Non-target Site Resistance Mechanisms Research Articles

A novel mutation Trp-2027-Gly in acetyl-CoA carboxylase confers resistance to cyhalofop-butyl in Chinese sprangletop (Leptochloa chinensis).

Chinese sprangletop [Leptochloa chinensis (L.) Nees] control is threatened by resistance to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. In this study, a L. chinensis population, HFLJ18, that survived cyhalofop-butyl [aryloxyphenoxypropionate (APP) herbicide, CyB] treatment was collected from a rice field in Lujiang County, Anhui Province, China. This study aimed to evaluate the susceptibility of HFLJ18 to herbicides with different modes-of-action and investigate the potential mechanisms of resistance to CyB. The HFLJ18 population exhibited high levels of resistance to CyB (10.92-fold) and showed resistance to the ACCase inhibitors metamifop (4.63-fold) and fenoxaprop-P-ethyl (8.39-fold), but was susceptible to clethodim, pinoxaden, florpyrauxifen-benzyl, oxadiazon and pretilachlor. Target gene sequencing revealed a novel Trp-to-Gly substitution at codon position 2027 of ACCase in the resistant plants. Molecular docking revealed that the spatial structure of ACCase changed significantly following the substitution, as indicated by reduced H-bonds. A newly derived cleaved amplified polymorphic sequence (dCAPS) marker was subsequently developed to detect the Trp-2027-Gly mutation in the ACCase of L. chinensis. Additionally, pretreatment with the cytochrome P450 (P450) inhibitor piperonyl butoxide (PBO) and the glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) did not reverse resistance to CyB, suggesting that nontarget-site resistance mechanisms were not involved in CyB resistance in the HFLJ18 population. Overall, the resistance to CyB in the HFLJ18 population derived from the mutation of ACCase gene, and to the best of our knowledge, this is the first report of the ACCase Trp-2027-Gly mutation conferring resistance to ACCase-inhibiting herbicides in grass species. © 2024 Society of Chemical Industry.

Occurrence and Mechanisms Conferring Multiple Resistance to ALS-Inhibiting and Auxins Mimics Herbicides in Papaver rhoeas from Tunisia

Herbicide-resistant corn poppy (Papaver rhoeas L.) is one of the most important broadleaved weeds and the number of resistant cases is still growing. The aims of this study were to confirm the resistance of P. rhoeas from Tunisia to ALS inhibitors and auxin mimics and investigate the mechanisms of Target-Site Resistance (TSR) and Non-Target Site Resistance (NTSR) involved. Dose–response trials to determine cross-resistance patterns for ALS inhibitors and auxin mimics were conducted in a greenhouse. In this study, multiple resistance to tribenuron-methyl and dicamba but not to 2,4-D was found in P. rhoeas populations. Cross-resistance to imazamox was confirmed as well. Sequence analysis of the ALS gene detected target-site mutations in codon 197 of the ALS gene, namely, Pro197His, Pro197Thr, Pro197Leu, and Pro197Asn. In this study, the metabolism experiments with malathion (a cytochrome P450 inhibitor) showed that malathion reduced resistance to imazamox, indicating that P450 is involved in the resistance. TSR and NTSR mechanisms to ALS inhibitors likely coexist. The findings of this study revealed a significant synergistic interaction between malathion and dicamba in particular populations, suggesting that the resistance to auxin mimics can be conferred by enhanced metabolism.

Metabolic Resistance to Protoporphyrinogen Oxidase-Inhibitor Herbicides in a Palmer amaranth Population from Kansas.

