Acetohydroxyacid Synthase Inhibitors Research Articles

Mutations on the A and C genomes of Brassica napus confer herbicide resistance to acetohydroxyacid synthase inhibitors

ABSTRACT The point mutations conferring resistance to the sulfonylurea herbicide, chlorsulfuron, are reported for two Brassica napus lines (19c and 30a) developed by seed mutagenesis using ethyl methanesulfonate. Crosses with the herbicide-susceptible wild-type line generated F2 and backcrossed progeny and confirmed that chlorsulfuron resistance was inherited as a dominant mutation at a single locus in both lines. F2 progeny from crossing 19c and 30a segregated in a 15 resistant: 1 susceptible ratio, establishing that the two herbicide-resistant loci are independently inherited and that the mutations for resistance occur at different loci. PCR amplification and DNA sequencing of regions from the acetohydroxyacid synthase gene revealed two separate point mutations at different nucleotides in the same codon. This results in the substitution of proline-197 for leucine in 19c and proline-197 for serine in 30a. The different amino acid substitutions in the acetohydroxyacid synthase enzyme explain the differing responses of 19c and 30a to sulfonylurea and imidazolinone herbicides. Based on attempts at introgression of chlorsulfuron resistance to a wide range of forage brassicas, we conclude that the 19c mutation resides in the Brassica C genome, whereas the 30a mutation is in the Brassica A genome.

Chemical Synthesis, Herbicidal Activity, Crop Safety, and Molecular Basis of ortho-Fluoroalkoxy Substituted Sulfonylureas as Novel Acetohydroxyacid Synthase Inhibitors.

In the face of increasing resistance to the currently used commercial herbicides and the lack of success in identifying new herbicide targets, alternative herbicides need to be developed to control unwanted monocotyledon grasses in food crops. Here, a panel of 29 novel sulfonylurea-based compounds with ortho-fluoroalkoxy substitutions at the phenyl ring were designed and synthesized. Pot assays demonstrated that two of these compounds, 6d and 6u, have strong herbicidal activities against Echinochloa crus-galli, Eleusine indica, Alopecurus aequalis, and Alopecurus japonicus Steudel at a dosage of 15 g ha-1. Furthermore, these two compounds exhibited <5% inhibition against wheat at a dosage of 30 g ha-1 under post-emergence conditions. 6u also exhibited <5% inhibition against rice at a dosage of 30 g ha-1 under both post-emergence and pre-emergence conditions. A kinetics study demonstrated that 6d and 6u are potent inhibitors of Arabidopsis thaliana acetohydroxyacid synthase (AHAS; EC 2.2.1.6) with potent Ki values of 18 ± 1.1 and 11.9 ± 4.0 nM, respectively. The crystal structure of 6u in complex with A. thaliana (At)AHAS has also been determined at 2.7 Å resolution. These new compounds represent new alternative herbicide choices to protect wheat or rice from invading grasses.

Fecundity and survival of flumetsulam-resistant and -susceptible Ranunculus acris plants with and without exposure to the herbicide

ABSTRACT Variable efficacy of the acetohydroxyacid synthase (AHAS) inhibitor herbicide, flumetsulam, against dairy pasture populations of Ranunculus acris, has been attributed to resistance. We compared, over two years, the fitness of two resistant (R) clones and nine susceptible (S) clones treated or not with flumetsulam by measuring seed yields and survival of plants. Without the herbicide, all plants grew and survived. The seed yields of the R and S plants without the herbicide were similar (327 and 217 seeds/plant, respectively in Year 1, and 257 and 323 seeds/plant in Year 2) indicating no fitness penalty to the resistance. With the herbicide, growth and survival of the R plants was unaffected, whereas all S plants were defoliated before seedhead formation but 96% regrew from their rhizomes in Year 2. Seed yields of the treated R and S plants in Year 1 were 347 and 0 seeds/plant, respectively and 243 and 205 seeds/plant in Year 2. Survival of flumetsulam-susceptible R. acris plants following spraying would maintain susceptible AHAS alleles in the population, while without a fitness penalty, resistant plants would persist in non-spraying years facilitating further selection when the herbicide is used again.

