A high diversity of non-target site resistance mechanisms to acetolactate-synthase (ALS) inhibiting herbicides has evolved within and among field populations of common ragweed (Ambrosia artemisiifolia L.).

BACKGROUND: Non-target site resistance (NTSR) to herbicides is a polygenic trait that threatens the chemical control of agricultural weeds. NTSR involves differential regulation of plant secondary metabolism pathways, but its precise genetic determinisms remain fairly unclear. Full-transcriptome sequencing had previously been implemented to identify NTSR genes. However, this approach had generally been applied to a single weed population, limiting our insight into the diversity of NTSR mechanisms. Here, we sought to explore the diversity of NTSR mechanisms in common ragweed (Ambrosia artemisiifolia L.) by investigating six field populations from different French regions where NTSR to acetolactate-synthase-inhibiting herbicides had evolved. RESULTS: A de novo transcriptome assembly (51,242 contigs, 80.2% completeness) was generated as a reference to seek genes differentially expressed between sensitive and resistant plants from the six populations. Overall, 4,609 constitutively differentially expressed genes were identified, of which none were common to all populations, and only 197 were shared by several populations. Similarly, population-specific transcriptomic response was observed when investigating early herbicide response. Gene ontology enrichment analysis highlighted the involvement of stress response and regulatory pathways, before and after treatment. The expression of 121 candidate constitutive NTSR genes including CYP71, CYP72, CYP94, oxidoreductase, ABC transporters, gluco and glycosyltransferases was measured in 220 phenotyped plants. Differential expression was validated in at least one ragweed population for 28 candidate genes. We investigated whether expression patterns at some combinations of candidate genes could predict phenotype. Within populations, prediction accuracy decreased when applied to an additional, independent plant sampling. Overall, a wide variety of genes linked to NTSR was identified within and among ragweed populations, of which only a subset was captured in our experiments. CONCLUSION: Our results highlight the complexity and the diversity of NTSR mechanisms that can evolve in a weed species in response to herbicide selective pressure. They strongly point to a non-redundant, population-specific evolution of NTSR to ALS inhibitors in ragweed. It also alerts on the potential of common ragweed for rapid adaptation to drastic environmental or human-driven selective pressures.

Prevalence, spatial structure and evolution of resistance to acetolactate-synthase (ALS) inhibitors and 2,4-D in the major weed Papaver rhoeas (L.) assessed using a massive, country-wide sampling.

BACKGROUND: Corn poppy (Papaver rhoeas) is the most damaging broadleaf weed in France. Massively parallel amplicon sequencing was used to investigate the prevalence, mode of evolution and spread of resistance-endowing ALS alleles in 422 populations randomly sampled throughout poppy's range in France. Bioassays were used to detect resistance to the synthetic auxin 2,4-D in 43 of these populations. RESULTS: A total of 21 100 plants were analysed and 24 mutant ALS alleles carrying an amino-acid substitution involved or potentially involved in resistance were identified. The vast majority (97.6%) of the substitutions occurred at codon Pro197, where all six possible single-nucleotide non-synonymous substitutions plus four double-nucleotide substitutions were identified. Changes observed in the enzymatic properties of the mutant ALS isoforms could not explain the differences in prevalence among the corresponding alleles. Sequence read analysis showed that mutant ALS alleles had multiple, independent evolutionary origins, and could have evolved several times independently within an area of a few kilometres. Finally, 2,4-D resistance was associated with mutant ALS alleles in individual plants in one third of the populations assayed. CONCLUSION: The intricate geographical mosaic of mutant ALS alleles observed is the likely result of the combination of huge population sizes, multiple independent mutation events and human-mediated spread of resistance. Our work highlights the ability of poppy populations and individual plants to accumulate different ALS alleles and as yet unknown mechanisms conferring resistance to synthetic auxins. This does not bode well for the continued use of chemical herbicides to control poppy. © 2023 Society of Chemical Industry.

A new insight into arable weed adaptive evolution: mutations endowing herbicide resistance also affect germination dynamics and seedling emergence.