Palmer amaranth has evolved target and nontarget site resistance to protoporphyrinogen oxidase-inhibitor herbicides in the United States. Recently, a population (KCTR) from a long-term conservation tillage study in Kansas was found to be resistant to herbicides from six sites of action, including to PPO-inhibitors, even with this herbicide group being minimally used in this field. This research investigated the level of resistance to postemergence PPO-inhibitors, target- and nontarget-site resistance mechanism(s), and efficacy of pre-emergence chemistries. The greenhouse experiments confirmed 6.1- to 78.9-fold resistance to lactofen in KCTR, with the level of resistance increasing when KCTR was purified for the resistance trait. PPO2 sequences alignment revealed the absence of known mutations conferring resistance to PPO-inhibitors in KCTR Palmer amaranth, and differential expression of the PPO2 gene did not occur. KCTR metabolized fomesafen faster than the susceptible population, indicating that herbicide detoxification is the mechanism conferring resistance in this population. Further, treatment with the cytochrome P450-inhibitor malathion followed by lactofen restored the sensitivity of KCTR to this herbicide. Despite being resistant to POST applied PPO-inhibitors, KCTR Palmer amaranth was completely controlled by the labeled rate of the PRE applied PPO-inhibitors fomesafen, flumioxazin, saflufenacil, sulfentrazone, and oxadiazon. The overall results suggest that P450-mediated metabolism confers resistance to PPO-inhibitors in KCTR, rather than alterations in the PPO2, which were more commonly found in other Palmer amaranth populations. Future work will focus on identifying the fomesafen metabolites and on unravelling the genetic basis of metabolic resistance to PPO-inhibitor herbicides in KCTR Palmer amaranth.

RNA and protein biomarkers for detecting enhanced metabolic resistance to herbicides mesosulfuron-methyl and fenoxaprop-ethyl in black-grass (Alopecurus myosuroides).

The evolution of non-target site resistance (NTSR) to herbicides leads to a significant reduction in herbicide control of agricultural weed species. Detecting NTSR in weed populations prior to herbicide treatment would provide valuable information for effective weed control. While not all NTSR mechanisms have been fully identified, enhanced metabolic resistance (EMR) is one of the better studied, conferring tolerance through increased herbicide detoxification. Confirming EMR towards specific herbicides conventionally involves detecting metabolites of the active herbicide molecule in planta, but this approach is time-consuming and requires access to well-equipped laboratories. In this study, we explored the potential of using molecular biomarkers to detect EMR before herbicide treatment in black-grass (Alopecurus myosuroides). We tested the reliability of selected biomarkers to predict EMR and survival after herbicide treatments in both reference and 27 field-derived black-grass populations collected from sites across the UK. The combined analysis of the constitutive expression of biomarkers and metabolism studies confirmed three proteins, namely, AmGSTF1, AmGSTU2 and AmOPR1, as differential biomarkers of EMR toward the herbicides fenoxaprop-ethyl and mesosulfuron in black-grass. Our findings demonstrate that there is potential to use molecular biomarkers to detect EMR toward specific herbicides in black-grass without reference to metabolism analysis. However, biomarker development must include testing at both transcript and protein levels in order to be reliable indicators of resistance. This work is a first step towards more robust resistance biomarker development, which could be expanded into other herbicide chemistries for on-farm testing and monitoring EMR in uncharacterised black-grass populations. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Insight into the herbicide resistance patterns in Lolium rigidum populations in Tunisian and Moroccan wheat regions.

Rigid ryegrass (Lolium rigidum Gaud.) is one of the most troublesome weeds in Moroccan and Tunisian cereal crop fields. In total, 19 rigid ryegrass field populations were randomly selected in northern wheat crop areas of Morocco and Tunisia to examine the patterns of herbicide resistance to acetolactate synthase (ALS)- and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. Greenhouse experiments confirmed reduced sensitivity to ALS- and/or ACCase-inhibiting herbicides in all L. rigidum populations. The occurrence of target-site resistance (TSR) was tested using high-throughput genotyping. The advent of next-generation sequencing (NGS) has enabled easy identification of causal mutations and confirmed the presence of ALS and ACCase mutations at specific codons conferring TSR. Thirteen populations showed resistance to ALS-inhibiting herbicides associated with point mutations in positions Pro-197-Thr, Pro-197-Ser, Pro-197-Leu, Pro-197-Gln and Trp-574-Leu, while resistance to ACCase-inhibiting herbicides was detected in 18 populations in positions Asp-2078-Val, Trp-2027-Cys, Ile-1781-Leu, Gly-2096-Ala, and Ile-2041-Asn of the enzymes conferring TSR. Additionally, dose-response experiments with pyroxsulam applied after the inhibition of cytochrome P450 monooxygenase by malathion showed an increase in sensitivity in two out of seven highly resistant (HR) rigid ryegrass populations. This demonstrates the presence of non-target-site resistance (NTSR) in some ryegrass populations. Further evidence of NTSR was investigated in dose-response experiments with pyroxsulam, following pretreatment with the glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole (NBD-Cl), which partially reversed resistance in only a few individuals of two L. rigidum populations. Hence, our study confirms the existence of multiple and cross-resistance to ALS- and ACCase-inhibiting herbicides in L. rigidum from Morocco and Tunisia with both TSR and NTSR mechanisms. These results emphasize local resistance management as an important tool to detect and mitigate gene flow from rigid ryegrass populations where resistance has evolved.

