional negative cross-resistance to other G2 or G14 and G27 herbicides indicates the need for a detailed understanding of resistance mechanisms to effectively address G2 resistance issues.

Blackgrass (Alopecurus myosuroides)

Alopecurus myosuroides populations from the United Kingdom display large variations in resistance among the G3 (dinitroaniline) herbicides. One population (Peldon) has a high level of resistance to pendimethalin and lower resistance to oryzalin and butralin, but is sensitive to trifluralin, ethalfluralin, and isopropalin (Moss Reference Moss1990). This population was presumed to have NTSR to the dinitroaniline herbicides. Further studies showed this population was resistant to dinitroanilines with a ring-methyl substitution but sensitive to those containing a 4-trifluoromethyl group (James et al. Reference James, Kemp and Moss1995). It was postulated that resistance was the result of enhanced metabolism of dinitroaniline herbicides at the 4-methyl position. In contrast, some TSR mutations provide broad cross-resistance to the dinitroaniline herbicides (Chen et al. Reference Chen, Yu, Patterson, Sayer and Powles2021).

Rigid ryegrass (Lolium rigidum)

Lolium rigidum in Australia has evolved resistance to trifluralin through TSR and NTSR mechanisms. A population (SLR31) without TSR (Fleet et al. Reference Fleet, Malone, Preston and Gill2018) had higher resistance (8- to 15-fold) to trifluralin, pendimethalin, and ethalfluralin, but lower resistance (2- to 4-fold) to oryzalin and isopropalin (McAlister et al. Reference McAlister, Holtum and Powles1995). This population was subsequently shown to be resistant due to enhanced metabolism of trifluralin by P450s (Chen et al. Reference Chen, Goggin, Han, Busi, Yu and Powles2018). Populations of L. rigidum with TSR have higher levels of trifluralin resistance than those with only NTSR (Chen et al. Reference Chen, Chu, Han, Goggin, Yu, Sayer and Powles2020; Fleet et al. Reference Fleet, Malone, Preston and Gill2018). An L. rigidum population with Val-202-Phe and Thr-239-Ile mutations in α-tubulin had 30-fold resistance to trifluralin and oryzalin, but only 12-fold resistance to pendimethalin and ethalfluralin (Chen et al. Reference Chen, Chu, Han, Goggin, Yu, Sayer and Powles2020). However, populations of L. rigidum with both TSR and NTSR to the dinitroaniline herbicides occur and tend to have higher levels of resistance to trifluralin (Chen et al. Reference Chen, Chu, Han, Goggin, Yu, Sayer and Powles2020, Reference Chen, Yu, Owen, Han, Patterson, Sayer and Powles2022).

Significantly, most L. rigidum populations in Australia remain sensitive to pronamide. Random collections of field populations from South Australia identified high frequencies of resistance to trifluralin; however, all populations were completely controlled by pronamide (Figure 2). This finding indicates that none of the mechanisms present in populations selected with trifluralin confer field-effective cross-resistance to pronamide in L. rigidum. The lack of cross-resistance to pronamide has led to this herbicide being frequently used to control L. rigidum populations resistant to trifluralin in pulse crops in southern Australia (Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2018).

Figure 2. Emergence of Lolium rigidum populations collected randomly from crop fields in southeastern South Australia. Each population was treated with the Group 3 herbicides, trifluralin and pronamide, preemergence. These findings demonstrate resistance to trifluralin but not pronamide in the field-collected populations above. Untreated (left); treated with 800 g ha⁻¹ trifluralin (middle); treated with 500 g ha⁻¹ pronamide (right). Each tray contains samples collected from 20 fields with the sample from an individual field sown in a single cell. The same 20 populations are planted in each tray. Each herbicide rate used represents the lowest labeled rate for weed management in no-till grain crop production in Australia.

Poa annua and Goosegrass [Eleusine indica (L.) Gaertn.]

Poa annua has evolved resistance to prodiamine, dithiopyr, and pronamide (McCullough et al. Reference McCullough, Yu and Czarnota2017). Little is known about resistance mechanisms to prodiamine or dithiopyr in P. annua. In some pronamide-resistant populations, mutations have not been identified in α- or β-tubulin (Barua et al. Reference Barua, Boutsalis, Malone, Gill and Preston2020; Singh et al. Reference Singh, Dos Reis, Reynolds, Elmore and Bagavathiannan2021). Resistance to pronamide is associated with reduced herbicide translocation in some populations (Ignes et al. Reference Ignes, McCurdy, McElroy, Castro, Ferguson, Meredith, Rutland, Stewart and Tseng2023; McCullough et al. Reference McCullough, Yu and Czarnota2017). A Thr-239-Ile mutation in α-tubulin was identified in several P. annua populations with pronamide resistance (Ignes et al. Reference Ignes, McCurdy, McElroy, Castro, Ferguson, Meredith, Rutland, Stewart and Tseng2023). However, this mutation was identified in a sensitive population, indicating the Thr-239-Ile mutation is not the sole factor for resistance. This conclusion is supported by a dinitroaniline-resistant E. indica population containing a Thr-239-Ile mutation in α-tubulin, which had high resistance to trifluralin and oryzalin but sensitivity to pronamide (Anthony et al. Reference Anthony, Waldin, Ray, Bright and Hussey1998).

Summary and Implications for Future Weed Management

As considerable variation exists in the mechanisms and levels of resistance to individual G3 herbicides in resistant weeds, this variability provides an opportunity to use some G3 herbicides in the short term to control weed populations that are resistant to other members of the G3 group. For example, the lack of cross-resistance to pronamide has led to this herbicide being frequently used to control L. rigidum populations resistant to trifluralin in pulse crops in southern Australia (Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2018). However, we acknowledge that the ability of outcrossing weed species (such as L. rigidum and A. myosuroides) to accumulate resistance alleles through cross-pollination means these strategies may need to be monitored and altered as necessary to control more complex G3 resistance patterns that may evolve in the future.

Amaranthaceae Family: Amaranthus tuberculatus, Amaranthus palmeri, and Bassia scoparia

Brassicaceae Family: Wild Mustard (Sinapis arvensis L.), Raphanus raphanistrum, and Oriental Mustard (Sisymbrium orientale L.)

Weed species in the Brassicaceae family have evolved resistance to G4 herbicides in Canada and Australia (Heap Reference Heap2024). An S. arvensis population found in a wheat (Triticum aestivum L.)/barley (Hordeum vulgare L.) field with prolonged history of G4 herbicide use in Canada was cross-resistant to 2,4-D, dicamba and picloram (Peniuk et al. Reference Peniuk, Romano and Hall1993). Several R. raphanistrum populations in Australia have evolved resistance to 2,4-D and MCPA (Heap Reference Heap2024). Although both herbicides belong to the phenoxy subclass of G4 herbicides, resistance mechanisms to these herbicides are not the same (Goggin et al. Reference Goggin, Cawthray and Powles2016, Reference Goggin, Kaur, Owen and Powles2018; Jugulam et al. Reference Jugulam, DiMeo, Veldhuis, Walsh and Hall2013) but likely involve alterations in auxin signaling pathways as well as cellular transport and whole-plant translocation. Two S. orientale populations from southern Australia are resistant to 2,4-D (Dang et al. Reference Dang, Malone, Boutsalis, Gill and Preston2017); these two populations display a high level of 2,4-D resistance (67- to 81-fold), although cross-resistance to other G4 herbicides was not reported. Both TSR and NTSR mechanisms were found in this population (Dang et al. Reference Dang, Malone, Boutsalis, Gill and Preston2017, Reference Dang, Malone, Boutsalis, Krishnan, Gill and Preston2018; Figueiredo et al. Reference Figueiredo, Küpper, Malone, Petrovic, Figueiredo, Campagnola and Gaines2022).

