Incidence of resistance to ALS and ACCase inhibitors in Echinochloa species and soil microbial composition in Northern Italy.

The increasing amount of weeds surviving herbicide represents a very serious problem for crop management. The interaction between microbial community of soil and herbicide resistance, along with the potential evolutive consequences, are still poorly known and need to be investigated to better understand the impact on agricultural management. In our study, we analyzed the microbial composition of soils in 32 farms, located in the Northern Italy rice-growing area (Lombardy) with the aim to evaluate the relationship between the microbial composition and the incidence of resistance to acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) inhibiting herbicides in Echinochloa species. We observed that the coverage of weeds survived herbicide treatment was higher than 60% in paddy fields with a low microbial biodiversity and less than 5% in those with a high microbial biodiversity. Fungal communities showed a greater reduction in richness than Bacteria. In soils with a reduced microbial diversity, a significant increase of some bacterial and fungal orders (i.e. Lactobacillales, Malasseziales and Diaporthales) was observed. Interestingly, we identified two different microbial profiles linked to the two conditions: high incidence of herbicide resistance (H-HeR) and low incidence of herbicide resistance (L-HeR). Overall, the results we obtained allow us to make hypotheses on the greater or lesser probability of herbicide resistance occurrence based on the composition of the soil microbiome and especially on the degree of biodiversity of the microbial communities.

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.

Ecological weed management and square planting influenced the weed management, and crop productivity in direct-seeded rice.

Herbicide use may pose a risk of environmental pollution or evolution of resistant weeds. As a result, an experiment was carried out to assess the influence of different non-chemical weed management tactics (one hoeing (HH) at 12 DAS followed by (fb) one hand weeding at 30 DAS, one HH at 12 DAS fb Sesbania co-culture and its mulching, one HH at 12 DAS fb rice straw mulching @ 4t ha-1, one HH at 12 DAS fb rice straw mulching @ 6 t ha-1) on weed control, crop growth and yield, and economic returns in direct-seeded rice (DSR). Experiment was conducted during kharif season in a split-plot design and replicated thrice. Zero-till seed drill-sown crop (PN) had the lowest weed density at 25 days after sowing (DAS), while square planting geometry (PS) had the lowest weed density at 60 DAS. PS also resulted in a lower weed management index (WMI), agronomic management index (AMI), and integrated weed management index (IWMI), as well as higher growth attributes, grain yield (4.19 t ha-1), and net return (620.98 US$ ha-1). The cultivar Arize 6444 significantly reduced weed density and recorded higher growth attributes, yield, and economic return. In the case of weed management treatments, one HH at 12 DAS fb Sesbania co-culture and its mulching had the lowest weed density, Shannon-weinner index and eveness at 25 DAS. However, one hoeing at 12 DAS fb one hand weeding at 30 DAS (HH + WH) achieved the highest grain yield (4.85 t ha-1) and net returns (851.03 US$ ha-1) as well as the lowest weed density at 60 DAS. PS × HH + WH treatment combination had the lowest weed persistent index (WPI), WMI, AMI, and IWMI, and the highest growth attributes, production efficiency, and economic return.

Optimizing herbicide selection for pre-emergence control of itchgrass and cypressvine morningglory in sugarcane.

The aim of this study was to assess the efficacy of herbicides in association to control Rottboellia exaltata and Ipomoea quamoclit during pre-emergence while also to evaluate the potential impact on the sugarcane. The experimental design employed a randomized block with seven treatments and four replications. The treatments were: 1 - no herbicide application; 2 - indaziflam + sulfentrazone; 3 - indaziflam + diclosulam; 4 - indaziflam + tebuthiuron; 5 - flumioxazin + diclosulam, 6 - flumioxazin + pyroxasulfone and 7 - clomazone + sulfentrazone. The evaluated parameters were: percentage of weeds control, green coverage percentage (Canopeo® system), weed biomass (g m-2), itchgrass height, and sugarcane tiller. Several herbicide associations have been proven effective alternatives for managing itchgrass and cypressvine morningglory. The most successful treatments for itchgrass control were indaziflam + tebuthiuron (100%) and indaziflam + diclosulam (97%), whereas for cypressvine morningglory, the betters were indaziflam + sulfentrazone (97%), indaziflam + diclosulam (98%), indaziflam + tebuthiuron (97%), flumioxazin + diclosulam (94%), and clomazone + sulfentrazone (96%). All treatments reduced the weed biomass, with indaziflam + tebuthiuron being the safest option for protecting sugarcane.