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.)

BackgroundNon-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.ResultsA 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.ConclusionOur 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.

Comprehensive insights into herbicide resistance mechanisms in weeds: a synergistic integration of transcriptomic and metabolomic analyses.

Omics techniques, including genomics, transcriptomics, proteomics, and metabolomics have smoothed the researcher's ability to generate hypotheses and discover various agronomically relevant functions and mechanisms, as well as their implications and associations. With a significant increase in the number of cases with resistance to multiple herbicide modes of action, studies on herbicide resistance are currently one of the predominant areas of research within the field of weed science. High-throughput technologies have already started revolutionizing the current molecular weed biology studies. The evolution of herbicide resistance in weeds (particularly via non-target site resistance mechanism) is a perfect example of a complex, multi-pathway integration-induced response. To date, functional genomics, including transcriptomic and metabolomic studies have been used separately in herbicide resistance research, however there is a substantial lack of integrated approach. Hence, despite the ability of omics technologies to provide significant insights into the molecular functioning of weeds, using a single omics can sometimes be misleading. This mini-review will aim to discuss the current progress of transcriptome-based and metabolome-based approaches in herbicide resistance research, along with their systematic integration.

Quizalofop resistance in weedy rice (Oryza sativa L.) is mainly conferred by an Ile1781Leu mutation

Weedy rice (Oryza sativa L.) is an economically important weed species in rice (Oryza sativa L.) cropping systems. Two weedy rice samples (acc7 and acc8) suspected to be resistant to quizalofop-ethyl (quizalofop) were collected in Arkansas. In this research, susceptibility to quizalofop and resistance mechanisms have been explored. Dose-response assays displayed a resistance index of 42- and 58-fold for the acc7 and acc8, respectively. Experiments with metabolism inhibitors demonstrated that NBD-Cl (4-chloro-7-nitrobenzofurazan) increased quizalofop efficacy slightly in acc8, whereas malathion did not improve effectiveness in resistant samples. Sequencing of the ACCase gene displayed an Ile1781Leu substitution in the resistant samples, like the mutation present in Provisia™ rice. In addition, an allele-specific PCR was developed to genotype the Ile1781Leu mutation. The gene copy number of ACCase showed similar values among samples. In the resistant plants, a KASP (Kompetitive Allele Specific PCR) assay to detect the ALSS653D (acetolactate synthase) and HIS1 (HPPD Inhibitor Sensitive 1) traits revealed that 37.5% of plants carried the ALSS653D trait, whereas 25% showed the HIS1 allele. In summary, a target-site mutation is the main resistance mechanism to quizalofop in weedy rice. Results also suggest the presence of herbicide metabolism (a non-target site resistance mechanism) mediated by glutathione-S-transferases (GSTs) in one resistant sample.

Asp376Glu Mutation and Enhanced Metabolism Controlling the Resistance to ALS-Inhibiting Herbicides in Ixophorus unisetus (J. Presl) Schltdl from the Bajio, Mexico