A Novel AHAS-Inhibiting Herbicide Candidate for Controlling Leptochloa chinensis: A Devastating Weedy Grass in Rice Fields.

Given the prevalence of the malignant weed Chinese Sprangletop (Leptochloa chinensis (L.) Nees) in rice fields, the development of novel herbicides against this weed has aroused wide interest. Here, we report a novel diphenyl ether-pyrimidine hybrid, DEP-5, serving as a systematic pre/postemergence herbicide candidate for broad-spectrum weed control in rice fields, specifically for L. chinensis. Notably, DEP-5 exhibits over 80% herbicidal activity against the resistant biotypes even at 37.5 g a.i./ha under greenhouse conditions and has complete control of L. chinensis at 150 g a.i./ha in the rice fields. We uncover that DEP-5 acts as a noncompetitive inhibitor of acetohydroxyacid synthase (AHAS) with an inhibition constant (Ki) of 39.4 μM. We propose that DEP-5 binds to AHAS in two hydrophobic-driven binding modes that differ from commercial AHAS inhibitors. Overall, these findings demonstrate that DEP-5 has great potential to be developed into a herbicide for L. chinensis control and inspire fresh concepts for novel AHAS-inhibiting herbicide design.

Salmonella mutant strains resistant to herbicides - Acetohydroxyacid synthase inhibitors and their use at the Ames test

Cytotoxicity of some pesticides is a disadvantage for the Salmonella/microsome assay with regard to the equivalence assessment of pesticide technical grade active ingredients to the original products and detection of low-level impurities. The technical grade active ingredients (TGAIs) of pesticides from certain chemical classes were found to be toxic for Salmonella typhimurium strains. Among the highly cytotoxic compounds were sulfonylureas, which include 20 active ingredients. In addition, this class includes active pharmaceutical ingredients used for the manufacture of antidiabetics drugs.A traditional selection methodology was applied using the cultivation of S. typhimurium TA100 in the presence of high concentrations of thifensulfuronmethyl (TFSM) to obtain a resistant test strain insusceptible to sulfonylurea toxic effect. Two strains resistant not only to sulfonylureas (SFU) but also triazolepyrimidines were received. The first mutant strain (deposited as S. typhimurium VKPM B-14099 in the Russian National Collection of Industrial Microorganisms) demonstrated the TA100 phenotypic characteristics: hisG46, rfa, ΔuvrB-bio, pKM101. The second strain (deposited as S. typhimurium VKPM B-14359) showed the TA1535 phenotypic characteristics and probably lost the R-factor due to the selection using the poor Gm-media with TFSM. Positive controls caused pronounced mutagenic effects (±S9) in both strains, consequently the mutants did not lose the ability to respond to induction of the reverse gene mutations. The maximum non-cytotoxic concentrations of SFUs and triazole-pyrimidines for the Ames test strains did not exceed 0.05–0.125 mg/plate, while no evidence of cytotoxicity was observed for the mutants up to 5.0 mg/plate. Electron microscopy of the ultrathin sections of Salmonella cells grown with and without TFSM showed an obvious difference in the structure of the cell wall and cytoplasm in mutant and parental cultures. The concurrent resistance both to SFU and triazolepyrimidines was assumed to be mediated by the same mechanism of action of the pesticides from these classes – inhibition of acetohydroxyacid synthase. To confirm this hypothesis, the tests in the presence of branched-chain amino acids were carried out. The enrichment of agar with isoleucine prevented the toxic effects of SFU and triazolepyrimidines for all Ames test strains used in the study, while strong cytotoxicity was observed in the presence of valine and leucine.Considering the tolerance of strains both to SFU and triazolpyrimidines and the results with branched-chain amino acids, the modification of target acetohydroxyacid synthase was supposed the key to the acquired resistance. The new strains resistant to sulfonylureas and triazole-pyrimidines expands the possibilities to reveal mutagenic impurities that may occur in TGAIs in small amounts.