Compositae Family: Yellow Starthistle (Centaurea solstitialis L.) and Tall Fleabane [Conyza sumatrensis (Retz.) E. Walker]

A population of C. solstitialis resistant to picloram was discovered in Washington State in 1988 following 10 years of repeated picloram treatments in a pasture (Callihan et al. Reference Callihan, Schirman and Northam1990). This population displayed cross-resistance to foliar-applied clopyralid, fluroxypyr, and dicamba, but not triclopyr or 2,4-D (Fuerst et al. Reference Fuerst, Sterling, Norman, Prather, Irzyk, Wu and Callihan1996). Interestingly, this population is>3.5-fold more resistant to clopyralid than picloram, although both herbicides belong to the pyridine-carboxylate subfamily (Fuerst et al. Reference Fuerst, Sterling, Norman, Prather, Irzyk, Wu and Callihan1996). Furthermore, inheritance studies indicate that clopyralid resistance in C. solstitialis is controlled by a single nuclear recessive gene (Sabba et al. Reference Sabba, Ray, Lownds and Sterling2003). It was postulated that pyridine-carboxylate–specific resistance may be due to alterations in specific binding site(s) of these herbicides (Sabba et al. Reference Sabba, Ray, Lownds and Sterling2003; Walsh et al. Reference Walsh, Neal, Merlo, Honma, Hicks, Wolff, Matsumura and Davies2006), which is discussed in more detail in the “Summary and Implications for Future Weed Management” for Group 4.

Corn Poppy (Papaver rhoeas L.)

Intense selection pressure from 2,4-D and tribenuron-methyl (a G2 herbicide; SU) resulted in the evolution of 2,4-D– and tribenuron-methyl–resistant P. rhoeas in Spain (Rey-Caballero et al. Reference Rey-Caballero, Menéndez, Giné-Bordonaba, Salas, Alcántara and Torra2016; Torra et al. Reference Torra, Rojano-Delgado, Rey-Caballero, Royo-Esnal, Salas and De Prado2017). Cross-resistance to other G4 herbicides is not common in the populations studied to date, although two 2,4-D–resistant P. rhoeas populations exhibited resistance to dicamba and aminopyralid (Rey-Caballero et al. Reference Rey-Caballero, Menéndez, Giné-Bordonaba, Salas, Alcántara and Torra2016). The level of 2,4-D resistance in P. rhoeas varied from 7- to 40-fold, depending on population (Rey-Caballero et al. Reference Rey-Caballero, Menéndez, Giné-Bordonaba, Salas, Alcántara and Torra2016; Torra et al. Reference Torra, Rojano-Delgado, Rey-Caballero, Royo-Esnal, Salas and De Prado2017). Reduced translocation and rapid 2,4-D metabolism were reported resistance mechanisms (Rey-Caballero et al. Reference Rey-Caballero, Menéndez, Giné-Bordonaba, Salas, Alcántara and Torra2016; Torra et al. Reference Torra, Rojano-Delgado, Rey-Caballero, Royo-Esnal, Salas and De Prado2017, Reference Torra, Rojano-Delgado, Menéndez, Salas and de Prado2021).

Summary and Implications for Future Weed Management

Despite more than seven decades of commercial use of G4, evolution resistance to this group has been relatively low (44 species total) compared with, for example, resistance in the G1, G2, and G5 groups. Both TSR and NTSR mechanisms have been reported (Figueiredo et al. Reference Figueiredo, Leibhart, Reicher, Tranel, Nissen, Westra, Bernards, Kruger, Gaines and Jugulam2018, Reference Figueiredo, Küpper, Malone, Petrovic, Figueiredo, Campagnola and Gaines2022; Goggin et al. Reference Goggin, Kaur, Owen and Powles2018; Leclere et al. Reference LeClere, Wu, Westra and Sammons2018; Shyam et al. Reference Shyam, Peterson and Jugulam2022). Levels of resistance or cross-resistance patterns to G4 herbicides within and among dicot species and populations is complicated, in part because multiple target-site proteins (six TIR1/AFBs) have been identified in Arabidopsis (Walsh et al. Reference Walsh, Neal, Merlo, Honma, Hicks, Wolff, Matsumura and Davies2006), although the precise number of target sites in most weed species is unknown. The relatively large number of potential G4 herbicide target sites, which display either specific or overlapping binding properties with G4 herbicide families in Arabidopsis (Walsh et al. Reference Walsh, Neal, Merlo, Honma, Hicks, Wolff, Matsumura and Davies2006), contributes to the complexity of cross-resistance within G4 compared with single site-of-action HRAC groups (i.e., G2, G9, and G27). For example, multiple alleles conferring resistance could evolve for each TIR1/AFB gene homologue in the same population or plant (Mithila et al. Reference Mithila, Hall, Johnson, Kelley and Riechers2011; Peterson et al. Reference Peterson, McMaster, Riechers, Skelton and Stahlmann2016).

Cross-resistance levels and patterns of resistance to G4 herbicides also depend on weed species or population and mechanism of resistance (TSR, NTSR, or a combination of both), which may be exacerbated by widespread use of G4 herbicides in numerous cropping systems (Geddes et al. Reference Geddes, Pittman, Hall, Topinka, Sharpe, Leeson and Beckie2023). However, potential fitness costs resulting from TSR mechanisms or impaired auxin signaling in response to G4 herbicides (Figueiredo et al. Reference Figueiredo, Küpper, Malone, Petrovic, Figueiredo, Campagnola and Gaines2022), combined with the recessive nature of some G4 resistance traits (Mithila et al. Reference Mithila, Hall, Johnson, Kelley and Riechers2011; Sabba et al. Reference Sabba, Ray, Lownds and Sterling2003), may limit the evolution and spread of additional G4-resistant species and populations.

Groundsel (Senecio vulgaris)

The first weed to evolve resistance to the G5 herbicides was S. vulgaris in 1968 (Ryan Reference Ryan1970). This S. vulgaris population evolved resistance to atrazine and simazine (triazines) applied preemergence or postemergence (0% control with both herbicides at 6.7 kg ai ha⁻¹); in contrast, this population was sensitive to chloroxuron and fluometuron (ureas) applied postemergence (100% control). This seminal research on G5 resistance concluded that this S. vulgaris population evolved resistance to triazines but not ureas.