BACKGROUND AND AIMS: Selective pressures exerted by agriculture on populations of arable weeds foster the evolution of adaptive traits. Germination and emergence dynamics and herbicide resistance are key adaptive traits. Herbicide resistance alleles can have pleiotropic effects on a weed's life cycle. This study investigated the pleiotropic effects of three acetyl-coenzyme A carboxylase (ACCase) alleles endowing herbicide resistance on the seed-to-plant part of the life cycle of the grass weed Alopecurus myosuroides. METHODS: In each of two series of experiments, A. myosuroides populations with homogenized genetic backgrounds and segregating for Leu1781, Asn2041 or Gly2078 ACCase mutations which arose independently were used to compare germination dynamics, survival in the soil and seedling pre-emergence growth among seeds containing wild-type, heterozygous and homozygous mutant ACCase embryos. KEY RESULTS: Asn2041 ACCase caused no significant effects. Gly2078 ACCase major effects were a co-dominant acceleration in seed germination (1·25- and 1·10-fold decrease in the time to reach 50 % germination (T50) for homozygous and heterozygous mutant embryos, respectively). Segregation distortion against homozygous mutant embryos or a co-dominant increase in fatal germination was observed in one series of experiments. Leu1781 ACCase major effects were a co-dominant delay in seed germination (1·41- and 1·22-fold increase in T50 for homozygous and heterozygous mutant embryos, respectively) associated with a substantial co-dominant decrease in fatal germination. CONCLUSIONS: Under current agricultural systems, plants carrying Leu1781 or Gly2078 ACCase have a fitness advantage conferred by herbicide resistance that is enhanced or counterbalanced, respectively, by direct pleiotropic effects on the plant phenology. Pleiotropic effects associated with mutations endowing herbicide resistance undoubtedly play a significant role in the evolutionary dynamics of herbicide resistance in weed populations. Mutant ACCase alleles should also prove useful to investigate the role played by seed storage lipids in the control of seed dormancy and germination.

Isolation and characterisation of 11 polymorphic microsatellite markers in Papaver rhoeas L. (Corn Poppy), a major annual plant species from cultivated areas.

Papaver rhoeas, an annual plant species in the Papaveraceae family, is part of the biodiversity of agricultural ecosystems and also a noxious agronomic weed. We developed microsatellite markers to study the genetic diversity of P. rhoeas, using an enriched microsatellite library coupled with 454 next-generation sequencing. A total of 13,825 sequences were obtained that yielded 1795 microsatellite loci. After discarding loci with less than six repeats of the microsatellite motif, automated primer design was successful for 598 loci. We tested 74 of these loci for amplification with a total of 97 primer pairs. Thirty loci passed our tests and were subsequently tested for polymorphism using 384 P. rhoeas plants originating from 12 populations from France. Of the 30 loci, 11 showed reliable polymorphism not affected by the presence of null alleles. The number of alleles and the expected heterozygosity ranged from 3 to 7.4 and from 0.27 to 0.73, respectively. A low but significant genetic differentiation among populations was observed (F(ST) = 0.04; p < 0.001). The 11 validated polymorphic microsatellite markers developed in this work will be useful in studies of genetic diversity and population structure of P. rhoeas, assisting in designing management strategies for the control or the conservation of this species.

Lab meets field: Accelerated selection and field monitoring concur that non-target-site-based resistance evolves first in the dicotyledonous, allergenic weed Ambrosia artemisiifolia.

Assessing weed capacity to evolve herbicide resistance before resistance occurs in the field is of major interest for chemical weed control. We used herbicide selection followed by controlled crosses to provoke accelerated evolution of resistance to imazamox (imidazolinones) and tribenuron (sulfonyurea), two acetolactate-synthase (ALS) inhibitors targeting Ambrosia artemisiifolia. In natural populations with no herbicide application records, some plants were initially resistant to metsulfuron (sulfonylurea), a cereal herbicide. Non-target-site-based resistance (NTSR) to metsulfuron was substantially increased from these plants within two generations. NTSR to imazamox and/or tribenuron emerged in metsulfuron-selected G1 progenies and was strongly reinforced in G2 progenies selected by imazamox or tribenuron. NTSR to the herbicides assayed was endowed by partly overlapping and partly specific pathways. Herbicide sensitivity bioassays conducted over 62 ALS-inhibitor-sprayed fields identified emerging resistance to imazamox and/or tribenuron in 14 A. artemisiifolia populations. Only NTSR was detected in 13 of these populations. In the last population, NTSR was present together with a mutant, herbicide-resistant ALS allele bearing an Ala-205-Thr substitution. NTSR was thus by far the predominant type of resistance to ALS inhibitors in France. This confirmed accelerated selection results and demonstrated the relevance of this approach to anticipate resistance evolution in a dicotyledonous weed.