Target and nontarget mechanisms of AHAS inhibitor cross-resistance patterns in Cyperus difformis

Weed resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides has been a critical issue for rice growers worldwide since the early 1990's. In California, resistance to bensulfuron-methyl was first detected in Cyperus difformis in 1993. Since then, populations of most major weeds of rice in California have been reported to show resistance to at least one AHAS inhibitor. We sought to describe the magnitude and mechanisms of AHAS inhibitor cross-resistance in California populations of C. difformis. Sixty-two populations were collected and screened for cross-resistance to bensulfuron-methyl (BEN), halosulfuron-methyl (HAL), bispyribac‑sodium (BIS), and penoxsulam (PEN), revealing six major patterns of cross-resistance. Representative C. difformis populations from each cross-resistance pattern were then subjected to dose-response, cytochrome P450 inhibition, AHAS gene sequencing, and metabolic studies with the same herbicides as in the screening. Dose-response confirmed the detected resistances in the representative populations, and suggested that the majority of observed resistance was dose-dependent. Cytochrome P450 inhibition via malathion revealed evidence of increased metabolic activity in resistant populations to BEN, BIS, and PEN. AHAS gene sequencing revealed amino acid substitutions in five of six populations: R3 (Pro197-Ser), R4 (Pro97-His), R10 (Asp376), R41 (Ala122-Asn), and R18 (Trp574-Leu). Metabolic studies confirmed evidence of increased activity of cytochrome P450s in all populations. Metabolic BEN and HAL analysis did not yield similar results to malathion inhibition, suggesting different P450's or other pathways. Taken together, the results of the studies confirm the complexity of AHAS inhibitor cross-resistance in C. difformis, and the presence of both target-site and metabolic resistance in most of the representative populations underscores the importance of proper herbicide selection, rotation, and scouting in fields.

Effect of mutations on acetohydroxyacid synthase (AHAS) function in Cyperus difformis L.

Cyperus difformis L. is a troublesome weed in paddy fields and has attracted attention due to its resistance to acetohydroxyacid synthase (AHAS) inhibitors. It was found that the amino acid mutation in AHAS was the primary cause for the resistance of Cyperus difformis. However, the effect of different mutations on AHAS function is not clear in Cyperus difformis. To confirm the effect of mutations on AHAS function, six biotypes were collected, including Pro197Arg, Pro197Ser, Pro197Leu, Asp376Glu, Trp574Leu and wild type, from Hunan, Anhui, Jiangxi and Jiangsu provinces, China and the function of AHAS was characterized. The AHAS in vitro inhibition assay results indicated that the mutations decreased the sensitivity of AHAS to pyrazosulfuron-ethyl, in which the I50 (the half maximal inhibitory concentration) of wild type AHAS was 0.04 μmol L–1 and Asp376Glu, Pro197Leu, Pro197Arg, Pro197Ser and Trp574Leu mutations were 3.98, 11.50, 40.38, 38.19 and 311.43 μmol L–1, respectively. In the determination of enzyme kinetics parameters, the Km and the maximum reaction velocity (Vmax) of the wild type were 5.18 mmol L–1 and 0.12 nmol mg–1 min–1, respectively, and the Km values of AHAS with Asp376Glu, Trp574Leu, Pro197Leu and Pro197Ser mutations were 0.38–0.93 times of the wild type. The Km value of the Pro197Arg mutation was 1.14 times of the wild type, and the Vmax values of the five mutations were 1.17–3.33-fold compared to the wild type. It was found that the mutations increased the affinity of AHAS to the substrate, except for the Pro197Arg mutation. At a concentration of 0.0032–100 mmol L–1 branched-chain amino acids (BCAAs), the sensitivity of the other four mutant AHAS biotypes to feedback inhibition decreased, except for the Pro197Arg mutation. This study elucidated the effect of different mutations on AHAS function in Cyperus difformis and provided ideas for further study of resistance development.