Design, Synthesis, and Biological Activity of Novel Triketone-Containing Phenoxy Nicotinyl Inhibitors of HPPD.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a crucial target enzyme in albino herbicides. The inhibition of HPPD activity interferes with the synthesis of carotenoids, blocking photosynthesis and resulting in bleaching and necrosis. To develop herbicides with excellent activity, a series of 3-hydroxy-2-(6-substituted phenoxynicotinoyl)-2-cyclohexen-1-one derivatives were designed via active substructure combination. The title compounds were characterized via infrared spectroscopy, 1H and 13C nuclear magnetic resonance spectroscopies, and high-resolution mass spectrometry. The structure of compound III-17 was confirmed via single-crystal X-ray diffraction. Preliminary tests demonstrated that some compounds had good herbicidal activity. Crop safety tests revealed that compound III-29 was safer than the commercial herbicide mesotrione in wheat and peanuts. Moreover, the compound exhibited the highest inhibitory activity against Arabidopsis thaliana HPPD (AtHPPD), with a half-maximal inhibitory concentration of 0.19 µM, demonstrating superior activity compared with mesotrione (0.28 µM) in vitro. A three-dimensional quantitative structure-activity relationship study revealed that the introduction of smaller groups to the 5-position of cyclohexanedione and negative charges to the 3-position of the benzene ring enhanced the herbicidal activity. A molecular structure comparison demonstrated that compound III-29 was beneficial to plant absorption and conduction. Molecular docking and molecular dynamics simulations further verified the stability of the complex formed by compound III-29 and AtHPPD. Thus, this study may provide insights into the development of green and efficient herbicides.

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.

Metabolism-Based Nontarget-Site Mechanism Is the Main Cause of a Four-Way Resistance in Shortawn Foxtail (Alopecurus aequalis Sobol.).

Alopecurus aequalis Sobol. is a predominant grass weed in Chinese winter wheat fields, posing a substantial threat to crop production owing to its escalating herbicide resistance. This study documented the initial instance of an A. aequalis population (AHFT-3) manifesting resistance to multiple herbicides targeting four distinct sites: acetyl-CoA carboxylase (ACCase), acetolactate synthase, photosystem II, and 1-deoxy-d-xylulose-5-phosphate synthase. AHFT-3 carried an Asp-to-Gly mutation at codon 2078 of ACCase, with no mutations in the remaining three herbicide target genes, and exhibited no overexpression of any target gene. Compared with the susceptible population AHFY-3, AHFT-3 metabolized mesosulfuron-methyl, isoproturon, and bixlozone faster. The inhibition and comparison of herbicide-detoxifying enzyme activities indicated the participation of cytochrome P450s in the resistance to all four herbicides, with glutathione S-transferases specifically linked to mesosulfuron-methyl. Three CYP72As and a Tau class glutathione S-transferase, markedly upregulated in resistant plants, potentially played pivotal roles in the multiple-herbicide-resistance phenotype.

Differential Resistance to Acetyl-CoA Carboxylase Inhibitors in Rice: Insights from Two Distinct Target-Site Mutations.