A high diversity of mechanisms endows ALS-inhibiting herbicide resistance in the invasive common ragweed (Ambrosia artemisiifolia L.).

Ambrosia artemisiifolia L. (common ragweed) is a globally invasive, allergenic, troublesome arable weed. ALS-inhibiting herbicides are broadly used in Europe to control ragweed in agricultural fields. Recently, ineffective treatments were reported in France. Target site resistance (TSR), the only resistance mechanism described so far for ragweed, was sought using high-throughput genotyping-by-sequencing in 213 field populations randomly sampled based on ragweed presence. Additionally, non-target site resistance (NTSR) was sought and its prevalence compared with that of TSR in 43 additional field populations where ALS inhibitor failure was reported, using herbicide sensitivity bioassay coupled with ALS gene Sanger sequencing. Resistance was identified in 46 populations and multiple, independent resistance evolution demonstrated across France. We revealed an unsuspected diversity of ALS alleles underlying resistance (9 amino-acid substitutions involved in TSR detected across 24 populations). Remarkably, NTSR was ragweed major type of resistance to ALS inhibitors. NTSR was present in 70.5% of the resistant plants and 74.1% of the fields harbouring resistance. A variety of NTSR mechanisms endowing different resistance patterns evolved across populations. Our study provides novel data on ragweed resistance to herbicides, and emphasises that local resistance management is as important as mitigating gene flow from populations where resistance has arisen.

Geographical variation in resistance to acetyl-coenzyme A carboxylase-inhibiting herbicides across the range of the arable weed Alopecurus myosuroides (black-grass).

*The geographical structure of resistance to herbicides inhibiting acetyl-coenzyme A carboxylase (ACCase) was investigated in the weed Alopecurus myosuroides (black-grass) across its geographical range to gain insight into the process of plant adaptation in response to anthropogenic selective pressures occurring in agricultural ecosystems. *We analysed 297 populations distributed across six countries in A. myosuroides' main area of occupancy. The frequencies of plants resistant to two broadly used ACCase inhibitors and of seven mutant, resistant ACCase alleles were assessed using bioassays and genotyping, respectively. *Most of the resistance was not endowed by mutant ACCase alleles. Resistance and ACCase allele distribution patterns were characterized by mosaicism. The prevalence of resistance and of ACCase alleles differed among countries. *Resistance clearly evolved by redundant evolution of a set of resistance alleles or genes, most of which remain unidentified. Resistance in A. myosuroides was shaped by variation in the herbicide selective pressure at both the individual field level and the national level.

Harnessing the power of next-generation sequencing technologies to the purpose of high-throughput pesticide resistance diagnosis.

BACKGROUND: Next Generation Sequencing (NGS) technologies offer tremendous possibilities for high-throughput pesticide resistance diagnosis via massive genotyping-by-sequencing. Herein, we used Illumina sequencing combined with a simple, non-commercial bioinformatics pipe-line to seek mutations involved in herbicide resistance in two weeds. RESULTS: DNA was extracted from 96 pools of 50 plants for each species. Three amplicons encompassing 15 ALS (acetolactate-synthase) codons crucial for herbicide resistance were amplified from each DNA extract. Above 18 and 20 million quality 250-nucleotide sequence reads were obtained for groundsel (Senecio vulgaris, tetraploid) and ragweed (Ambrosia artemisiifolia, diploid), respectively. Herbicide resistance-endowing mutations were identified in 45 groundsel and in eight ragweed field populations. The mutations detected and their frequencies assessed by NGS were checked by individual plant genotyping or Sanger sequencing. NGS results were fully confirmed, except in three instances out of 12 where mutations present at a frequency of 1% were detected below the threshold set for reliable mutation detection. CONCLUSION: Analyzing 9600 plants requested 192 DNA extractions followed by 1728 PCRs and two Illumina runs. Equivalent results obtained by individual analysis would have necessitated 9600 individual DNA extractions followed by 216 000 genotyping PCRs, or by 121 500 PCRs and 40 500 Sanger sequence runs. This clearly demonstrates the interest and power of NGS-based detection of pesticide resistance from pools of individuals for diagnosing resistance in massive numbers of individuals. © 2019 Society of Chemical Industry.

Cross-resistance patterns to ACCase-inhibiting herbicides conferred by mutant ACCase isoforms in Alopecurus myosuroides Huds. (black-grass), re-examined at the recommended herbicide field rate.