Facile synthesis, crystal structure, quantum calculation, and biological evaluations of novel selenenyl sulfide compounds as potential agrochemicals.

In order to design compounds with fresh molecular skeleton to break through the limitation of available agrochemicals, a series of 36 novel selenenyl sulfide compounds were chemically synthesized, and their biological activities were fully evaluated against tobacco mosaic virus (TMV), 14 plant pathogenic fungi, three insect species and plant acetohydroxyacid synthase (AHAS). All the target compounds were characterized by proton nuclear magnetic resonance (1 H-NMR), carbon-13 (13 C)-NMR, selenium-77 (77 Se)-NMR, and high-resolution mass spectrometry (HRMS). The crystal structure of 10j indicated that the Se-S bond was successfully constructed. Compounds 10d, 10h, 10s, 10u, 10aa, 10ac, 10ae, 10ag, and 10ai exhibited 40%, 43%, 39%, 41%, 47%, 46%, 47%, 42%, and 39% anti-TMV activities at 500 mg L-1 , better than that of ribavirin. The median effective concentration (EC50 ) against Sclerotinia sclerotiorum of 10ac was 6.69 mg L-1 and EC50 values against Physalospora piricola and Pyricularia grisea of 10z were 12.25 mg L-1 and 15.27 mg L-1 , respectively, superior to the corresponding values of chlorothalonil. Compounds 10c and 10v demonstrated 100% larvicidal activity against Culex pipiens pallens at 5mg L-1 , while 10a displayed 100% insecticidal activity against Mythimna separata at 200 mg L-1 . Compounds 10c, 10j, and 10o showed > 60% inhibitions against plant AHAS at 10μmol L-1 . From the quantum calculation, highest occupied molecular orbital (HOMO) was considered as a factor that affects the anti-TMV activity. The preliminary results suggested that more efforts should be devoted to exploring the selenenyl sulfides for the discovery of new leads of antiviral agent, fungicide, insecticide or AHAS inhibitors as potential agrochemicals for crop protection. © 2023 Society of Chemical Industry.

Asymmetric Synthesis of Trifluoroethyl-Based, Chiral 3-Benzyloxy-1- and -2-Phenyl-quinazolinones of Biomedicinal Interest by Radical Type Cross-Coupling to Olefins

Several 2-substituted (H, Ph, and S-Me) and 1-substituted (H, Ph, and Bn), 3-hydroxy-1,3-quinazolin(di)ones were utilized for the first time as radical trapping agents in asymmetric 1,2-oxytrifluoromethylation of styrenes catalyzed by chiral vanadyl methoxide complexes bearing 3,5-disubstituted-N-salicylidene-t-leucinate templates. The effects of catalysts and solvents on the asymmetric induction were systematically examined. The best and complementary scenarios involved the use of vanadyl complexes V(O)-1 and V(O)-2, which bear 3-(2,5-dimethyl)phenyl-5-bromophenyl and 3-t-butyl-5-bromophenyl groups in an i-propanol solvent at ambient temperature. The corresponding (R)-cross-coupling products by V(O)-1 were obtained in 45-71% (for 2-substituted series) and 59-93% yields (for 1-substituted series) for p-/m-methylstyrenes and m-halo/CF3/CO2Me-styrenes in 38-63% ees (the best in 2-H case) and 60-84% ees (the best in 1-benzyl cases), respectively. The corresponding (S)-cross-coupling products by V(O)-2 were obtained in 28-55% (for 2-substituted series) and 45-72% yields (for 1-substituted series) for the same substrate class in 50-91% ees (85-91% ees in 2-phenyl cases) and 64-75% ees (up to 74-75% ees for each 1-H, Ph, and Bn cases), respectively. Theoretical calculations were carried out to explain the origin and extent of enantiocontrols. They both may serve as potential inhibitors of acetohydroxyacid synthase and epidermal growth factor receptor (EGFR) kinases.