Weeds present a significant challenge to agricultural productivity, and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides have proven to be effective in managing weed populations in rice fields. To develop ACCase-inhibiting herbicide-resistant rice, we generated mutants of rice ACCase (OsACC) featuring Ile-1792-Leu or Gly-2107-Ser substitutions through ethyl methyl sulfonate (EMS) mutagenesis. The Ile-1792-Leu mutant displayed cross-resistance to aryloxyphenoxypropionate (APP) and phenylpyrazoline (DEN) herbicides, whereas the Gly-2107-Ser mutants primarily exhibited cross-resistance to APP herbicides with diminished resistance to the DEN herbicide. In vitro assays of the OsACC activity revealed an increase in resistance to haloxyfop and quizalofop, ranging from 4.84- to 29-fold in the mutants compared to that in wild-type. Structural modeling revealed that both mutations likely reduce the binding affinity between OsACC and ACCase inhibitors, thereby imparting resistance. This study offers insights into two target-site mutations, contributing to the breeding of herbicide-resistant rice and presenting alternative weed management strategies in rice cultivation.

The ilv2 gene, encoding acetolactate synthase for branched chain amino acid biosynthesis, is required for plant pathogenicity by Leptosphaeria maculans.

BACKGROUND: Control of blackleg disease of canola caused by the fungus Leptosphaeria maculans relies on strategies such as the inhibition of growth with fungicides. However, other chemicals are used during canola cultivation, including fertilizers and herbicides. There is widespread use of herbicides that target the acetolactate synthase (ALS) enzyme involved in branched chain amino acid synthesis and low levels of these amino acids within leaves of Brassica species. In L. maculans the ilv2 gene encodes ALS and thus ALS-inhibiting herbicides may inadvertently impact the fungus. METHODS AND RESULTS: Here, the impact of a commercial herbicide targeting ALS and mutation of the homologous ilv2 gene in L. maculans was explored. Exposure to herbicide had limited impact on growth in vitro but reduced lesion sizes in plant disease experiments. Furthermore, the mutation of the ilv2 gene via CRISPR-Cas9 gene editing rendered the fungus non-pathogenic. CONCLUSION: Herbicide applications can influence disease outcome, but likely to a minor extent.

Chalcones-Features, Identification Techniques, Attributes, and Application in Agriculture.

This review article is a comprehensive and current overview on chalcones, covering their sources, identification methods, and properties with a particular focus on their applications in the agricultural sector. The widespread use of synthetic pesticides has not only led to increased resistance among weeds and pests, resulting in economic losses, but it has also raised significant health concerns due to the overuse of these chemicals. In line with the European Green Deal 2030 and its Farm to Fork strategy, there is a targeted 50% reduction in the use of chemical pesticides by 2030, emphasizing a shift towards natural alternatives that are more environmentally sustainable and help in the restoration of natural resources. Chalcones and their derivatives, with their herbicidal, fungicidal, bactericidal, and antiviral properties, appear to be ideal candidates. These naturally occurring compounds have been recognized for their beneficial health effects for many years and have applications across multiple areas. This review not only complements the previous literature on the agricultural use of chalcones but also provides updates and introduces methods of detection such as chromatography and MALDI technique.

Discovery of Novel Pyridazine Herbicides Targeting Phytoene Desaturase with Scaffold Hopping.

Phytoene desaturase (PDS) is a critical functional enzyme in blocking ζ-carotene biosynthesis and is one of the bleaching herbicide targets. At present, norflurazon (NRF) is the only commercial pyridazine herbicide targeting PDS. Therefore, developing new and diverse pyridazine herbicides targeting PDS is urgently required. In this study, diflufenican (BF) was used as the lead compound, and a scaffold-hopping strategy was employed to design and synthesize some pyridazine derivatives based on the action mode of BF and PDS. The preemergence herbicidal activity tests revealed that compound 6-chloro-N-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenoxy)pyridazine-4-carboxamide (B1) with 2,4-diF substitution in the benzeneamino ring showed 100% inhibition rates against the roots and stems of Echinochloa crus-galli and Portulaca oleracea at 100 µg/mL, superior to the inhibition rates of BF. Meanwhile, compound B1 demonstrated excellent postemergence herbicidal activity against broadleaf weeds, which was similar to that of BF (inhibition rate of 100%) but superior to that of NRF. This indicated that 6-Cl in the pyridazine ring is the key group for postemergence herbicidal activity. In addition, compound B1 could induce downregulation of PDS gene expression, 15-cis-phytoene accumulation, and Y(II) deficiency and prevent photosynthesis. Therefore, B1 can be considered as a promising candidate for developing high-efficiency PDS inhibitors.