A study was carried out to determine the ALS (acetolactate synthase)-inhibitor herbicide resistance in the Mexican grass Ixophorus unisetus, a troublesome weed in corn crops in Mexico. First, the resistance was confirmed in field screening assays. Eight populations that survived nicosulfuron treatment at a field rate of 40 g ai ha−1 were labeled as putative-resistant. Dose–response trials demonstrated a high resistance in the eight populations (GR50 from 140.33 to 245.46 g ai ha−1). The synergism of malathion plus nicosulfuron demonstrated that the non-target-site resistance (NTSR) mechanism based on cytochrome P450 (Cyt-P450) was involved in five populations of I. unisetus. Molecular studies revealed that a single-nucleotide change occurs in the amino acid at position 376 (from GAT to GAG), which codifies from Asp-376 to Glu-376. This is the first time that Asp-376-Glu has been reported in this species. Assays in vitro and in vivo demonstrated I. unisetus cross-resistance to flucarbazone, penoxsulam, bispyribac-Na, and imazamox. No multiple resistance was found in two resistant populations exposed to different herbicides. Our results indicate that the lack of good control over Mexican grass in corn with ALS inhibitors is due to target-site mutation and NTSR mechanisms (Cyt-P450-mediated metabolism). A strategy should be established in Mexican fields to continue controlling this weed, including mechanical control practices and a good combination of the available pre- and post-emergence herbicides.

Nontarget Site-Based Resistance to Fenoxaprop-P-ethyl and Candidate Genes Involved in Alopecurus japonicus

Nontarget-site resistance (NTSR) is a complex multigenic trait that is associated with the potential mechanisms of herbicide resistance which pose a serious threat to global crop protection. However, the NTSR mechanisms of Alopecurus japonicus, a malignant weed infesting wheat fields, are less characterized. In this study, we used RNA-sequencing transcriptome and enzyme activity detection to investigate the NTSR mechanisms and candidate genes involved in fenoxaprop-P-ethyl (FE) in a previously identified resistant population compared to the sensitive population of A. japonicus. Transcriptome analysis identified nine upregulated genes, which were constitutively overexpressed and upregulated by FE application in the resistant population, and the results were validated using quantitative real-time PCR. These genes including one cytochrome P450 monooxygenase (P450) gene (CYP75B4), one ATP-binding cassette (ABC) transporter gene (ABCG36), one laccase (LAC) gene (LAC15), one 9-cis-epoxycarotenoid dioxygenase (NCED) gene (NCED5), two purple acid phosphatase (PAP) genes (PAP4, PAP15), one sucrose phosphate synthase (SPS) gene (SPS3), one protein related to disease resistance gene (RGA3) and one immune protein gene (R1B-17). The activity assay of LAC, NCED, PAP and SPS revealed that the activities of these enzymes in the resistant population were significantly higher than those in the sensitive population at 0 h and after FE application at 12 h, 24 h and 72 h. Nevertheless, whether LAC, NCED, PAP and SPS genes were involved in herbicide metabolism needs to be further validated. Our results revealed that CYP, ABC transporter and LAC genes may participate in A. japonicus resistance. These genes identified in the present study provide new insights into the resistance mechanism of weeds in response to herbicide. Our study also implies the complexity of the NTSR mechanisms of weeds.

Comparative proteomic and physiological analyses reveal tribenuron-methyl phytotoxicity and nontarget-site resistance mechanisms in Brassicanapus.

The application of herbicides is the most effective strategy for weed control and the development of herbicide-resistant crops will facilitate the weed management. The acetolactate synthase-inhibiting herbicide, tribenuron-methyl (TBM), is broadly used for weed control. However, its application in rapeseed field is restricted since rapeseed is sensitive to TBM. Herein, an integrated study of cytological, physiological and proteomic analysis of the TBM-resistant rapeseed mutant M342 and its wild-type (WT) plants was conducted. After TBM spraying, M342 showed improved tolerance to TBM, and proteins implicated in non-target-site resistance (NTSR) to herbicides had a significantly higher level in M342 as compared with the WT. Differentially accumulated proteins (DAPs) between these two genotypes were enriched in glutathione metabolism and oxidoreduction coenzyme metabolic process, which protected the mutant from oxidative stress triggered by TBM. Important DAPs related to stress or defence response were up-accumulated in M342regardless of the TBM treatment, which might serve as the constitutive part of NTSR to TBM. These results provide new clues for further exploration of the NTSR mechanism in plants and establish a theoretical basis for the development of herbicide-resistant crops.

Target-site and non-target site resistance mechanisms are associated with iodosulfuron resistance in Lolium perenne L.