New possibilities of the Ames test for evaluation of mutagenicity of technical products of active ingredients of pesticides

Introduction. The Ames test is the one of the most popular methods for mutagenicity evaluation of environmental factors. In some cases, this method is suggested to be the only and sufficient assay for the first stage of the equivalence assessment of pesticide technical grade active ingredients (TGAI) to the original products. A limitation of the Ames test is related to the impossibility of an objective equivalence assessment of some cytotoxic TGAIs, in particular, sulfonylureas, and triazolpyrimidines. Based on the mode of action of the pesticides belongs to these chemical classes, we suggested a modification of the plate incorporation method protocol of the Ames test to the increase of maximal non-cytotoxic concentration up to the 5 mg/plate recommended by regulatory documents. Materials and methods. The five strains of Salmonella typhimurium TA98, TA100, TA1535, TA97, TA102 were used. The modification of the protocol included a supplementation of the top agar with isoleucine (1-5 mM). Results. The maximum non-cytotoxic concentrations of thifensulfuron-methyl and florasulam using the standard top agar did not exceed 0.05-0.125 mg/plate. The enrichment of the top agar with isoleucine allowed evaluating the mutagenicity of the substances up to the maximal recommended concentration of 5.0 mg/plate. The number of spontaneous revertants was within the historical limits of the laboratory control obtained under standard conditions. Positive controls showed pronounced mutagenic effects in case of all strains with and without metabolic activation (p≤0.05). Limitations. Mutagenicity was evaluated only for TGAIs, which are acetohydroxyacid synthase inhibitors. Conclusion. The application of the modified Ames test protocol for mutagenicity assessment of TGAIs from the classes of sulfonylureas and triazolpyrimidines under supplementation of the top agar with isoleucine is a more objective way to evaluate their mutagenicity. The proposed protocol expands the possibilities of revealing dangerous mutagenic impurities that may occur in TGAIs in the small quantities, and after entering the environment can cause the gain in the mutation level in living organisms.

In silico modeling of the AHAS inhibition of an augmented series of pyrimidine herbicides and design of novel derivatives

Pyrimidine compounds comprise a class of acetohydroxyacid synthase (AHAS) inhibitors, thus possessing herbicidal activity. Due to the ongoing development of resistance by weeds to current herbicides, the design of new agrochemical candidates is often required. This work reports the proposition of unprecedented pyrimidines as herbicides guided by quantitative structure-activity relationship (QSAR) modeling. Multivariate image analysis (MIA) descriptors for 66 pyrimidine derivatives obtained from different sources were regressed against inhibitory activity data, and the resulting QSAR models were found to be reliable and predictive (r2 = 0.88 ± 0.07, q2 = 0.53 ± 0.06, and r2pred = 0.51 ± 0.10 in a bootstrapping experiment using electronegativity-based descriptors). The chemical features responsible for the herbicidal activities were analyzed through MIA contour maps that describe the substituent effects on the response variables, whereas the interaction between the proposed compounds and AHAS was analyzed through docking studies. From the proposed compounds, at least five pyrimidine derivatives exhibited promising performance as AHAS inhibitors compared to the known analogs.

In silico screening leads to novel scaffolds with both antifungal and anti-NLRP3 inflammasome activity

Due to structural similarities that exist between established inhibitors of the NLRP3-inflammasome, sulfonylureas Glyburide and MCC-950, and herbicidal-sulfonylureas, that specifically target fungal acetohydroxyacid synthase (AHAS), we sought to determine the potential for compounds to block both inflammation and inhibit fungal growth. In silico screening of ∼250,000 compounds was used to identify a prioritized list of chemical structures capable of inhibiting both targets. Prioritization of the top 1% of scores identified ∼70 compounds with a diverse set of scaffolds for testing in vitro. Selected hits were used to assess anti-inflammatory function in a THP-1 challenge model with LPS+ATP and resulting IC50 values were obtained. MIC and hyphal-growth assays were conducted to determine potential antifungal activity using media depleted of branched chain amino acids isoleucine and valine, to confirm on target AHAS inhibition. Identification of hits that exhibited low micromolar activity for NLRP3 and AHAS inhibition were selected for SAR study. In vitro testing of the analogs along with molecular docking led to increased knowledge for lead optimization of the potential hits. In silico screening has resulted in IC50 (IL-1β release) and MIC50 (fungal growth) values with low μM potency against several Candida species. In vivo validation will further confirm the potential of the scaffolds for further synthetic-modification for the rationale design of novel dual-purpose drugs

