Enhanced Herbicide Metabolism and Target Site Mutation Enabled the Multiple Resistance to Cyhalofop-butyl, Florpyrauxifen-benzyl, and Penoxsulam in Echinochloa crus-galli.

This study investigated the multiple herbicide resistance (MHR) mechanism of one Echinochloa crus-galli population that was resistant to florpyrauxifen-benzyl (FPB), cyhalofop-butyl (CHB), and penoxsulam (PEX). This population carried an Ala-122-Asn mutation in the acetolactate synthase (ALS) gene but no mutation in acetyl-CoA carboxylase (ACCase) and transport inhibitor response1 (TIR1) genes. The metabolism rate of PEX was 2-fold higher, and the production of florpyrauxifen-acid and cyhalofop-acid was lower in the resistant population. Malathion and 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) could reverse the resistance, suggesting that cytochrome P450 (CYP450) and glutathione S-transferase (GST) contribute to the enhanced metabolism. According to RNA-seq and qRT-PCR validation, two CYP450 genes (CYP71C42 and CYP71D55), one GST gene (GSTT2), two glycosyltransferase genes (rhamnosyltransferase 1 and IAAGLU), and two ABC transporter genes (ABCG1 and ABCG25) were induced by CHB, FPB, and PEX in the resistant population. This study revealed that the target mutant and enhanced metabolism were involved in the MHR mechanism in E. crus-galli.

Identification of novel genes associated with herbicide tolerance in Lentil (Lens culinaris ssp. culinaris Medik.).

Weeds pose a major constraint in lentil cultivation, leading to decrease farmers' revenues by reducing the yield and increasing the management costs. The development of herbicide tolerant cultivars is essential to increase lentil yield. Even though herbicide tolerant lines have been identified in lentils, breeding efforts are still limited and lack proper validation. Marker assisted selection (MAS) can increase selection accuracy at early generations. Total 292 lentil accessions were evaluated under different dosages of two herbicides, metribuzin and imazethapyr, during two seasons at Marchouch, Morocco and Terbol, Lebanon. Highly significant differences among accessions were observed for days to flowering (DF) and maturity (DM), plant height (PH), biological yield (BY), seed yield (SY), number of pods per plant (NP), as well as the reduction indices (RI) for PH, BY, SY and NP. A total of 10,271 SNPs markers uniformly distributed along the lentil genome were assayed using Multispecies Pulse SNP chip developed at Agriculture Victoria, Melbourne. Meta-GWAS analysis was used to detect marker-trait associations, which detected 125 SNPs markers associated with different traits and clustered in 85 unique quantitative trait loci. These findings provide valuable insights for initiating MAS programs aiming to enhance herbicide tolerance in lentil crop.

Target-site and non-target-site resistance mechanisms confer mesosulfuron-methyl resistance in Alopecurus aequalis.

BACKGROUND: Shortawn foxtail (Alopecurus aequalis Sobol.) is a noxious weed in China. The resistance of A. aequalis developed rapidly due to the long-term application of acetolactate synthase (ALS)-inhibiting herbicides. Here, a suspected mesosulfuron-methyl-resistant A. aequalis population, Aa-R, was collected from a wheat field in China. RESULTS: A dose‒response test showed that the Aa-R population has evolved a high level of resistance to mesosulfuron-methyl, and its growth was suppressed by imazamox, pyroxsulam and bispyribac-sodium. ALS gene sequence analysis revealed that a known resistance-related mutation (Pro-197-Thr) was present in the Aa-R population. Moreover, ALS gene overexpression was detected in the Aa-R population. The mesosulfuron-methyl resistance could be reversed by cytochrome P450 monooxygenase (CYP450) and glutathione S-transferase (GST) inhibitors. In addition, enhanced metabolism of mesosulfuron-methyl was detected in the Aa-R population compared with the susceptible population. NADPH-cytochrome P450 reductase and GST activities were strongly inducible in the Aa-R population. One CYP450 gene, CYP74A2, and one GST gene, GST4, were constitutively upregulated in the Aa-R population. Molecular docking results showed the binding affinity of CYP74A2 and GST4 for the tested ALS-inhibiting herbicides, respectively. CONCLUSION: This study confirmed that target-site resistance and non-target-site resistance involving CYP450 and GST were the main mechanisms involved in resistance in the mesosulfuron-methyl-resistant A. aequalis population.