BACKGROUND: Target-site-based resistance to acetyl-CoA carboxylase (ACCase) inhibitors in Alopecurus myosuroides Huds. is essentially due to five substitutions (Isoleucine-1781-Leucine, Tryptophan-2027-Cysteine, Isoleucine-2041-Asparagine, Aspartate-2078-Glycine, Glycine-2096-Alanine). Recent studies suggested that cross-resistance patterns associated with each mutation using a seed-based bioassay may not accurately reflect field resistance. The authors aimed to connect the presence of mutant ACCase isoform(s) in A. myosuroides with resistance to five ACCase inhibitors (fenoxaprop, clodinafop, haloxyfop, cycloxydim, clethodim) sprayed at the recommended field rate. RESULTS: Results from spraying experiments and from seed-based bioassays were consistent for all mutant isoforms except the most widespread, Leucine-1781. In spraying experiments, Leucine-1781 ACCase conferred resistance to clodinafop and haloxyfop. Some plants containing Leucine-1781 or Alanine-2096 ACCase, but not all, were also resistant to clethodim. CONCLUSION: Leucine-1781, Cysteine-2027, Asparagine-2041 and Alanine-2096 ACCases confer resistance to fenoxaprop, clodinafop and haloxyfop at field rates. Leucine-1781 ACCase also confers resistance to cycloxydim at field rate. Glycine-2078 ACCase confers resistance to all five herbicides at field rates. Only Glycine-2078 ACCase confers clethodim resistance under optimal application conditions. It may be that Leucine-1781 and Alanine-2096 ACCases may also confer resistance to clethodim in the field if the conditions are not optimal for herbicide efficacy, or at reduced clethodim field rates.

Genetic basis, evolutionary origin and spread of resistance to herbicides inhibiting acetolactate synthase in common groundsel (Senecio vulgaris).

BACKGROUND: Following control failure by herbicides inhibiting acetolactate synthase (ALS) in French wheat fields and vineyards, we aimed to confirm resistance evolution and investigate the evolutionary origin and spread of resistance in the tetraploid species Senecio vulgaris (common groundsel), a widespread, highly mobile weed. RESULTS: Sequencing of two ALS homeologues in S. vulgaris enabled the first identification and characterisation of ALS-based resistance in this species. Cross-resistance patterns associated with Leu-197 and Ser-197 ALS1 were established using eight herbicides. Sequencing and genotyping showed that ALS-based resistance evolved by multiple, independent appearances of mutant ALS1 and ALS2 alleles followed by spread. Spread of a mutant ALS1 allele issued from one particular appearance event was observed over 60 km. Independent resistance appearance events and easy seed dispersion are the most likely reasons for populations of S. vulgaris containing different mutant ALS alleles. Accumulation of different alleles probably due to sexual reproduction was observed in the same plant. CONCLUSION: Mutant ALS alleles and possibly other mechanisms cause resistance to ALS inhibitors in S. vulgaris. Management strategies should aim at limiting S. vulgaris establishment and seed set. Considering the mobility of this species, control coordination at a regional level is clearly necessary if resistance spread is to be contained.

Using next-generation sequencing to detect mutations endowing resistance to pesticides: application to acetolactate-synthase (ALS)-based resistance in barnyard grass, a polyploid grass weed.

BACKGROUND: Next-generation sequencing (NGS) technologies offer tremendous possibilities for accurate detection of mutations endowing pesticide resistance, yet their use for this purpose has not emerged in crop protection. This study aims at promoting NGS use for pesticide resistance diagnosis. It describes a simple procedure accessible to virtually any scientist and implementing freely accessible programs for the analysis of NGS data. RESULTS: Three PCR amplicons encompassing seven codons of the acetolactate-synthase gene crucial for herbicide resistance were sequenced using non-quantified pools of crude DNA extracts from 40 plants in each of 28 field populations of barnyard grass, a polyploid weed. A total of 63,959 quality NGS sequence runs were obtained using the 454 technology. Three herbicide-resistance-endowing mutations (Pro-197-Ser, Pro-197-Leu and/or Trp-574-Leu) were identified in seven populations. The NGS results were confirmed by individual plant Sanger sequencing. CONCLUSION: This work demonstrated the feasibility of NGS-based detection of pesticide resistance, and the advantages of NGS compared with other molecular biology techniques for analysing large numbers of individuals. NGS-based resistance diagnosis has the potential to play a substantial role in monitoring resistance, maintaining pesticide efficacy and optimising pesticide applications.