Novel Mutation in the Acetohydroxyacid Synthase (AHAS), Gene Confers Imidazolinone Resistance in Chickpea Cicer arietinum L. Plants.

Chickpea (Cicer arietinum L.) is an important crop in crop-rotation management in Israel. Imidazolinone herbicides have a wide spectrum of weed control, but chickpea plants are sensitive to acetohydroxyacid synthase (AHAS; also known as acetolactate synthase [ALS]) inhibitors. Using the chemical mutagen ethyl methanesulfonate (EMS), we developed a chickpea line (M2033) that is resistant to imidazolinone herbicides. A point mutation was detected in one of the two genes encoding the AHAS catalytic subunit of M2033. The transition of threonine to isoleucine at position 192 (203 according to Arabidopsis) conferred resistance of M2033 to imidazolinones, but not to other groups of AHAS inhibitors. The role of this substitution in the resistance of line M2033 was proven by genetic transformation of tobacco plants. This resistance showed a single-gene semidominant inheritance pattern. Conclusion: A novel mutation, T192I (T203I according to Arabidopsis), providing resistance to IMI herbicides but not to other groups of AHAS inhibitors, is described in the AHAS1 protein of EMS-mutagenized chickpea line M2033.

Evidence for the Application of Emerging Technologies to Accelerate Crop Improvement - A Collaborative Pipeline to Introgress Herbicide Tolerance Into Chickpea.

Accelerating genetic gain in crop improvement is required to ensure improved yield and yield stability under increasingly challenging climatic conditions. This case study demonstrates the effective confluence of innovative breeding technologies within a collaborative breeding framework to develop and rapidly introgress imidazolinone Group 2 herbicide tolerance into an adapted Australian chickpea genetic background. A well-adapted, high-yielding desi cultivar PBA HatTrick was treated with ethyl methanesulfonate to generate mutations in the ACETOHYDROXYACID SYNTHASE 1 (CaAHAS1) gene. After 2 years of field screening with imidazolinone herbicide across >20 ha and controlled environment progeny screening, two selections were identified which exhibited putative herbicide tolerance. Both selections contained the same single amino acid substitution, from alanine to valine at position 205 (A205V) in the AHAS1 protein, and KASP™ markers were developed to discriminate between tolerant and intolerant genotypes. A pipeline combining conventional crossing and F2 production with accelerated single seed descent from F2:4 and marker-assisted selection at F2 rapidly introgressed the herbicide tolerance trait from one of the mutant selections, D15PAHI002, into PBA Seamer, a desi cultivar adapted to Australian cropping areas. Field evaluation of the derivatives of the D15PAHI002 × PBA Seamer cross was analyzed using a factor analytic mixed model statistical approach designed to accommodate low seed numbers resulting from accelerated single seed descent. To further accelerate trait introgression, field evaluation trials were undertaken concurrent with crop safety testing trials. In 2020, 4 years after the initial cross, an advanced line selection CBA2061, bearing acetohydroxyacid synthase (AHAS) inhibitor tolerance and agronomic and disease resistance traits comparable to parent PBA Seamer, was entered into Australian National Variety Trials as a precursor to cultivar registration. The combination of cross-institutional collaboration and the application of novel pre-breeding platforms and statistical technologies facilitated a 3-year saving compared to a traditional breeding approach. This breeding pipeline can be used as a model to accelerate genetic gain in other self-pollinating species, particularly food legumes.