The Cys-2088-Arg mutation in the ACCase gene and enhanced metabolism confer cyhalofop-butyl resistance in Chinese sprangletop (Leptochloa chinensis).

Acetyl-CoA carboxylase (ACCase)-inhibiting herbicides are among the most commonly used herbicides to control grassy weeds, especially Leptochloa chinensis, in rice fields across China. Herein, we collected a suspected resistant (R) population of L. chinensis (HFLJ16) from Lujiang county in Anhui Province. Whole plant dose response tests showed that, compared with the susceptible (S) population, the R population showed high resistance to cyhalofop-butyl (22-fold) and displayed cross-resistance to metamifop (9.7-fold), fenoxaprop-P-ethyl (18.7-fold), quizalofop-P-ethyl (7.6-fold), clodinafop-propargyl (12-fold) and clethodim (8.4-fold). We detected an amino acid substitution (Cys-2088-Arg) in the ACCase of resistant L. chinensis. However, ACCase gene expression levels were not significantly different (P > 0.05) between R plants and S plants, without or with cyhalofop-butyl treatment. Furthermore, pretreatment with piperonyl butoxide (PBO, a cytochrome P450 monooxygenase (CYP450) inhibitor) or 4-chloro-7-nitrobenzoxadiazole (NBD-Cl, a glutathione-S-transferase (GST) inhibitor), inhibited the resistance of the R population to cyhalofop-butyl significantly (by approximately 60% and 26%, respectively). Liquid chromatography tandem mass spectrometry analysis showed that R plants metabolized cyhalofop-butyl and cyhalofop acid (its metabolite) significantly faster than S plants. Three CYP450 genes, one GST gene, and two ABC transporter genes were induced by cyhalofop-butyl and were overexpressed in the R population. Overall, GST-associated detoxification, CYP450 enhancement, and target-site gene mutation are responsible for the resistance of L. chinensis to cyhalofop-butyl.

An Asp376Glu substitution and P450s-involved metabolism endow resistance to ALS inhibitors in an Ammannia auriculata Willd. Population.

Ammannia auriculata Willd. is a noxious broadleaf weed, commonly infesting rice ecosystems across southern China. A putative resistant A. auriculata population (AHSC-5) was sampled from a rice field of Anhui Province, where bensulfuron-methyl (BM) was unable to control its occurrence. This study aimed to determine the sensitivities of the AHSC-5 population to common-use herbicides, and to investigate the underlying resistance mechanisms. The bioassays showed that the AHSC-5 population was 138.1-fold resistant to BM, compared with the susceptible population (JSGL-1). Pretreatment of malathion reduced the resistance index to 19.5. ALS sequencing revealed an Asp376Glu substitution in the AHSC-5 population, and in vitro ALS activity assays found that 50% activity inhibition (I50) of BM in AHSC-5 was 75.4 times higher than that of JSGL-1. Moreover, the AHSC-5 population displayed cross-resistance to pyrazosulfuron-ethyl (10.6-fold), bispyribac­sodium (3.6-fold), and imazethapyr (2.2-fold), and was in the process of evolving multiple resistance to synthetic auxin herbicides fluroxypyr (2.3-fold) and florpyrauxifen-benzyl (3.1-fold). This study proved the BM resistance in A. auriculata caused by the Asp376Glu mutation and P450-regulated metabolism. This multi-resistant population can still be controlled by penoxsulam, MCPA, bentazone, and carfentrazone-ethyl, which aids in developing targeted and effective weed management strategies.