Jimsonweed (Datura stramonium L.)

A population of D. stramonium that evolved resistance to atrazine and simazine (triazines) was sensitive to prometryn (triazine), metribuzin (triazinone), and terbacil (uracil) (Yerkes Reference Yerkes1995). Resistance in this population was influenced by the herbicide family (triazine vs. triazinone and uracil) and active ingredient within the triazine family (resistant to atrazine and simazine but sensitive to prometryn).

Common Lambsquarters (Chenopodium album L.)

The first documented case of G5-resistant C. album was in Ontario, Canada, in 1974 (Bandeen and McLaren Reference Bandeen and McLaren1976). This population evolved resistance to atrazine, cyanazine, simazine, and cyprazine (triazines) but was sensitive to metribuzin (triazinone) and linuron, metobromuron, and chlorbromuron (ureas). The authors concluded this G5-resistant C. album population was chemical family specific; it was resistant to triazines but sensitive to triazinones and ureas. The mechanism of resistance in this biotype has not been elucidated. In contrast, a C. album population from eastern Canada with the Ser-264-Gly mutation evolved resistance to the G5 herbicide metribuzin (triazinone) but was sensitive to linuron (urea) (McKenzie-Gopsill et al. Reference McKenzie-Gopsill, Graham, Laforest, Ibarra, Hann and Wagg2020). A C. album population from Germany with a Leu-218-Val substitution evolved resistance to the triazinones but was sensitive to terbuthylazine (triazine) (Thiel and Varrelmann Reference Thiel and Varrelmann2014); all C. album populations tested were more resistant to metribuzin than metamitron, both triazinone herbicides. Thus, G5-resistant C. album varies by population. The Ontario population is resistant to the triazine herbicides but sensitive to triazinone and urea herbicides; the eastern Canada population is resistant to triazinones but sensitive to linuron; and the population from Germany is the opposite of the Ontario population, resistant to triazinones but sensitive to triazines. These research findings clearly demonstrate it is an oversimplification to merely refer to G5-resistant C. album.

Figure 3. Control of Amaranthus tuberculatus with metribuzin (560 g ai ha⁻¹) with Group 5 resistance due to enhanced metabolism (left) and an altered target site (right) from two fields in Ontario, Canada.

Amaranthus spp

Wild radish (Raphanus raphanistrum)

A population of R. raphanistrum in Australia evolved resistance to atrazine and simazine (triazines) and metribuzin (triazinone) but was sensitive to diuron (urea) (Hashem et al. Reference Hashem, Dhammu, Powles, Bowran, Piper and Cheam2001; Walsh et al. Reference Walsh, Powles, Beard, Parkin and Porter2004). Further research by Lu et al. (Reference Lu, Yu, Han, Owen and Powles2019) determined metribuzin resistance was conferred by TSR (Ser-264-Gly) and NTSR (enhanced metabolism resulting in reduced translocation). The level of resistance in R. raphanistrum is amino acid substitution-specific; populations possessing the Ser-264-Gly substitution exhibit a higher level of resistance compared with populations possessing the Phe-274-Val substitution (Lu et al. Reference Lu, Yu, Han, Owen and Powles2018).

Shepherd’s Purse [Capsella bursa-pastoris (L.) Medik.]

Capsella bursa-pastoris evolved TSR to G5 herbicides via a Phe-255-Ile substitution in the D1 protein (Perez-Jones et al. Reference Perez-Jones, Intanon and Mallory-Smith2009). This substitution conferred resistance to hexazinone (triazinone) but not atrazine (triazine), terbacil (uracil), and diuron (urea) in C. bursa-pastoris; the authors concluded resistance was chemical family or active ingredient specific (Perez-Jones et al. Reference Perez-Jones, Intanon and Mallory-Smith2009).

Velvetleaf (Abutilon theophrasti Medik.)

Resistance in A. theophrasti to atrazine (triazine) was confirmed in populations from Maryland (Ritter Reference Ritter1986) and Wisconsin (Gray et al. Reference Gray, Stoltenberg and Balke1995) collected in 1986 and 1990, respectively. The mechanism of resistance was enhanced metabolism via GST activity (Gray et al. Reference Gray, Stoltenberg and Balke1995). Resistance factors for these populations were: atrazine, ≥99; simazine, ≥100; ametryne, ≤1.6; cyanazine, ≤1.1; metribuzin, ≤1.1; linuron, ≤1.0; and terbacil, ≤0.8. Group 5 resistance in A. theophrasti is thus active ingredient (within chemical family) specific (triazines: atrazine and simazine vs. ametrine and cyanazine) as well as chemical family specific (triazine vs. triazinone, urea, and uracil) (Gray et al. Reference Gray, Stoltenberg and Balke1995).

Other Examples of G5 Resistance

A population of S. orientale with a Ser-264-Gly amino acid substitution in D1 was resistant to atrazine (triazine) (≥300-fold) but sensitive to diuron (urea) (Dang et al. Reference Dang, Malone, Boutsalis, Gill and Preston2017). A S. arvensis population evolved resistance to atrazine (triazine) but was sensitive to diuron (urea) (Ali et al. Reference Ali, McLaren and Machado1986). An A. hybridus population with the Ser-264-Gly substitution in D1 was 1,000-fold resistant to atrazine (triazine) but sensitive to diuron (urea) (Gronwald Reference Gronwald, Powles and Holtum1994). A silvergrass [Vulpia bromoides (L.) Gray] population from western Australian with the Ser-264-Gly substitution in D1 evolved resistance to atrazine and simazine (triazines) (594-fold) and metribuzin (triazinone) but was sensitive to diuron (urea) (Ashworth et al. Reference Ashworth, Han, Knell and Powles2016). Collectively, these research findings demonstrate G5 resistance is chemical family (triazine or triazinone vs. urea) specific or active ingredient (atrazine vs. diuron) specific.

G5 Resistance and Negative Cross-Resistance to G6 Herbicides

Summary and Implications for Future Weed Management

The G5 herbicides are important for annual broadleaf weed control in numerous crops. The chemical family/active ingredient cross-resistance patterns within G5 herbicides allows knowledgeable weed management practitioners to exploit this knowledge for the benefit of crop producers globally. For example, G5-resistant C. album in Ontario, Canada, can be successfully controlled in soybean [Glycine max (L.) Merr.] with metribuzin but not atrazine (Bandeen and McLaren Reference Bandeen and McLaren1976); in contrast, G5-resistant C. album in Prince Edward Island, ON, Canada, can be successfully controlled in potato (Solanum tuberosum L.) with linuron, a G5 herbicide, but not metribuzin (McKenzie-Gopsill et al. Reference McKenzie-Gopsill, Graham, Laforest, Ibarra, Hann and Wagg2020). Precise knowledge of the cross-resistance patterns within a group can result in improved weed control, reduced weed seed return to the soil, and delayed evolution of herbicide resistance.