Triazolopyrimidine herbicides are potent inhibitors of Aspergillus fumigatus acetohydroxyacid synthase and potential antifungal drug leads

Aspergillus fumigatus is a fungal pathogen whose effects can be debilitating and potentially fatal in immunocompromised patients. Current drug treatment options for this infectious disease are limited to just a few choices (e.g. voriconazole and amphotericin B) and these themselves have limitations due to potentially adverse side effects. Furthermore, the likelihood of the development of resistance to these current drugs is ever present. Thus, new treatment options are needed for this infection. A new potential antifungal drug target is acetohydroxyacid synthase (AHAS; EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, and a target for many commercial herbicides. In this study, we have expressed, purified and characterised the catalytic subunit of AHAS from A. fumigatus and determined the inhibition constants for several known herbicides. The most potent of these, penoxsulam and metosulam, have Ki values of 1.8 ± 0.9 nM and 1.4 ± 0.2 nM, respectively. Molecular modelling shows that these compounds are likely to bind into the herbicide binding pocket in a mode similar to Candida albicans AHAS. We have also shown that these two compounds inhibit A. fumigatus growth at a concentration of 25 µg/mL. Thus, AHAS inhibitors are promising leads for the development of new anti-aspergillosis therapeutics.

Discovery of ortho-Alkoxy Substituted Novel Sulfonylurea Compounds That Display Strong Herbicidal Activity against Monocotyledon Grasses.

In the present study, we have designed and synthesized a series of 42 novel sulfonylurea compounds with ortho-alkoxy substitutions at the phenyl ring and evaluated their herbicidal activities. Some target compounds showed excellent herbicidal activity against monocotyledon weed species. When applied at 7.5 g ha-1, 6-11 exhibited more potent herbicidal activity against barnyard grass (Echinochloa crus-galli) and crab grass (Digitaria sanguinalis) than commercial acetohydroxyacid synthase (AHAS; EC 2.2.1.6) inhibitors triasulfuron, penoxsulam, and nicosulfuron at both pre-emergence and postemergence conditions. 6-11 was safe for peanut for postemergence application at this ultralow dosage, suggesting that it could be considered a potential herbicide candidate for peanut fields. Although 6-11 and triasulfuron share similar chemical structures and have close Ki values for plant AHAS, a significant difference has been observed between their LUMO maps from DFT calculations, which might be a possible factor that leads to their different behaviors toward monocotyledon weed species.

Harzianic Acid from Trichoderma afroharzianum Is a Natural Product Inhibitor of Acetohydroxyacid Synthase.

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthetic pathway and is a validated target for herbicide and fungicide development. Here we report harzianic acid (HA, 1) produced by the biocontrol fungus Trichoderma afroharzianum t-22 (Tht22) as a natural product inhibitor of AHAS. The biosynthetic pathway of HA was elucidated with heterologous reconstitution. Guided by a putative self-resistance enzyme in the genome, HA was biochemically demonstrated to be a selective inhibitor of fungal AHAS, including those from phytopathogenic fungi. In addition, HA can inhibit a common resistant variant of AHAS in which the active site proline is mutated. Structural analysis of AHAS complexed with HA revealed the molecular basis of competitive inhibition, which differs from all known commercial AHAS inhibitors. The alternative binding mode also rationalizes the selectivity of HA, as well as effectiveness toward resistant mutants. A proposed role of HA biosynthesis by Tht22 in the rhizosphere is discussed based on the data.