Pro197Ser and the new Trp574Leu mutations together with enhanced metabolism contribute to cross-resistance to ALS inhibiting herbicides in Sinapis alba.

White mustard, (Sinapis alba), a problematic broadleaf weed in many Mediterranean countries in arable fields has been detected as resistant to tribenuron-methyl in Tunisia. Greenhouse and laboratory studies were conducted to characterize Target-Site Resistance (TSR) and the Non-Target Site Resistance (NTSR) mechanisms in two suspected white mustard biotypes. Herbicide dose-response experiments confirmed that the two S. alba biotypes were resistant to four dissimilar acetolactate synthase (ALS)-pinhibiting herbicide chemistries indicating the presence of cross-resistance mechanisms. The highest resistance factor (>144) was attributed to tribenuron-methyl herbicide and both R populations survived up to 64-fold the recommended field dose (18.7 g ai ha-1). In this study, the metabolism experiments with malathion (a cytochrome P450 inhibitor) showed that malathion reduced resistance to tribenuron-methyl and imazamox in both populations, indicating that P450 may be involved in the resistance. Sequence analysis of the ALS gene detected target site mutations in the two R biotypes, with amino acid substitutions Trp574Leu, the first report for the species, and Pro197Ser. Molecular docking analysis showed that ALSPro197Ser enzyme cannot properly bind to tribenuron-methyl's aromatic ring due to a reduction in the number of hydrogen bonds, while imazamox can still bind. However, Trp574Leu can weaken the binding affinity between the mutated ALS enzyme and both herbicides with the loss of crucial interactions. This investigation provides substantial evidence for the risk of evolving multiple resistance in S. alba to auxin herbicides while deciphering the TSR and NTSR mechanisms conferring cross resistance to ALS inhibitors.

A Critical Review of Conventional and Modern Approaches to Develop Herbicide-Resistance in Rice.

Together with rice, weeds strive for nutrients and space in farmland, resulting in reduced rice yield and quality. Planting herbicide-resistant rice varieties is one of the effective ways to control weeds. In recent years, a series of breakthroughs have been made to generate herbicide-resistant germplasm, especially the emergence of biotechnological tools such as gene editing, which provides an inherent advantage for the knock-out or knock-in of the desired genes. In order to develop herbicide-resistant rice germplasm resources, gene manipulation has been conducted to enhance the herbicide tolerance of rice varieties through the utilization of techniques such as physical and chemical mutagenesis, as well as genome editing. Based on the current research and persisting problems in rice paddy fields, research on the generation of herbicide-resistant rice still needs to explore genetic mechanisms, stacking multiple resistant genes in a single genotype, and transgene-free genome editing using the CRISPR system. Current rapidly developing gene editing technologies can be used to mutate herbicide target genes, enabling targeted genes to maintain their biological functions, and reducing the binding ability of target gene encoded proteins to corresponding herbicides, ultimately resulting in herbicide-resistant crops. In this review article, we have summarized the utilization of conventional and modern approaches to develop herbicide-resistant cultivars in rice as an effective strategy for weed control in paddy fields, and discussed the technology and research directions for creating herbicide-resistant rice in the future.

Diverse ALS mutations and cross-and multiple-resistance to ALS and EPSPS inhibitors in flucarbazone­sodium-resistant Bromus japonicus populations from Hebei province, China.