Background on the Evolution of Glyphosate Resistance in Weeds

The G9 consists of one herbicide family (glycine) and one active ingredient (glyphosate) (HRAC 2024). Glyphosate was commercialized in 1974 and is a systemic, nonselective herbicide (Jaworski Reference Jaworski1972). The precise mechanism of action of glyphosate is competitive inhibition of phosphoenolpyruvate in the binary complex of shikimate-3-phosphate: 3-phosphoshikimate-1-carboxyvinyltransferase, EPSP synthase (EPSPS; EC 2.5.1.19) (Kishore and Shah Reference Kishore and Shah1988). Compared with other single site-of-action herbicides, glyphosate was initially considered a low-risk compound with respect to evolved weed resistance (Bradshaw et al. Reference Bradshaw, Padgette, Kimball and Well1997), primarily because it is poorly metabolized by plants and binds tightly to a few conserved amino acids in EPSPS (Duke and Powles Reference Duke and Powles2008; Sammons et al. Reference Sammons, Heering, Danicola, Glick and Elmore2007). Consequently, TSR due to EPSPS mutations was deemed unlikely as it would incur a severe fitness cost. However, glyphosate resistance is now documented in 28 dicot and 31 grass weeds and is third-most in terms of number of species (Heap Reference Heap2024). The unprecedented pressure exerted by extensive use of glyphosate has selected for the most diverse types of resistance mechanisms (TSR and NTSR) compared with other herbicides (Gaines et al. Reference Gaines, Duke, Morran, Rigon, Tranel, Küpper and Dayan2020), as discussed in the following sections.

Glyphosate resistance due to single, double, and triple TSR mutations has been identified in 20 weed species (Heap Reference Heap2024). The most common EPSPS mutation is the Pro-106-Ser mutation, followed by the Pro-106-Arg, Pro-106-Thr, and Pro-106-Leu. The Pro-106-Ser mutation has been documented in E. indica, A. palmeri, horseweed [E. canadensis (L.) Cronquist], and sourgrass [Digitaria insularis (L.) Mez ex Ekman], among others (Beres et al. Reference Beres, Giese, Mackey, Owen, Page and Snow2020; Kaundun et al. Reference Kaundun, Zelaya, Dale, Lycett, Carter, Sharples and McIndoe2008, Reference Kaundun, Jackson, Hutchings, Galloway, Marchegiani, Howell, Carlin, Tuesca and Moreno2019a, 2019b). Another rare mutation conferring glyphosate resistance is the Thr-102-Ser change found in a coatbuttons (Tridax procumbens L.) population (Li et al. Reference Li, Peng, Han, Nyporko, Kulynych, Yu and Powles2018). Additionally, double Thr-102-Ile/Pro-106-Ser mutations in E. indica and hairy beggarticks (Bidens pilosa L.) and double Thr-102-Ile/Pro-106-Thr mutations in greater beggar’s ticks (Bidens subalternans DC) endow TSR to glyphosate (Alcántara-de la Cruz et al. 2016; Takano et al. Reference Takano, Fernandes, Adegas, Oliveira, Westra, Gaines and Dayan2020). Recently, a triple Thr-102-Ile/Ala-103-Val/Pro-106-Ser mutant was associated with glyphosate resistance in smooth pigweed (Amaranthus hybridus L.), although the precise role of the Ala-103-Val mutation is yet to be established (Perotti et al. Reference Perotti, Larran, Palmieri, Martinatto, Alvarez, Tuesca and Permingeat2019). In contrast to weed populations with single Pro-106-Ser/Thr/Ala/Leu and Thr-102-Ser mutations that confer relatively low levels of glyphosate resistance (<10-fold), weed populations containing the double Thr-102-Ile/Pro-106-Ser, Thr-102-Ile/Pro-106-Thr or triple Thr-102-Ile/Ala-103-Val/Pro-106-Ser mutations confer high resistance levels (Baek et al. Reference Baek, Bobadilla, Giacomini, Montgomery, Murphy, Tranel and Knaak2021).

An additional and unusual TSR mechanism to glyphosate consists of EPSPS gene overexpression (Gaines et al. Reference Gaines, Duke, Morran, Rigon, Tranel, Küpper and Dayan2020). This mechanism was first identified in A. palmeri (Gaines et al. Reference Gaines, Zhang, Wang, Bukuna, Chisholm, Shaner, Nissen, Patzoldt, Tranel, Culpepper, Grey, Webster, Vencill, Sammons and Jiang2010) and is now documented in three other dicot and six grass species, including A. tuberculatus, B. scoparia, L. perenne ssp. multiflorum, and Australian fingergrass (Chloris truncata R. Br.) (Baek et al. Reference Baek, Bobadilla, Giacomini, Montgomery, Murphy, Tranel and Knaak2021). Other NTSR mechanisms consist of reduced uptake and impaired transport. Reduced glyphosate absorption has been detected in several species, including B. pilosa, A. palmeri, johnsongrass [Sorghum halepense (L.) Pers.], tall windmill grass (Chloris elata Desv.), and Judd’s grass [Leptochloa virgata (L.) P. Beauv.] (Alcántara-de la Cruz et al. 2016; Brunharo et al. Reference Brunharo, Patterson, Carrijo, de Melo, Nicolai, Gaines, Nissen and Christoffoleti2016; de Carvalho et al. Reference de Carvalho, PLdCA, González-Torralva, Cruz-Hipolito, Rojano-Delgado, De Prado, Gil-Humanes, Barro and de Castro MD2012; Dominguez-Valenzuela et al. Reference Dominguez-Valenzuela, Gherekhloo, Fernández-Moreno, Cruz-Hipolito, Alcántara-de la Cruz, Sánchez-González and De Prado2017; Michitte et al. Reference Michitte, De Prado, Espinoza, Ruiz-Santaella and Gauvrit2007; Vila-Aiub et al. Reference Vila-Aiub, Balbi, Distéfano, Fernández, Hopp, Yu and Powles2012). Reduced glyphosate absorption alone typically does not result in control failures. Impaired transport was identified in the second glyphosate-resistant L. rigidum sample collected from Australia (Powles et al. Reference Powles, Lorraine-Colwill, Dellow and Preston1998) and can endow low, moderate, or high levels of glyphosate resistance. In most instances, the precise genetic basis underlying impaired transport remains undetermined.