Multiple origins for black-grass (Alopecurus myosuroides Huds) target-site-based resistance to herbicides inhibiting acetyl-CoA carboxylase.

We have investigated the process of evolution of target-site-based resistance to herbicides inhibiting acetyl-CoA carboxylase (ACCase) in nine French populations of black-grass (Alopecurus myosuroides Huds). To date, two different ACCase resistant alleles are known. One contains an isoleucine-to-leucine substitution at position 1781, the second contains an isoleucine-to-asparagine substitution at position 2041. Using phylogenetic analysis of ACCase sequences, we showed that 1781Leu ACCase alleles evolved from four independent origins in the nine black-grass populations studied, while 2041Asn ACCase alleles evolved from six independent origins. No geographical structure of black-grass populations was revealed. This implies that these populations, although geographically distant, are, or have until recently been, connected by gene flows. Comparison of biological data obtained from herbicide sensitivity bioassay and molecular data showed that distinct resistance mechanisms often exist in a single black-grass population. Accumulation of different resistance mechanisms in a single plant was also demonstrated. We conclude that large-scale evolution of resistance to herbicides in black-grass is a complex phenomenon, resulting from the independent selection of various resistance mechanisms in local black-grass populations undergoing contrasted herbicide and agronomical selection pressures, and connected by gene flows whose parameters remain to be determined.

Weed response to herbicides: regional-scale distribution of herbicide resistance alleles in the grass weed Alopecurus myosuroides.

Effective herbicide resistance management requires an assessment of the range of spatial dispersion of resistance genes among weed populations and identification of the vectors of this dispersion. In the grass weed Alopecurus myosuroides (black-grass), seven alleles of the acetyl-CoA carboxylase (ACCase) gene are known to confer herbicide resistance. Here, we assessed their respective frequencies and spatial distribution on two nested geographical scales (the whole of France and the French administrative district of Côte d'Or) by genotyping 13 151 plants originating from 243 fields. Genetic variation in ACCase was structured in local populations at both geographical scales. No spatial structure in the distribution of resistant ACCase alleles and no isolation by distance were detected at either geographical scale investigated. These data, together with ACCase sequencing and data from the literature, suggest that evolution of A. myosuroides resistance to herbicides occurred at the level of the field or group of adjacent fields by multiple, independent appearances of mutant ACCase alleles that seem to have rather restricted spatial propagation. Seed transportation by farm machinery seems the most likely vector for resistance gene dispersal in A. myosuroides.

Molecular bases for sensitivity to acetyl-coenzyme A carboxylase inhibitors in black-grass.

In grasses, residues homologous to residues Ile-1,781 and Ile-2,041 in the carboxyl-transferase (CT) domain of the chloroplastic acetyl-coenzyme A (CoA) carboxylase (ACCase) from the grass weed black-grass (Alopecurus myosuroides [Huds.]) are critical determinants for sensitivity to two classes of ACCase inhibitors, aryloxyphenoxypropionates (APPs) and cyclohexanediones. Using natural mutants of black-grass, we demonstrated through a molecular, biological, and biochemical approach that residues Trp-2,027, Asp-2,078, and Gly-2,096 are also involved in sensitivity to ACCase inhibitors. In addition, residues Trp-2,027 and Asp-2,078 are very likely involved in CT activity. Using three-dimensional modeling, we found that the side chains of the five residues are adjacent, located at the surface of the inside of the cavity of the CT active site, in the vicinity of the binding site for APPs. Residues 1,781 and 2,078 are involved in sensitivity to both APPs and cyclohexanediones, whereas residues 2,027, 2,041, and 2,096 are involved in sensitivity to APPs only. This suggests that the binding sites for these two classes of compounds are overlapping, although distinct. Comparison of three-dimensional models for black-grass wild-type and mutant CTs and for CTs from organisms with contrasted sensitivity to ACCase inhibitors suggested that inhibitors fitting into the cavity of the CT active site of the chloroplastic ACCase from grasses to reach their active sites may be tight. The three-dimensional shape of this cavity is thus likely of high importance for the efficacy of ACCase inhibitors.

Molecular bases for sensitivity to tubulin-binding herbicides in green foxtail.