Japanese brome (Bromus japonicus) has become one of the main weeds in wheat fields in Hebei province of China and causes a large decrease of wheat production. A total of 44 putative resistant and 2 susceptible Japanese brome populations were collected in the 2021/2022 crop season from Hebei province of China to determine resistance levels to flucarbazone­sodium and to investigate the diversity of acetolactate synthase (ALS) mutations, as well as to confirm the cross-and multiple-resistance levels to ALS and EPSPS (5-enolpyruvate shikimate-3-phosphate synthetase) inhibitors. Whole plant bioassay results showed that 15 out of 44 populations tested or 34% were resistant to flucarbazone­sodium. The resistance indices of Japanese brome to flucarbazone­sodium ranged from 43 to 1977. The resistant populations were mainly distributed in Baoding and Shijiazhuang districts, and there was only one resistant population in Langfang district. Resistant Japanese brome had diverse ALS mutations, including Pro-197-Ser, -Thr, -Arg and Asp-376-Glu. The incidence of Pro-197-Ser mutation was the highest at 68%. Application of the CYP450 inhibitor malathion suggested that CYP450 was involved in metabolic resistance in a population without an ALS mutation. The population with Pro-197-Thr mutation evolved weak cross-resistance to mesosulfuron-methyl and pyroxsulam, and it is in the process of evolving multiple-resistance to glyphosate.

Genome-wide study of glutathione transferases and their regulation in flufenacet susceptible and resistant black-grass (Alopecurus myosuroides Huds.).

BACKGROUND: Glutathione transferases (GSTs) are enzymes with a wide range of functions, including herbicide detoxification. Up-regulation of GSTs and their detoxification activity enables the grass weed black-grass (Alopecurus myosuroides Huds.) to metabolize the very-long-chain fatty acid synthesis inhibitor flufenacet and other herbicides leading to multiple herbicide resistance. However, the genomic organization and regulation of GSTs genes is still poorly understood. RESULTS: In this genome-wide study the location and expression of 115 GSTs were investigated using a recently published black-grass genome. Particularly, the most abundant GSTs of class tau and phi were typically clustered and often followed similar expression patterns but possessed divergent upstream regulatory regions. Similarities were found in the promoters of the most up-regulated GSTs, which are located next to each other in a cluster. The binding motif of the E2F/DP transcription factor complex in the promoter of an up-regulated GST was identical in susceptible and resistant plants, however, adjacent sequences differed. This led to a stronger binding of proteins to the motif of the susceptible plant, indicating repressor activity. CONCLUSIONS: This study constitutes the first analysis dealing with the genomic investigation of GST genes found in black-grass and their transcriptional regulation. It highlights the complexity of the evolution of GSTs in black-grass, their duplication and divergence over time. The large number of GSTs allows weeds to detoxify a broad spectrum of herbicides. Ultimately, more research is needed to fully elucidate the regulatory mechanisms of GST expression. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Dual plastid targeting of protoporphyrinogen oxidase 2 in Amaranthaceae promotes herbicide tolerance.

Plant tetrapyrrole biosynthesis (TPB) takes place in plastids and provides the chlorophyll and heme required for photosynthesis and many redox processes throughout plant development. TPB is strictly regulated, since accumulation of several intermediates causes photodynamic damage and cell death. Protoporphyrinogen oxidase (PPO) catalyzes the last common step before TPB diverges into chlorophyll and heme branches. Land plants possess two PPO isoforms. PPO1 is encoded as a precursor protein with a transit peptide, but in most dicotyledonous plants PPO2 does not possess a cleavable N-terminal extension. Arabidopsis (Arabidopsis thaliana) PPO1 and PPO2 localize in chloroplast thylakoids and envelope membranes, respectively. Interestingly, PPO2 proteins in Amaranthaceae contain an N-terminal extension that mediates their import into chloroplasts. Here, we present multiple lines of evidence for dual targeting of PPO2 to thylakoid and envelope membranes in this clade and demonstrate that PPO2 is not found in mitochondria. Transcript analyses revealed that dual targeting in chloroplasts involves the use of two transcription start sites and initiation of translation at different AUG codons. Among eudicots, the parallel accumulation of PPO1 and PPO2 in thylakoid membranes is specific for the Amaranthaceae and underlies PPO2-based herbicide resistance in Amaranthus species.

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.

CRISPR/Cas9-mediated homology donor repair base editing system to confer herbicide resistance in maize (Zea mays L.).