Different glyphosate-resistance mechanisms can occur in the same populations and plants, especially when one mechanism is not sufficient to confer significant levels of glyphosate resistance (González-Torralva et al. Reference González-Torralva, Gil-Humanes, Barro, Brants and De Prado2012). For example, low levels of EPSPS overexpression combined with mutations at codon 106 act in concert to endow significant glyphosate resistance in junglerice [Echinochloa colona (L.) Link] (Alarcón-Reverte et al. Reference Alarcón-Reverte, García, Watson, Abdallah, Sabaté, Hernández, Dayan and Fischer2015) and E. indica (Gherekhloo et al. Reference Gherekhloo, Fernández-Moreno, Alcántara-de la Cruz, Sanchez-Gonzalez, Cruz-Hipolito, Dominguez-Valenzuela and De Prado2017). Reduced translocation and the Pro-106-Ser mutation endow resistance to A. tuberculatus and narrowleaf plantain (Plantago lanceolata L.) populations (Nandula et al. Reference Nandula, Ray, Ribeiro, Pan and Reddy2013; Ndou et al. Reference Ndou, Pieterse, Brand, Vorster, Louw and Phiri2021). Similarly, EPSPS overexpression and the Pro-106-Ser mutation act additively to confer glyphosate resistance in an A. palmeri population from Argentina (Kaundun et al. Reference Kaundun, Jackson, Hutchings, Galloway, Marchegiani, Howell, Carlin, Tuesca and Moreno2019a). Reduced absorption and translocation, EPSPS mutation at codon 106, and low levels of glyphosate metabolism, each conferring low levels of glyphosate resistance, have been identified in a D. insularis population from Brazil (de Carvalho et al. Reference de Carvalho, PLdCA, González-Torralva, Cruz-Hipolito, Rojano-Delgado, De Prado, Gil-Humanes, Barro and de Castro MD2012).

Because glyphosate is the only G9 member, characterizing resistance to this HRAC group should theoretically be straightforward compared with other herbicide groups consisting of structurally diverse active ingredients with distinct binding sites and NTSR profiles (e.g., G1 and G2). However, complexity arises because phenotypic expression and fold resistance to glyphosate can depend on herbicide rate, plant growth stage, environmental conditions, heterozygosity at EPSPS (codon 106), and ploidy of the species, especially for weed populations characterized by low-to-moderate resistance levels, as discussed in the following sections for several weed species.

Goosegrass (Eleusine indica)

Glyphosate rate dependency for phenotypic resistance expression was noted in two E. indica populations, one from Tennessee and one from the Philippines, with the Pro-106-Ser mutation in EPSPS (Huffman et al. Reference Huffman, Riggins, Steckel and Tranel2016; Kaundun et al. Reference Kaundun, Zelaya, Dale, Lycett, Carter, Sharples and McIndoe2008). Resistance indices associated with the single target-site mutation were low (2.1- to 3.4-fold) in homozygous resistant SS106 versus wild-type PP106 genotypes derived from the same parental populations in both studies. At a glyphosate rate (1.0 kg ae ha⁻¹) that completely killed a PP106 subpopulation, 72% of homozygous mutant SS106 E. indica plants (13-cm tall) survived glyphosate (Kaundun et al. Reference Kaundun, Zelaya, Dale, Lycett, Carter, Sharples and McIndoe2008). Compared with heterozygous PS106 individuals, a larger proportion of homozygous resistant SS106 mutant plants survived and accumulated greater dry biomass following a discriminating glyphosate rate of 350 g ae ha⁻¹ in the greenhouse (Huffman et al. Reference Huffman, Riggins, Steckel and Tranel2016).

Palmer amaranth (Amaranthus palmeri)

A similar observation was made for a unique A. palmeri population from Argentina characterized by the Pro-106-Ser mutation coupled with a low level (1.8-fold) of EPSPS overexpression, but surprisingly in the absence of EPSPS gene duplication (Kaundun et al. Reference Kaundun, Jackson, Hutchings, Galloway, Marchegiani, Howell, Carlin, Tuesca and Moreno2019a). Approximately 75% of heterozygous PS106 and 88% homozygous SS106 plants survived a reduced rate of glyphosate-potassium (400 g ai ha⁻¹). However, survivorship of PS106 and SS106 individuals decreased to 40% and 73%, respectively, at the recommended glyphosate use rate of 800 g ai ha⁻¹ (Kaundun et al. Reference Kaundun, Jackson, Hutchings, Galloway, Marchegiani, Howell, Carlin, Tuesca and Moreno2019a).

Effect of Plant Growth Stage, Environmental Factors, and Ploidy Level on Glyphosate Resistance

Summary and Implications for Future Weed Management

Although investigations of cross-resistance are not possible, because glyphosate is the only G9 herbicide member, it is clear that the magnitude and diversity of glyphosate resistance within a species, population, or individual plant is affected by numerous TSR and NTSR mechanisms as well as plant growth stage, environmental conditions, and genetics (zygosity and ploidy). Resistant weed populations evolved relatively quickly during the past 30 yr due to the intense selection pressure exerted by widespread glyphosate use in glyphosate-resistant cropping systems, combined with fewer applications of preemergence and postemergence herbicides from other HRAC groups (Landau et al. Reference Landau, Bradley, Burns, Flessner, Gage, Hager, Ikley, Jha, Jhala, Johnson, Johnson, Lancaster, Legleiter, Lingenfelter and Loux2023). Management of glyphosate-resistant biotypes is influenced by glyphosate rate, growth stage of the weed, and temperature at the time of application.

Waterhemp (Amaranthus tuberculatus) and Palmer amaranth (Amaranthus palmeri)

Multiple TSR and NTSR mechanisms have been identified in several Amaranthus species that confer resistance to the G14 herbicides applied postemergence (Barker et al. Reference Barker, Pawlak, Duke, Beffa, Tranel, Wuerffel, Young, Porri, Liebl, Aponte, Findley, Betz, Lerchl, Culpepper, Bradley and Dayan2023). Relatively few G14-resistance mechanisms have been identified in Amaranthus tuberculatus, with ΔGly210 and/or the Arg-128-Gly/Ile as the only TSR mechanisms elucidated (Nie et al. Reference Nie, Mansfield, Harre, Young, Steppig and Young2019; Patzoldt et al. Reference Patzoldt, Hager, McCormick and Tranel2006). NTSR (presumably metabolism-based, but not studied directly) to carfentrazone-ethyl postemergence in A. tuberculatus has been noted, but plants remained sensitive to other G14 herbicides postemergence (Obenland et al. Reference Obenland, Ma, Lygin and Riechers2019). Conversely, G14 resistance in A. palmeri is considerably more complex, with the presence of multiple target-site mutations identified in the target protein PPO2: ΔGly210, Arg-128-Gly/Met, Gly-399-Ala, and the recently detected Val-361-Ala mutation (Nie et al. Reference Nie, Harre and Young2023) conferring cross-resistance to nearly all postemergence G14 herbicides, except trifludimoxazin (Porri et al. Reference Porri, Betz, Seebruck, Knapp, Johnen, Witschel, Aponte, Liebl, Tranel and Lerchl2023). Metabolism-based NTSR (Varanasi et al. Reference Varanasi, Brabham and Norsworthy2018) and enhanced PPO2 expression have also been discovered alone or in combination with various TSR mutations (Rangani et al. Reference Rangani, Porri, Salas-Perez, Lerchl, Karaikal, Velásquez and Roma-Burgos2023), further complicating and contributing to G14 resistance postemergence. From a practical standpoint, TSR mutations such as ΔGly210, Gly-399-Ala, and Arg-128-Gly/Met have been discovered in the same population, plant, and even in the same PPO2 allele (Noguera et al. Reference Noguera, Rangani, Heiser, Bararpour, Steckel, Betz, Porri, Lerchl, Zimmermann, Nichols and Roma-Burgos2021), thus creating new challenges for resistance management recommendations.