ABSTRACT Resistance to iodosulfuron, a herbicide targeting acetolactate synthase (ALS), was identified and characterised in a population of Lolium perenne L. from Canterbury, New Zealand. The resistant population was 264 times more resistant to iodosulfuron than a susceptible population, based on a ratio of the rate corresponding to a 50% reduction in resistant and susceptible plant survival (R/S LD50). The resistant population was also 14-times more resistant to nicosulfuron, another ALS-inhibitor of the same class. However, no resistance to imazapyr, an ALS-inhibitor from a different chemical class, was detected in the resistant population. Pre-treatment with an inhibitor of cytochrome P450 reduced the level of iodosulfuron resistance to 46-fold in the resistant population, suggesting enhanced herbicide metabolism, a non-target site mechanism, contributed to resistance of this herbicide. In addition, a proline to leucine substitution at codon 197 was detected, suggesting a target-site-based mechanism was also associated with resistance to iodosulfuron. This is the first report on the resistance mechanisms to ALS-inhibiting herbicides identified in Lolium perenne in New Zealand and highlights the importance of integrated weed management approaches to reduce the likelihood of ALS-inhibitor herbicide resistance becoming more widespread.

Multiple Resistance Mechanisms Involved in Glyphosate Resistance in Eleusine indica

Glyphosate is a non-selective herbicide and is widely used for weed control in non-cultivated land in China. One susceptible (S) and five putative glyphosate-resistant (R1, R2, R3, R4, and R5) Eleusine indica biotypes were selected to investigate their resistance levels and the potential resistance mechanisms. Based on the dose-response assays, the R3 and R5 biotypes showed a low-level (2.4 to 3.5-fold) glyphosate resistance, and the R1, R2, and R4 biotypes exhibited a moderate- to high-level (8.6 to 19.2-fold) resistance, compared with the S biotype. The analysis of the target-site resistance (TSR) mechanism revealed that the P106A mutation and the heterozygous double T102I + P106S mutation were found in the R3 and R4 biotypes, respectively. In addition, the similar EPSPS gene overexpression was observed in the R1, R2, and R5 biotypes, suggesting that additional non-target-site resistance (NTSR) mechanisms may contribute to glyphosate resistance in R1 and R2 biotypes. Subsequently, an RNA-Seq analysis was performed to identify candidate genes involved in NTSR. In total, ten differentially expressed contigs between untreated S and R1 or R2 plants, and between glyphosate-treated S and R1 or R2 plants, were identified and further verified with RT-qPCR. One ATP-binding cassette (ABC) transporter gene, one aldo-keto reductases (AKRs) gene and one cytochrome P450 monooxygenase (CytP450) gene were up-regulated in R1 or R2 plants. These results indicated that EPSPS overexpression, single or double mutation was a common TSR mechanisms in E. indica. Additional NTSR mechanisms could play an essential role in glyphosate resistance. Three genes, ABCC4, AKR4C10, and CYP88, could serve as important candidate genes and deserve further functional studies.

Enhanced Metabolism Evolved High-Level Resistance to Fenoxaprop-P-Ethyl in Alopecurus japonicus

Alopecurus japonicus has been a serious weed across China and developed resistance to the acetyl-CoA carboxylase (ACCase)-inhibiting herbicide. The A. japonicus ACCase gene mutations accounting for target-site resistance (TSR) have been clarified, while non-target-site resistance (NTSR) is not distinct. Here, dose–response testing indicated that an A. japonicus population (R) was resistant to four ACCase-inhibiting herbicides fenoxaprop-P-ethyl, sethoxydim, clethodim, and pinoxaden. Compared with herbicide-susceptible A. japonicus (S), no known resistant mutations for TSR in ACCase were identified in the R population using sequencing. To investigate the NTSR mechanism, increased metabolism of fenoxaprop-P-ethyl was detected in the R population using high-performance liquid chromatography (HPLC) analysis. Notably, resistance cannot be reversed by P450 and GST inhibitors. RNA-seq was performed to further explore the resistance mechanisms, and eight candidate contigs (four glycosyl transferases (GT) and four ATP-binding cassette (ABC) transporters) were chosen and their expression patterns were validated using RT-qPCR. Three GT and three ABC transporter contigs were constitutively upregulated in the R population. In short, six contigs expressed highly in the R population causing enhanced fenoxaprop-P-ethyl metabolism appear to be involved in fenoxaprop-P-ethyl resistance.