Weed infestation is a significant concern to crop yield loss, globally. The potent broad-spectrum glyphosate (N-phosphomethyl-glycine) has a widely utilized herbicide, acting on the shikimic acid pathway within chloroplast by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This crucial enzyme plays a vital role in aromatic amino acid synthesis. Repurposing of CRISPR/Cas9-mediated gene-editing was the inflection point for generating novel crop germplasm with diverse genetic variations in essential agronomic traits, achieved through the introduction of nucleotide substitutions at target sites within the native genes, and subsequent induction of indels through error-prone non-homologous end-joining DNA repair mechanisms. Here, we describe the development of efficient herbicide-resistant maize lines by using CRISPR/Cas9 mediated site-specific native ZmEPSPS gene fragment replacement via knock-out of conserved region followed by knock-in of desired homologous donor repair (HDR-GATIPS-mZmEPSPS) with triple amino acid substitution. The novel triple substitution conferred high herbicide tolerance in edited maize plants. Transgene-free progeny harbouring the triple amino acid substitutions revealed agronomic performances similar to that of wild-type plants, suggesting that the GATIPS-mZmEPSPS allele substitutions are crucial for developing elite maize varieties with significantly enhanced glyphosate resistance. Furthermore, the aromatic amino acid contents in edited maize lines were significantly higher than in wild-type plants. The present study describing the introduction of site-specific CRISPR/Cas9- GATIPS mutations in the ZmEPSPS gene via genome editing has immense potential for higher tolerance to glyphosate with no yield penalty in maize.

Fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon resistance status in Beckmannia syzigachne from wheat fields across Anhui Province, China.

Severe infestations of American sloughgrass (Beckmannia syzigachne (Steud.) Fernald) in wheat fields throughout Anhui Province, China, pose a significant threat to local agricultural production. This study aims to evaluate the susceptibility of 37 B. syzigachne populations collected from diverse wheat fields in Anhui Province to three commonly used herbicides: fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon. Single-dose testing revealed that out of the 37 populations, 31, 26, and 11 populations had either evolved or were evolving resistance to fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon, respectively. Among them, 25 populations displayed concurrent resistance to both fenoxaprop-P-ethyl and mesosulfuron-ethyl, while eight exhibited resistance to all three tested herbicides. Whole-plant bioassays confirmed that approximately 84% of the fenoxaprop-P-ethyl-resistant populations manifested high-level resistance (resistance index (RI) ≥10); 62% of the mesosulfuron-ethyl-resistant populations and 82% of the isoproturon-resistant populations exhibited low- to moderate-level resistance (2 ≤ RI <10). Three distinct target-site mutations were identified in 27% of fenoxaprop-P-ethyl-resistant populations, with no known resistance mutations detected in the remaining herbicide-resistant populations. The inhibition of cytochrome P450s (P450s) and/or glutathione S-transferases (GSTs) substantially increased susceptibility in the majority of resistant populations lacking mutations at the herbicide target site. In conclusion, resistance to fenoxaprop-P-ethyl and mesosulfuron-ethyl was widespread in B. syzigachne within Anhui Province's wheat fields, while resistance to isoproturon was rapidly evolving due to its escalating usage. Target-site mutations were present in approximately one-third of fenoxaprop-P-ethyl-resistant populations, and alternative mechanisms involving P450s and/or GSTs could explain the resistance observed in most of the remaining populations.

Response characterization and target site mechanism study in glyphosate-resistant populations of Lolium multiflorum L. from Brazil.