Resistance to preemergence G14 herbicides is more nuanced. In numerous greenhouse trials, research investigating control of G14-resistant Amaranthus has demonstrated that R/S ratios for fomesafen are much lower preemergence (when using field soil or a mixture of field soil, peat, and sand) compared with postemergence (Lillie et al. Reference Lillie, Giacomini and Tranel2020; Wuerffel et al. Reference Wuerffel, Young, Matthews and Young2015a). Field trials conducted on G14-resistant A. tuberculatus with the ΔGly210 mutation demonstrated that commercially acceptable control can be achieved when G14 herbicides, including diphenyl ethers such as fomesafen, are applied preemergence (Falk et al. Reference Falk, Shoup, Al-Khatib and Peterson2006; Houston et al. Reference Houston, Barber, Norsworthy and Roberts2021; Wuerffel et al. Reference Wuerffel, Young, Tranel and Young2015b). Research on A. palmeri has concluded that preemergence fomesafen may not adequately control G14-resistant populations (regardless of the mechanism), but if a preemergence G14 herbicide is desired, then flumioxazin, sulfentrazone, or saflufenacil provides the greatest levels of control (Rangani et al. Reference Rangani, Porri, Salas-Perez, Lerchl, Karaikal, Velásquez and Roma-Burgos2023; Schwartz-Lazaro et al. Reference Schwartz-Lazaro, Norsworthy, Scott and Barber2017; Umphres et al. Reference Umphres, Steckel and Mueller2018). Although growers applying G14 herbicides preemergence may achieve acceptable control depending on the species and G14 herbicide used, selection pressure still occurs as the herbicide concentration decreases in the soil, thereby shortening the length of residual control (Lillie et al. Reference Lillie, Giacomini and Tranel2020; Wuerffel et al. Reference Wuerffel, Young, Tranel and Young2015b).

Interestingly, G14 herbicides applied preemergence are seemingly less effective for G14-resistant A. palmeri compared with A. tuberculatus, possibly due to the greater number of resistance mechanisms, and combinations thereof, present in A. palmeri. Furthermore, Rangani et al. (Reference Rangani, Porri, Salas-Perez, Lerchl, Karaikal, Velásquez and Roma-Burgos2023) noted that individual TSR mutations in PPO2 are necessary, but alone may not be sufficient, to overcome soil-applied G14 herbicides in resistant Amaranthus populations. Multiple “small-effect” TSR mechanisms, such as increased PPO2 overexpression and combinations of target-site mutations in an individual plant or PPO2 allele (Porri et al. Reference Porri, Noguera, Betz, Sälinger, Brändle, Bowe, Lerchl, Meyer, Knapp and Roma-Burgos2022), and/or NTSR mechanisms such as enhanced metabolism (Borgato et al. Reference Borgato, Thiagarayaselvam, Peterson, Hay, Dille and Jugulam2024; Varanasi et al. Reference Varanasi, Brabham and Norsworthy2018), may lead to increased chances of seedling survival to preemergence-applied G14 herbicides (Rangani et al. Reference Rangani, Porri, Salas-Perez, Lerchl, Karaikal, Velásquez and Roma-Burgos2023). For example, populations of A. palmeri heterozygous for the ΔGly210 mutation were more sensitive than individuals homozygous for the ΔGly210 mutation or heterozygous for the ΔGly210 and Gly-399-Ala mutations in PPO2 (mutations most likely in different alleles; Carvalho-Moore et al. Reference Carvalho-Moore, Rangani, Heiser, Findley, Bowe and Roma-Burgos2021). This finding provides further support of the hypothesis that combinations of multiple G14-resistance mechanisms (TSR and/or NTSR) likely reduce efficacy of preemergence G14 herbicides in certain A. palmeri populations.

Summary and Implications for Future Weed Management

Evolved resistance to G14 herbicides has become more complex in recent years, especially in Amaranthus species, in which several TSR and NTSR mechanisms have been identified in the same individual, and even the same allele, consequently conferring greater levels of G14 resistance compared with a single G14-resistance mechanism (Barker et al. Reference Barker, Pawlak, Duke, Beffa, Tranel, Wuerffel, Young, Porri, Liebl, Aponte, Findley, Betz, Lerchl, Culpepper, Bradley and Dayan2023; Noguera et al. Reference Noguera, Rangani, Heiser, Bararpour, Steckel, Betz, Porri, Lerchl, Zimmermann, Nichols and Roma-Burgos2021). Known G14-resistance mechanisms in Amaranthus confer cross-resistance to all G14 herbicides applied postemergence, with the exception of new, highly active G14 herbicides like trifludimoxazin (Porri et al. Reference Porri, Betz, Seebruck, Knapp, Johnen, Witschel, Aponte, Liebl, Tranel and Lerchl2023) or unknown NTSR mechanism(s) conferring carfentrazone-ethyl–specific resistance in A. tuberculatus (Obenland et al. Reference Obenland, Ma, Lygin and Riechers2019). Generally, G14 herbicides applied preemergence provide acceptable control of A. tuberculatus (Falk et al. Reference Falk, Shoup, Al-Khatib and Peterson2006; Wuerffel et al. Reference Wuerffel, Young, Tranel and Young2015b); however, multiple research groups have found this not to be the case in several A. palmeri populations (Schwartz-Lazaro et al. Reference Schwartz-Lazaro, Norsworthy, Scott and Barber2017; Umphres et al. Reference Umphres, Steckel and Mueller2018), possibly due to the greater number and diversity of TSR and NTSR G14-resistance mechanisms (Rangani et al. Reference Rangani, Porri, Salas-Perez, Lerchl, Karaikal, Velásquez and Roma-Burgos2023). In summary, it is clear that simply stating “G14 resistance” in Amaranthus spp. is inaccurate and misleading due to the active ingredient– and application timing–specific resistance profiles described.

Wild oats (Avena fatua)

Several A. fatua populations were classified as resistant to G15 herbicides (specifically, triallate) in the late 1980s and 1990s in North America (Heap Reference Heap2024). Triallate resistance in A. fatua has been associated with low resistance levels (R/S<3) to the G15 herbicide, pyroxasulfone, and G14 herbicide, sulfentrazone, despite this population lacking exposure to either herbicide (Mangin et al. Reference Mangin, Hall and Beckie2016). It was theorized that triallate, G1 herbicides, and/or G2 herbicides selected for cross-resistance to pyroxasulfone and sulfentrazone via enhanced metabolism (Mangin et al. Reference Mangin, Hall and Beckie2016).