Design, synthesis, herbicidal activity, in vivo enzyme activity evaluation and molecular docking study of acylthiourea derivatives as novel acetohydroxyacid synthase inhibitor

In order to develop novel herbicides, a series of novel acylthiourea derivatives containing methylsulfone were synthesized and characterized by melting point, elemental analysis, infrared spectroscopy, mass spectrometry and 1H NMR spectroscopy. Furthermore, the preliminary herbicidal activity of title compounds was determined against Echinochloa crusgal, Polypogon fugax, Digitaria adscendens, Brassica napus, Amaranthus retroflexus and Abutilon theophrasti. The results of the petri dish experiments showed that a small part of title compounds had a good inhibitory effect on Digitaria adscendens and Brassica napus. The results of the greenhouse herbicidal activity experiments indicated that the compounds 4p, 4 w and 4x exhibited more high herbicidal activity against Brassica napus and Digitaria adscendens than the commercial herbicide bensulfuron-methyl. Moreover, except for compound 4i, all test compounds performed well in crop safety evaluation. Next, acetohydroxyacid synthase (AHAS) enzyme activity experiments were carried out, and the results showed that compound 4x had the highest inhibition rate reaching 89.12%. Finally, in order to screen AHAS inhibitor, molecular docking was performed by using AHAS protein and target ligand molecules 4a-4x. The results of molecular docking were similar to bioassay experiments. Compound 4p, 4w and 4x were considered potential AHAS inhibitors.

Asp-376-Glu substitution endows target-site resistance to AHAS inhibitors in Limnocharis flava, an invasive weed in tropical rice fields.

Limnocharis flava (L.) Buchenau is a problematic weed in rice fields and water canals of Southeast Asia, and in Malaysia this invasive aquatic weed species has evolved multiple resistance to synthetic auxin herbicide and acetohydroxyacid synthase (AHAS) inhibitors. In this study, it was revealed that, a single nucleotide polymorphism (SNP) at amino acid position 376, where C was substituted to G at the third base of the same codon (GAC to GAG), resulting in Aspartate (Asp) substitution by Glutamate (Glu) was the contributing resistance mechanism in the L. flava population to AHAS inhibitors. In vitro assay further proved that, all the L. flava individuals carrying AHAS resistance mutation exhibited decreased-sensitivity to AHAS inhibitors at the enzyme level. In the bensulfuron-methyl whole-plant bioassay, high resistance indices (RI) of 328- and 437-fold were recorded in the absence and presence of malathion (the P450 inhibitor), respectively. Similarly, translocation and absorption of bensulfuron-methyl in both resistant and susceptible L. flava populations showed no remarkable differences, hence eliminated the possible co-existence of non-target-site resistance mechanism in the resistant L. flava. This study has confirmed another new case of a target-site resistant weed species to AHAS-inhibitors.

An integrated approach for the characterization of one‐ and two‐gene imazamox‐resistant wheat lines

AbstractIn wheat (Triticum aestivum L.), cultivars resistant to imidazolinone herbicides are an important tool for weed management. The target site of imidazolinones is acetohydroxyacid synthase (AHAS), an enzyme coded by three homeologous genes (ahasL‐A1, ahasL‐B1, and ahasL‐D1). This study aimed to evaluate the effect of the imidazolinone herbicide imazamox on a single‐gene (AhasL‐D1) resistant line (R1) and a two‐gene (AhasL‐B1 and AhasL‐D1) resistant line (R2) at multiple biological levels. These lines showed variation in the transcriptional levels of ahasL homeologs, but no differences in total AHAS activity. The relative contribution of resistant alleles to the ahasL transcript pool accounted for 47% in the R1 line and 77% in the R2 line. The in vitro AHAS inhibition of the R1 line was intermediate between the R2 and the susceptible lines. At an anatomical level, high imazamox concentrations induced alterations in the root tip features of R1 plants but did not affect R2 plants, whereas at in vivo AHAS activity and whole‐plant levels, both resistant lines showed high resistance. The herbicide dose that reduced shoot biomass of resistant lines by 50% was 35‐fold higher than that of the susceptible line. We conclude that homeolog variation allows a higher contribution of resistance genes to the ahasL transcript pool in the R1 and R2 lines. Although both resistant lines showed high whole‐plant resistance, the differential root damage induced by imazamox between wheat lines suggests that two‐gene resistant cultivars could be an effective option to avoid the negative impacts caused by herbicide residues in the soil.