Design, Synthesis, and Biological Evaluation of Pyridazinone-Containing Derivatives As Novel Protoporphyrinogen IX Oxidase Inhibitor.

Protoporphyrinogen IX oxidase (PPO, E.C. 1.3.3.4) plays a pivotal role in chlorophyll biosynthesis in plants, making it a prime target for herbicide development. In this study, we conducted an investigation aimed at discovering PPO-inhibiting herbicides. Through this endeavor, we successfully identified a series of novel compounds based on the pyridazinone scaffold. Following structural optimization and biological assessment, compound 10ae, known as ethyl 3-((6-fluoro-5-(6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate, emerged as a standout performer. It exhibited robust activity against Nicotiana tabacum PPO (NtPPO) with an inhibition constant (Ki) value of 0.0338 µM. Concurrently, we employed molecular simulations to obtain further insight into the binding mechanism with NtPPO. Additionally, another compound, namely, ethyl 2-((6-fluoro-5-(5-methyl-6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate (10bh), demonstrated broad-spectrum and highly effective herbicidal properties against all six tested weeds (Leaf mustard, Chickweed, Chenopodium serotinum, Alopecurus aequalis, Poa annua, and Polypogon fugax) at the dosage of 150 g a.i./ha through postemergence application in a greenhouse. This work identified a novel lead compound (10bh) that showed good activity in vitro and excellent herbicidal activity in vivo and had promising prospects as a new PPO-inhibiting herbicide lead.

Trade-offs constrain the success of glyphosate-free farming.

Glyphosate, the most widely used herbicide, is linked with environmental harm and there is a drive to replace it in agricultural systems. We model the impacts of discontinuing glyphosate use and replacing it with cultural control methods. We simulate winter wheat arable systems reliant on glyphosate and typical in northwest Europe. Removing glyphosate was projected to increase weed abundance, herbicide risk to the environment, and arable plant diversity and decrease food production. Weed communities with evolved resistance to non-glyphosate herbicides were not projected to be disproportionately affected by removing glyphosate, despite the lack of alternative herbicidal control options. Crop rotations with more spring cereals or grass leys for weed control increased arable plant diversity. Stale seedbed techniques such as delayed drilling and choosing ploughing instead of minimum tillage had varying effects on weed abundance, food production, and profitability. Ploughing was the most effective alternative to glyphosate for long-term weed control while maintaining production and profit. Our findings emphasize the need for careful consideration of trade-offs arising in scenarios where glyphosate is removed. Integrated Weed Management (IWM) with more use of cultural control methods offers the potential to reduce chemical use but is sensitive to seasonal variability and can incur negative environmental and economic impacts.

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.

A dew-responsive pectin-based herbicide for enhanced photodynamic inactivation.

5-aminolevulinic acid (5-ALA) has been fully demonstrated as a biodegradable, without resistance, and pollution-free pesticide. However, the lack of targeting and the poor adhesion result in a low utilization rate, limiting its practical application. Herein, a dew-responsive polymer pro-pesticide Pec-hyd-ALA was successfully synthesized by grafting 5-ALA onto the pectin (PEC) backbone via acid-sensitive acylhydrazone bonds. When the pro-pesticide is exposed to acid dew on plant surfaces at night, 5-ALA is released and subsequently converted to photosensitize (Protoporphyrin IX, PpIX)in plant cells, leading to its accumulation and promoting photodynamic inactivation (PDI). An inverted fluorescence microscope has verified the accumulation of tetrapyrrole in plant cells. In addition, the highly bio-adhesive PEC backbone effectively improved the wetting and retention of 5-ALA on leaves. The pot experiment also demonstrated the system's control effect on barnyard grass. This work provides a promising approach to improving the herbicidal efficacy of 5-ALA.