Rigid ryegrass (Lolium rigidum)

The first weed population with reported resistance to G15 herbicides (specifically, metolachlor and triallate) was an L. rigidum population in Southern Australia in 1982 (Heap Reference Heap2024). Resistance to pyroxasulfone and other G15 herbicides was subsequently discovered in L. rigidum as well (Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2018, Reference Brunton, Boutsalis, Gill and Preston2019; Burnet et al. Reference Burnet, Barr and Powles1994; Busi et al. Reference Busi, Gaines, Vila-Aiub and Powles2014). A pyroxasulfone-resistant L. rigidum population displayed cross-resistance to prosulfocarb and triallate (Busi and Powles Reference Busi and Powles2013), which could be selected for by either herbicide (Busi and Powles Reference Busi and Powles2016). Varying levels of resistance (between 8- and 66-fold) to the G15 herbicides triallate, EPTC, prosulfocarb, thiobencarb, S-metolachlor, metazachlor, and pyroxasulfone, as well as the G3 herbicide, trifluralin, have also been reported in L. rigidum from Australia (Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2018, Reference Brunton, Boutsalis, Gill and Preston2019). Despite G15 resistance, certain mixtures containing two different herbicides, such as pyroxasulfone plus triallate, applied PPI improved L rigidum control and wheat yield compared with a single PPI herbicide (Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2020).

Echinochloa Complex: Echinochloa crus-galli, Early Barnyardgrass [Echinochloa oryzoides (Ard.) Fritsch], and Rice Barnyardgrass [Echinochloa phyllopogon (Stapf) Koso-Pol.]

Resistance to the G15 herbicide, butachlor, has been reported in E. crus-galli in China (Heap Reference Heap2024), and several E. crus-galli populations resistant to butachlor and propanil (a PSII inhibitor) were identified in Asia (Juliano et al. Reference Juliano, Casimero and Llewellyn2010). Thiocarbamate-resistant E. phyllopogon and E. oryzoides populations discovered in rice fields in California were resistant to multiple herbicides, including G1, G2, G4 (quinclorac), and G13 (clomazone), but were not resistant to propanil, glyphosate, or glufosinate (Busi Reference Busi2014; Fischer et al. Reference Fischer, Ateh, Bayer and Hill2000). Interestingly, the addition of thiobencarb to bispyribac-sodium in a tank mixture led to synergistic control of bispyribac-sodium–resistant and bispyribac-sodium–sensitive E. phyllopogon populations (Fischer et al. Reference Fischer, Cheetham, Vidotto and De Prado2004).

Recent molecular-genetic studies of multiple-resistant E. phyllopogon (Dimaano et al. Reference Dimaano, Tominaga and Iwakami2022) indicated distinct enzymes confer resistance to G1 and G2 versus G15 herbicides. For example, transgenic Arabidopsis plants expressing the CYP81A12/21 genes demonstrated this P450 enzyme likely provides cross-resistance to G1 and G2 herbicides, but not the G15 herbicide, thiobencarb, implying that resistance in E. phyllopogon may involve a combination of cross- and multiple-resistance traits (Dimaano et al. Reference Dimaano, Tominaga and Iwakami2022).

Amaranthus spp

Waterhemp (Amaranthus tuberculatus) and Palmer amaranth (Amaranthus palmeri)

Two A. tuberculatus populations resistant to G15-inhibiting herbicides have been characterized in Illinois: one population from McLean County (MCR or SIR; Strom et al. Reference Strom, Gonzini, Mitsdarfer, Davis, Riechers and Hager2019) and one population from Champaign County (CHR; Evans et al. Reference Evans, Strom, Riechers, Davis, Tranel and Hager2019). Both resistant populations exhibited reduced control in the field with G15 herbicides from the isoxazoline and α-chloroacetamide chemical families (HRAC 2024); however, the greatest decline in efficacy (relative to sensitive populations) occurred with S-metolachlor, while acetochlor was affected the least (Strom et al. Reference Strom, Gonzini, Mitsdarfer, Davis, Riechers and Hager2019, Reference Strom, Jacobs, Seiter, Davis, Riechers and Hager2022). In greenhouse studies, resistance to S-metolachlor ranged from 18- to 64-fold in the MCR and CHR populations (Strom et al. Reference Strom, Gonzini, Mitsdarfer, Davis, Riechers and Hager2019). The primary mechanism of resistance to S-metolachlor in both populations is enhanced oxidative metabolism mediated by P450 activity (Strom et al. Reference Strom, Hager, Concepcion, Seiter, Davis, Morris, Kaundun and Riechers2021), although rapid glutathione conjugation likely plays a secondary role (Strom et al. Reference Strom, Hager, Seiter, Davis and Riechers2020, Reference Strom, Hager, Concepcion, Seiter, Davis, Morris, Kaundun and Riechers2021). However, the reason acetochlor remains most effective compared with other active ingredients in G15 for controlling these G15-resistant populations (Figure 4) has not been determined but warrants further research, including comparative metabolism studies and in vitro enzyme assays.

Figure 4. Representative images of variable Group 15 (G15) herbicide efficacy for controlling a G15-resistant Amaranthus tuberculatus population (CHR) and a sensitive (Urbana, IL) population, 28 d after treatment. Figure 4 was reproduced directly without changes from Strom et al. (Reference Strom, Jacobs, Seiter, Davis, Riechers and Hager2022).

Resistance to G15 herbicides in two A. palmeri populations has been reported in Arkansas (Brabham et al. Reference Brabham, Norsworthy, Houston, Varanasi and Barber2019) as well as in several populations collected in the U.S. Midsouth (Kouame et al. Reference Kouame, Bertucci, Savin, Bararpour, Steckel, Butts, Willett, Machado and Burgos2022). Greenhouse studies demonstrated 8- to 10-fold resistance to S-metolachlor in the two resistant populations from Arkansas compared with sensitive populations (Brabham et al. Reference Brabham, Norsworthy, Houston, Varanasi and Barber2019). Although cross-resistance to other G15 herbicides was not detected, the resistant A. palmeri populations from Arkansas displayed reduced sensitivity to acetochlor, dimethenamid-p, and pyroxasulfone (Brabham et al. Reference Brabham, Norsworthy, Houston, Varanasi and Barber2019).

Summary and Implications for Future Weed Management

Resistance to G15 herbicides has been slow to evolve compared with other herbicide groups (Heap Reference Heap2024). Several reasons have been posited concerning why resistance has been slow to evolve to the G15 herbicides, including the complexity and possible redundancy of VLCFAE enzymes in plants and potential fitness costs associated with TSR mechanisms (Beckie Reference Beckie2006; Böger Reference Böger2003; Busi Reference Busi2014; Jhala et al. Reference Jhala, Singh, Shergill, Singh, Jugulam, Riechers, Ganie, Selby, Werle and Norsworthy2024). All reported resistant weed populations to date with known G15-resistance mechanisms have exhibited rapid metabolic detoxification rates (with the exception of triallate-resistant A. fatua, in which lack of bioactivation confers resistance; Kern et al. Reference Kern, Peterson, Miller, Colliver and Dyer1996) due to oxidative and/or glutathione-mediated reactions compared with sensitive populations of the same species (Busi Reference Busi2014; Busi et al. Reference Busi, Porri, Gaines and Powles2018; Strom et al. Reference Strom, Hager, Seiter, Davis and Riechers2020). However, until specific Phase I or II genes and enzymes are discovered and biochemically characterized, we can only speculate that differential herbicide substrate specificities or expression patterns of these Phase I/II enzymes underlies the unique active ingredient–specific patterns of cross-resistance in weeds.