We investigated the molecular bases for resistance to several classes of herbicides that bind tubulins in green foxtail (Setaria viridis L. Beauv.). We identified two alpha- and two beta-tubulin genes in green foxtail. Sequence comparison between resistant and sensitive plants revealed two mutations, a leucine-to-phenylalanine change at position 136 and a threonine-to-isoleucine change at position 239, in the gene encoding alpha2-tubulin. Association of mutation at position 239 with herbicide resistance was demonstrated using near-isogenic lines derived from interspecific pairings between green foxtail and foxtail millet (Setaria italica L. Beauv.), and herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Association of mutation at position 136 with herbicide resistance was demonstrated using herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Both mutations were associated with recessive cross resistance to dinitroanilines and benzoic acids, no change in sensitivity to benzamides, and hypersensitivity to carbamates. Using three-dimensional modeling, we found that the two mutations are adjacent and located into a region involved in tubulin dimer-dimer contact. Comparison of three-dimensional alpha-tubulin models for organisms with contrasted sensitivity to tubulin-binding herbicides enabled us to propose that residue 253 and the vicinity of the side chain of residue 251 are critical determinants for the differences in herbicide sensitivity observed between organisms, and that positions 16, 24, 136, 239, 252, and 268 are involved in modulating sensitivity to these herbicides.

Nucleotide variability at the acetyl coenzyme A carboxylase gene and the signature of herbicide selection in the grass weed Alopecurus myosuroides (Huds.).

Acetyl coenzyme A carboxylase (ACCase) is the target of highly effective herbicides. We investigated the nucleotide variability of the ACCase gene in a sample of 18 black-grass (Alopecurus myosuroides [Huds.]) populations to search for the signature of herbicide selection. Sequencing 3,396 bp encompassing ACCase herbicide-binding domain in 86 individuals revealed 92 polymorphisms, which formed 72 haplotypes. The ratio of nonsynonymous versus synonymous substitutions was very low, in agreement with ACCase being a vital metabolic enzyme. Within black grass, most nonsynonymous substitutions were related to resistance to ACCase-inhibiting herbicides. Differentiation between populations was strong, in contrast to expectations for an allogamous, annual plant. Significant H tests revealed recent hitchhiking events within populations. These results were consistent with recent and local positive selection. We propose that, although they have only been used since at most 15 black-grass generations, ACCase-inhibiting herbicides have exerted a positive selection targeting resistant haplotypes that has been strong enough to have a marked effect upon ACCase nucleotide diversity. A minimum-spanning network of nonrecombinant haplotypes revealed multiple, independent apparitions of resistance-associated mutations. This study provides the first evidence for the signature of ongoing, recent, pesticide selection upon variation at the gene encoding the targeted enzyme in natural plant populations.

An isoleucine residue within the carboxyl-transferase domain of multidomain acetyl-coenzyme A carboxylase is a major determinant of sensitivity to aryloxyphenoxypropionate but not to cyclohexanedione inhibitors.

A 3,300-bp DNA fragment encoding the carboxyl-transferase domain of the multidomain, chloroplastic acetyl-coenzyme A carboxylase (ACCase) was sequenced in aryloxyphenoxypropionate (APP)-resistant and -sensitive Alopecurus myosuroides (Huds.). No resistant plant contained an Ile-1,781-Leu substitution, previously shown to confer resistance to APPs and cyclohexanediones (CHDs). Instead, an Ile-2,041-Asn substitution was found in resistant plants. Phylogenetic analysis of the sequences revealed that Asn-2,041 ACCase alleles derived from several distinct origins. Allele-specific polymerase chain reaction associated the presence of Asn-2,041 with seedling resistance to APPs but not to CHDs. ACCase enzyme assays confirmed that Asn-2,041 ACCase activity was moderately resistant to CHDs but highly resistant to APPs. Thus, the Ile-2,041-Asn substitution, which is located outside a domain previously shown to control sensitivity to APPs and CHDs in wheat (Triticum aestivum), is a direct cause of resistance to APPs only. In known multidomain ACCases, the position corresponding to the Ile/Asn-2,041 residue in A. myosuroides is occupied by an Ile or a Val residue. In Lolium rigidum (Gaud.), we found Ile-Asn and Ile-Val substitutions. The Ile-Val change did not confer resistance to the APP clodinafop, whereas the Ile-Asn change did. The position and the particular substitution at this position are of importance for sensitivity to APPs.