Baseline sensitivity of European Zymoseptoria tritici populations to the complex III respiration inhibitor fenpicoxamid.

Fenpicoxamid is a recently developed fungicide belonging to the quinone inside inhibitor (QiI) group. This is the first fungicide within this group to be active against the Zymoseptoria tritici, which causes Septoria tritici blotch on wheat. The occurrence of pre-existing resistance mechanisms was monitored, using sensitivity assays and Illumina sequencing, in Z. tritici populations sampled in multiple European countries before the introduction of fenpicoxamid. Although differences in sensitivity to all three fungicides tested (fenpicoxamid, fentin chloride and pyraclostrobin) existed between the isolate collections, no alterations associated with QiI resistance were detected. Among the isolates, a range in sensitivity to fenpicoxamid was observed (ratio between most sensitive/least sensitive=53.1), with differences between the most extreme isolates when tested in planta following limited fenpicoxamid treatment. Sensitivity assays using fentin chloride suggest some of the observed differences in fenpicoxamid sensitivity are associated with multi-drug resistance. Detailed monitoring of the wider European population using Illumina-based partial sequencing of the Z. tritici also only detected the presence of G143A, with differences in frequencies of this alteration observed across the region. This study provides a baseline sensitivity for European Z. tritici populations to fenpicoxamid. Target-site resistance appears to be limited or non-existing in European Z. tritici populations prior to the introduction of fenpicoxamid. Non-target site resistance mechanisms exist, but their impact in the field is predicted to be limited. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Non-Target-Site Resistance Mechanisms Endow Multiple Herbicide Resistance to Five Mechanisms of Action in Conyza bonariensis.

The repeated use of herbicides can lead to the selection of multiple resistance weeds. Some populations of Conyza bonariensis occurring in olive groves from southern Spain have developed resistance to various herbicides. This study determined the resistance levels to 2,4-D, glyphosate, diflufenican, paraquat, and tribenuron-methyl in a putative resistant (R) C. bonariensis population, and the possible non-target-site resistance (NTSR) mechanisms involved were characterized. Resistance factors varied as follows: glyphosate (8.9), 2,4-D (4.8), diflufenican (5.0), tribenuron-methyl (19.6), and paraquat (85.5). Absorption of 14C-glyphosate was up to 25% higher in the susceptible (S) population compared to the R one, but 14C-paraquat absorption was similar (up to 70%) in both populations. S plants translocated more than 60% of both 14C-glyphosate and 14C-paraquat toward shoots and roots, while R plants translocated less than 10%. The R population was able to metabolize 57% of the 2,4-D into nontoxic metabolites and 68% of the tribenuron-methyl into metsulfuron-methyl (10%), metsulfuron-methyl-hydroxylate (18%), and conjugate-metsulfuron-methyl (40%). Among the NTSR mechanisms investigated, absorption and translocation could be involved in glyphosate resistance, but only translocation for paraquat. Proofs of the presence of enhanced metabolism as a resistance mechanism were found for tribenuron-methyl and 2,4-D, but not for diflufenican. This research informs the first occurrence of multiple resistance to five herbicide classes (acetolactate synthase inhibitors, 5-enolpyruvylshikimate-3-phosphate synthase inhibitors, photosystem I electron diverters, photosystem II inhibitors, and synthetic auxin herbicides) in C. bonariensis.

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.

Negative cross-resistance to clomazone in imazethapyr-resistant Echinochloa crus-galli caused by increased metabolization