Italian ryegrass (Lolium multiflorum L.) is an invasive species widely spread in croplands worldwide. The intensive use of glyphosate has resulted in the selection of resistance to this herbicide in Italian ryegrass. This work characterized the response to glyphosate of Italian ryegrass populations from the South and Southwest regions of Paraná, Brazil. A total of 44 Italian ryegrass populations were collected in farming areas, and were classified for glyphosate resistance with 75% of populations resistant to gloyphosate. Of these, 3 resistant (VT05AR, MR20AR and RN01AR) and three susceptible (VT07AS, MR05AS and RN01AS) of these populations were selected to determine the resistance level and the involvement of the target site mechanisms for glyphosate resistance. Susceptible populations GR50 ranged from 165.66 to 218.17 g.e.a. ha-1 and resistant populations from 569.37 to 925.94, providing RI ranging from 2.88 and 4.70. No mutation in EPSPS was observed in the populations, however, in two (MR20AR and RN02AR) of the three resistant populations, an increase in the number of copies of the EPSPs gene (11 to 57×) was detected. The number of copies showed a positive correlation with the gene expression (R2 = 0.86) and with the GR50 of the populations (R2 = 0.81). The increase in EPSPS gene copies contributes to glyphosate resistance in Italian ryegrass populations from Brazil.

Inheritance and stacking effect of mutant ALS genes in Schoenoplectiella juncoides (Roxb.) Lye (Cyperaceae).

Schoenoplectiella juncoides, a noxious sedge weed in Japanese rice paddy, has two ALS genes, and ALS-inhibitor-resistant plants have a mutation in one of the ALS genes. The authors aimed (a) to quantitate the effect of the number of mutant alleles of ALS genes on whole-plant resistance of S. juncoides and (b) to clarify a mode of inheritance of the resistance by investigating resistance levels of the progenies of a hybrid between two S. juncoides plants with Trp574Leu substitution in different ALS. A dose-response analysis on the parental lines and the F1 population suggested that the two ALS genes contribute equally to whole-plant resistant levels. A dose-response study on the F2 population indicated that it could be classified into five groups based on the sensitivities to metsulfuron-methyl. The five groups (in ascending order of resistance levels) were considered to have zero, one, two, three, and four mutant alleles. The stacking effect of mutant alleles on resistance enhancement was more significant when the number of mutant alleles was low than when it was high; in other words, each additional mutant allele stacking increases plant resistance, but the effect saturates as the number of mutant alleles increases. A chi-square test supported that the segregation ratio of the five groups corresponds to 1:4:6:4:1 of Mendelian independence for the two ALS loci.

Gene flow and spontaneous mutations are responsible for imidazolinone herbicide-resistant weedy rice (Oryza sativa L.).

For more than two decades, weedy rice (Oryza sativa L.) has been controlled in rice fields by using imidazolinone (IMI) herbicide-resistant rice technology (Clearfield®). Outcrossing in weedy rice populations and spontaneous mutations are potential problems with herbicide-resistant crop management technologies, such as the IMI-resistant rice. The aim of this study was to characterize the mechanism of IMI herbicide resistance in weedy rice through dose-response bioassay study and evaluating amino acid substitutions in acetolactate synthase (ALS) protein. A total of 118 suspected IMI-resistant weedy rice samples, which survived in the field after an IMI herbicide application, were collected at harvest time from Türkiye in 2020 and 2021. Single-dose imazamox application experiment revealed that 38 plants survived herbicide treatment. The imazamox resistance of the surviving plants was confirmed by dose-response experiment. ALS gene region underwent a sanger DNA partial sequencing. No substitution was found in 10 samples, however, amino acid substitutions were found in 26 samples with S563N, one sample with S653T, and one sample with E630D. The S653N point is the same substitution point that serves as the origin of resistance for the Clearfield® rice varieties that are commonly cultivated in the region. It has been hypothesized that the gene flow from IMI-resistant rice may be the cause of resistance in the IMI resistant weedy rice samples with S653N. The other substitution, S653T, were considered spontaneous mutation to IMI resistance. Interestingly, the S653T mutation was detected for the first time in weedy rice. The mechanism of resistance of 10 resistant weedy rice was not confirmed in this study, however, it may be a non-target resistance or another mutation point in target site, but evidently, they did not acquire resistance by gene flow from IMI-resistant rice. It has been concluded that the effectiveness of IMI-resistant rice technology in controlling weedy rice has drastically decreased due to possible gene flow, spontaneous mutation and non-target resistance. In addition to cultural controls like clean seed, clean machinery and crop rotation, other herbicide-tolerant rice systems such as Provisia® and Roxy-RPS® rice are needed to create a diverse weedy rice management ensemble available for rice production and move towards sustainable rice farming.