Structure-Activity Relationships of Natural C-9-Methyl-Substituted 10-Membered Lactones and Their Semisynthetic Derivatives.

Fungal 10-membered lactones (TMLs), such as stagonolide A, herbarumin I, pinolidoxin, and putaminoxin, are promising candidates for the development of nature-derived herbicides. The aim of this study was to analyze the structure-activity relationships (SAR) of C-9-methyl-substituted TMLs with a multitarget bioassay approach to reveal compounds with useful (phytotoxic, entomotoxic, antimicrobial) or undesirable (cytotoxic) bioactivities. A new TML, stagonolide L (1), along with five known compounds (stagonolides D (2) and E (3), curvulides A (4) and B1/B2 (5a,b), and pyrenolide C (6)), were purified from cultures of the phytopathogenic fungus Stagonospora cirsii, and five semisynthetic derivatives of 3 and 4 (7-11) were obtained. The absolute configuration of 4 was revised to 2Z, 4S, 5S, 6R, and 9R. The identity of 5a,b and stagonolide H is discussed. The phytotoxicity of compound 4, the entomotoxicity of 5a,b, and nonselective toxicity of compound 6 are demonstrated. The latter confirms the hypothesis that the α,ß-unsaturated carbonyl group is associated with the high general toxicity of TML, regardless of its position in the ring and other substituents. The epoxide in compound 4 is important for phytotoxicity. The revealed SAR patterns will be useful for further rational design of TML-based herbicides including curvulide A analogs with a 4,5-epoxy group.

Investigation into the Synthesis, Bioactivity, and Mechanism of Action of the Novel 6-Pyrazolyl-2-picolinic Acid as a Herbicide.

A series of new 4-amino-3,5-dicholo-6-(5-aryl-substituted-1H-pyrazol-1-yl)-2-picolinic acid compounds were designed and prepared to discover herbicidal molecules. The inhibitory activities of all new compounds against the root growth ofArabidopsis thaliana were assayed. On the whole, the new synthesized compounds displayed good inhibition effects and had excellent herbicidal activities on root growth of weed at 500 µM. Importantly, a selection of compounds demonstrated comparable herbicidal properties to picloram. At the dosage of 250 g/ha, most of the compounds showed a 100% postemergence herbicidal activity to control Chenopodium album and Amaranthus retroflexus. Using compound V-2, the mechanism of action was investigated based on a phenotype study using AFB5-deficient Arabidopsis thaliana. It was found that the novel 6-pyrazolyl-2-picolinic acids were auxinic compounds. In addition, it was proposed that V-2 may be an immune activator due to its upregulation of defense genes and the increased content of jasmonic acid.

Understanding the Effects of Ligand Configuration on Protoporphyrinogen IX Oxidase with Rationally Designed 3-(N-Phenyluracil)but-2-enoates.

Protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) is a promising target for green herbicide discovery. However, the ligand configuration effects on PPO activity were still poorly understood. Herein, we designed 3-(N-phenyluracil)but-2-enoates using our previously developed active fragments exchange and link (AFEL) approach and synthesized a series of novel compounds with nanomolar ranges of Nicotiana tabacum PPO (NtPPO) inhibitory potency and promising herbicidal potency. Our systematic structure-activity relationship investigations showed that the E isomers of 3-(N-phenyluracil)but-2-enoates displayed improved bioactivity than their corresponding Z isomers. Using molecular simulation studies, we found that the E isomers showed a relatively lower entropy change and could sample more stable binding conformation to the receptor than the Z isomers. Our density functional theory (DFT) calculations showed that the E isomers showed higher chemical reactivity and lower electronic chemical potential than their corresponding Z isomers. Compound E-Ic emerged as the optimal compound with a Ki value of 3.0 nM against NtPPO, exhibiting a broader spectrum of weed control than saflufenacil at 37.5-75 g ai/ha and also safe to maize at 75 g ai/ha, which could be considered as a promising lead herbicide for further development.

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.

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.