Herbicide resistance is frequently reported in E. crus-galli globally with target and non-target site resistance mechanism to acetolactate synthase (ALS)-inhibiting herbicides. However, resistance to certain herbicides can result in increased sensitivity to other herbicides, a phenomenon called negative cross-resistance. The objective of this study is to identify the occurrence of negative cross-resistance (NCR) to the pro-herbicide clomazone in populations of E. crus-galli resistant to ALS inhibitors due to increased metabolization. Clomazone dose-response curves, with and without malathion, were performed in imazethapyr-resistant and -susceptible E. crus-galli biotypes. CYPs genes expression and antioxidant enzymes activity were also evaluated. The effective dose to reduce 50% (ED50) of dry shoot weight obtained in the clomazone dose-response curves of the metabolic based imazethapyr-resistant and -susceptible biotypes groups were 22.712 and 58.745 g ha−1, respectively, resulting in a resistance factor (RF) of 0.37, indicating the occurrence of NCR. The application of malathion prior to clomazone increased the resistance factor from 0.60 to 1.05, which indicate the reversion of the NCR. Some CYP genes evaluated were expressed in a higher level, ranging from 2.6–9.1 times according to the biotype and the gene, in the imazethapyr-resistant than in -susceptible biotypes following clomazone application. Antioxidant enzyme activity was not associated with NCR. This study is the first report of NCR directly related to the mechanism of resistance increased metabolization in plants. The occurrence of NCR to clomazone in E. crus-galli can help delay the evolution of herbicide resistance.

Point Mutations as Main Resistance Mechanism Together With P450-Based Metabolism Confer Broad Resistance to Different ALS-Inhibiting Herbicides in Glebionis coronaria From Tunisia.

Resistance to acetolactate synthase (ALS) inhibiting herbicides has recently been reported in Glebionis coronaria from wheat fields in northern Tunisia, where the weed is widespread. However, potential resistance mechanisms conferring resistance in these populations are unknown. The aim of this research was to study target-site resistance (TSR) and non-target-site resistance (NTSR) mechanisms present in two putative resistant (R) populations. Dose–response experiments, ALS enzyme activity assays, ALS gene sequencing, absorption and translocation experiments with radiolabeled herbicides, and metabolism experiments were carried out for this purpose. Whole plant trials confirmed high resistance levels to tribenuron and cross-resistance to florasulam and imazamox. ALS enzyme activity further confirmed cross-resistance to these three herbicides and also to bispyribac, but not to flucarbazone. Sequence analysis revealed the presence of amino acid substitutions in positions 197, 376, and 574 of the target enzyme. Among the NTSR mechanisms investigated, absorption or translocation did not contribute to resistance, while evidences of the presence of enhanced metabolism were provided. A pretreatment with the cytochrome P450 monooxygenase (P450) inhibitor malathion partially synergized with imazamox in post-emergence but not with tribenuron in dose–response experiments. Additionally, an imazamox hydroxyl metabolite was detected in both R populations in metabolism experiments, which disappeared with the pretreatment with malathion. This study confirms the evolution of cross-resistance to ALS inhibiting herbicides in G. coronaria from Tunisia through TSR and NTSR mechanisms. The presence of enhanced metabolism involving P450 is threatening the chemical management of this weed in Tunisian wheat fields, since it might confer cross-resistance to other sites of action.

Can Control of Glyphosate Susceptible and Resistant Conyza sumatrensis Populations Be Dependent on the Herbicide Formulation or Adjuvants?

In this work, we studied the effect of three glyphosate formulations (isopropylamine, ammonium and potassium salts) and two non-ionic adjuvants on the resistance response of two resistant (R1, R2) and one susceptible population of the highly invasive Asteraceae, Conyza sumatrensis, from Southern France vineyards. Only in R1, an amino acid substitution (Pro106Thr) was found in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The two adjuvants, in a similar fashion, significantly reduced GR50 values for every population and glyphosate formulation. Without adjuvants, glyphosate as potassium salt was the only formulation able to significantly reduce the GR50 values of every population. For every population, the two adjuvants improved, indistinguishably, leaf retention of the herbicidal solution and the potassium salt formulation led to the highest retention, both with and without the adjuvant added. Uptake responses paralleled those of retention and adjuvant addition was more effective in increasing foliar uptake of the lower performing formulations (isopropylamine and ammonium salts). The allocation pattern of glyphosate among plant compartments was only dependent on population, with R2 retaining most glyphosate in the treated leaf, clearly suggesting the occurrence of a Non-Target Site Resistance (NTSR) mechanism. Results indicate that control of weed populations possessing NTSR mechanisms of resistance to glyphosate may be improved through adequate selection of formulation and adjuvant use.