Waterhemp (Amaranthus tuberculatus)

Amaranthus tuberculatus has evolved resistance to G27 herbicides from three herbicide families: pyrazoles (topramezone), isoxazoles (isoxaflutole), and triketones (mesotrione, tembotrione, and syncarpic acid-3) applied postemergence (Concepcion et al. Reference Concepcion, Kaundun, Morris, Hutchings, Strom, Lygin and Riechers2021; Evans et al. Reference Evans, Strom, Riechers, Davis, Tranel and Hager2019; Hausman et al. Reference Hausman, Singh, Tranel, Riechers, Kaundun, Polge, Thomas and Hager2011; McMullan and Green Reference McMullan and Green2011; O’Brien et al. 2018; Oliveira et al. Reference Oliveira, Jhala, Gaines, Irmak, Amundsen, Scott and Knezevic2017; Shergill et al. Reference Shergill, Bish, Jugulam and Bradley2018). The resistance level to G27 herbicides is active ingredient specific. This specificity is exemplified by varying levels of resistance to mesotrione, tembotrione, and topramezone of 18-, 5-, and 2-fold, respectively (Oliveira et al. Reference Oliveira, Jhala, Gaines, Irmak, Amundsen, Scott and Knezevic2017). Variable results are corroborated by Evans et al. (Reference Evans, Strom, Riechers, Davis, Tranel and Hager2019), who reported the relative order of A. tuberculatus control and biomass reductions following postemergence field use rates was topramezone > tembotrione > mesotrione. However, the field use history of G27 herbicides may also play a significant role in determining cross-resistance (both levels and patterns) in A. tuberculatus. For example, the Nebraska population (NEB) was exposed to mesotrione and tembotrione (both triketones), while the Illinois population (MCR or SIR) was treated with mesotrione, tembotrione, and topramezone (Hausman et al. Reference Hausman, Singh, Tranel, Riechers, Kaundun, Polge, Thomas and Hager2011; Kaundun et al. Reference Kaundun, Hutchings, Dale, Howell, Morris, Kramer, Shivrain and Mcindoe2017). The NEB population is resistant to topramezone, which implies that triketone G27 herbicides can select for cross-resistance to pyrazole G27 herbicides.

Control of G27-resistant A. tuberculatus can be influenced by application timing. Resistance levels calculated with mesotrione were 9- or 13-fold applied preemergence (depending on the population or subpopulation used) and 10- or 35-fold applied postemergence (depending on the sensitive population used) for comparison (Hausman et al. Reference Hausman, Singh, Tranel, Riechers, Kaundun, Polge, Thomas and Hager2011, Reference Hausman, Tranel, Riechers, Maxwell, Gonzini and Hager2013, 2016), and 2.4- or 45-fold for preemergence or postemergence, respectively (Kaundun et al. Reference Kaundun, Hutchings, Dale, Howell, Morris, Kramer, Shivrain and Mcindoe2017). Early postemergence (5 cm; 4 to 5 true leaves) isoxaflutole and mesotrione treatments led to 10- and 32-fold resistance levels, respectively, in the Illinois SIR population and 4- and 7-fold resistance levels, respectively, in the NEB population compared with a G27-sensitive A. tuberculatus population (O’Brien et al. 2018). Group 27 resistance in A. tuberculatus can thus be affected by active ingredient (mesotrione vs. topramezone vs. tembotrione), application timing (preemergence vs. postemergence), and populations examined.

Wild radish (Raphanus raphanistrum)

Raphanus raphanistrum is resistant to triketones (mesotrione and tembotrione), isoxazole (isoxaflutole), and pyrazoles (topramezone and pyrasulfotole) (Busi et al. Reference Busi, Zhang, Goggin, Bryant and Beckie2022; Lu et al. Reference Lu, Liu, Li, Han, Zhou, Nyporko, Yu, Qiang and Powles2023). Busi et al. (Reference Busi, Zhang, Goggin, Bryant and Beckie2022) reported 50%, 60%, and 100% survival with the recommended postemergence field rates of mesotrione, topramezone, and pyrasulfotole, respectively, in two R. raphanistrum populations. Differences among R. raphanistrum populations in their responses to pyrasulfotole were also reported; populations 86-2020 and 91-2020 had resistance indices of 5.3 and 8.4, respectively (Busi et al. Reference Busi, Zhang, Goggin, Bryant and Beckie2022). Furthermore, population 86-2020 was more resistant to postemergence mesotrione (triketone) and topramezone (pyrazole) + MCPA, while population 91-2020 was more resistant to pyrasulfotole (pyrazole) + bromoxynil. Interestingly, mesotrione (triketone) preemergence controlled R. raphanistrum>99%, while pyrasulfotole (pyrazole) + bromoxynil and topramezone (pyrazole) + bromoxynil postemergence provided<90% control in the field (Busi et al. Reference Busi, Zhang, Goggin, Bryant and Beckie2022). Mesotrione preemergence controlled one G27-resistant R. raphanistrum population (91-2020) in the field, but this population was resistant to postemergence mesotrione in the greenhouse (Busi et al. Reference Busi, Zhang, Goggin, Bryant and Beckie2022).

Summary and Implications for Future Weed Management

Herbicides in G27 were commercialized in the late 1990s (Jhala et al. Reference Jhala, Kumar, Yadav, Jha, Jugulam, Dayan, Burton, Dale and Norsworthy2023) and thus have had a relatively “brief” time period to select for G27-resistant weed populations compared with all other HRAC groups. It is currently unknown whether weed resistance to G27 herbicides evolved solely due to selection pressure from G27 herbicides or whether previous usage patterns of other herbicides (before 1998) may have contributed to the magnitude and patterns of G27 resistance. Despite a noncommercial class of G27 herbicides (e.g., syncarpic acids) having known sites of oxidative metabolism chemically blocked, effective control of G27-resistant A. tuberculatus populations was not obtained with the herbicide, syncarpic acid-3 (Concepcion et al. Reference Concepcion, Kaundun, Morris, Hutchings, Strom, Lygin and Riechers2021) due to a previously unknown metabolic pathway involving Phase I (enzymes not yet characterized) and Phase II (GST) enzymes acting in concert. This unexpected metabolic capacity revealed an incredible, adaptive response possessed by some multiple-herbicide resistant A. tuberculatus populations to display cross-resistance, via detoxification, of compounds to which they had not been previously exposed. With the anticipated introduction of G27-resistant soybean cultivar technology (Siehl et al. Reference Siehl, Tao, Albert, Dong, Heckert, Madrigal, Lincoln-Cabatu, Lu, Fenwick, Bermudez, Sandoval, Horn, Green, Hale and Pagano2014) in the future, resistance and cross-resistance to G27 herbicides (current or in development) may be exacerbated unless proper stewardship of active ingredients is practiced. In summary, patterns and magnitude of G27 resistance in weeds can be population specific (as in R. raphanistrum), active ingredient specific (as in A. tuberculatus and R. raphanistrum), and application timing specific (as in A. tuberculatus and R. raphanistrum).