Trp-574-Leu mutation and metabolic resistance by cytochrome P450 gene conferred high resistance to ALS-inhibiting herbicides in Descurainia sophia.

Descurainia sophia (flixweed) is a troublesome weed in winter wheat fields in North China. Resistant D. sophia populations with different acetolactate synthetase (ALS) mutations have been reported in recent years. In addition, metabolic resistance to ALS-inhibiting herbicides has also been identified. In this study, we collected and purified two resistant D. sophia populations (R1 and R2), which were collected from winter wheat fields where tribenuron-methyl provided no control of D. sophia at 30 g a.i. ha-1. Whole plant bioassay and ALS activity assay results showed the R1 and R2 populations had evolved high-level resistance to tribenuron-methyl and florasulam and cross-resistance to imazethapyr and pyrithiobac­sodium. The two ALS genes were cloned from the leaves of R1 and R2 populations, ALS1 (2004 bp) and ALS2 (1998 bp). A mutation of Trp 574 to Leu in ALS1 was present in both R1 and R2. ALS1 and ALS2 were cloned from R1 and R2 populations respectively and transferred into Arabidopsis thaliana. Homozygous T3 transgenic seedlings with ALS1 of R1 or R2 were resistant to ALS-inhibiting herbicides and the resistant levels were the same. Transgenic seedlings with ALS2 from R1 or R2 were susceptible to ALS-inhibiting herbicides. Treatment with cytochrome P450 inhibitor malathion decreased the resistant levels to tribenuron-methyl in R1 and R2. RNA-Seq was used to identify target cytochrome P450 genes possibly involved in resistance to ALS-inhibiting herbicides. There were five up-regulated differentially expressed cytochrome P450 genes: CYP72A15, CYP83B1, CYP81D8, CYP72A13 and CYP71A12. Among of them, CYP72A15 had the highest expression level in R1 and R2 populations. The R1 and R2 populations of D. sophia have evolved resistance to ALS-inhibiting herbicides due to Trp 574 Leu mutation in ALS1 and possibly other mechanisms. The resistant function of CYP72A15 needs further research.

Distribution, frequency and molecular basis of clethodim and quizalofop resistance in brome grass (Bromus diandrus).

BACKGROUND: Brome grass (Bromus diandrus Roth) is prevalent in the southern and western cropping regions of Australia, where it causes significant economic damage. A targeted herbicide resistance survey was conducted in 2020 by collecting brome grass populations from 40 farms in Western Australia and subjecting these samples to comprehensive herbicide screening. One sample (population 172-20), from a field that had received 12 applications of clethodim over 20 years of continuous cropping, was found to be highly resistant to the acetyl-CoA carboxylase (ACCase)-inhibiting herbicides clethodim and quizalofop, and so the molecular basis of resistance was investigated. RESULTS: All 31 individuals examined from population 172-20 carried the same resistance-endowing point mutation causing an aspartate-to-glycine substitution at position 2078 in the translated ACCase protein sequence. A wild-type susceptible population and the resistant population had similar expression levels of plastidic ACCase genes. The level of resistance to quizalofop, either standalone or in mixture with clethodim, in population 172-20 was lower under cooler growing conditions. CONCLUSION: Target-site resistance to ACCase-inhibiting herbicides, conferred by one ACCase mutation, was selected in all tested brome plants infesting a field with a history of repeated clethodim use. This mutation appears to have been fixed in the infesting population. Notably, clethodim resistance in this population was not detected by the farmer, and a high future incidence of quizalofop resistance is anticipated. Herbicide resistance testing is essential for the detection of evolving weed resistance issues and to inform effective management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.