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.

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.

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 Herbicidal Evaluation of Pyrrolidinone-Containing 2-Phenylpyridine Derivatives as Novel Protoporphyrinogen Oxidase Inhibitors.

In this work, a series of pyrrolidinone-containing 2-phenylpyridine derivatives were synthesized and evaluated as novel protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors for herbicide development. At 150 g ai/ha, compounds 4d, 4f, and 4l can inhibit the grassy weeds of Echinochloa crus-galli (EC), Digitaria sanguinalis (DS), and Lolium perenne (LP) with a range of 60 to 90%. Remarkably, at 9.375 g ai/ha, these compounds showed 100% inhibition effects against broadleaf weeds of Amaranthus retroflexus (AR) and Abutilon theophrasti (AT), which were comparable to the performance of the commercial herbicides flumioxazin (FLU) and saflufenacil (SAF) and better than that of acifluorfen (ACI). Molecular docking analyses revealed significant hydrogen bonding and π-π stacking interactions between compounds 4d and 4l with Arg98, Asn67, and Phe392, respectively. Additionally, representative compounds were chosen for in vivo assessment of PPO inhibitory activity, with compounds 4d, 4f, and 4l demonstrating excellent inhibitory effects. Notably, compounds 4d and 4l induced the accumulation of reactive oxygen species (ROS) and a reduction in the chlorophyll (Chl) content. Consequently, compounds 4d, 4f, and 4l are promising lead candidates for the development of novel PPO herbicides.

New α-pyrone derivatives with herbicidal activity from the endophytic fungus Alternaria brassicicola.

Three pairs of undescribed enantiomeric α-pyrone derivatives (1a/1b-3a/3b) and six undescribed congeners (4-9), were obtained from the fungus Alternaria brassicicola that was isolated from the fresh leaves of Siegesbeckia pubescens Makino (Compositae). The structures of these new compounds were characterized by extensive NMR spectroscopic and HRESIMS data, and their absolute configurations were further elucidated by a modified Mosher's method, chemical conversion, single-crystal X-ray diffraction analysis, and ECD calculations. This is the first report of three pairs of enantiomeric α-pyrone derivatives from the fungus A. brassicicola, and these enantiomers were successfully acquired from scalemic mixtures via chiral HPLC. Compounds 1a/1b-3a/3b and 4-9 were evaluated for the herbicidal activity against Echinochloa crusgalli, Setaria viridis, Portulaca oleracea, and Taraxacum mongolicum. At a concentration of 100 µg/mL, compounds 1a and 1b could significantly inhibit the germination of monocotyledon weed seeds (E. crusgalli and S. viridis) with inhibitory ratios ranging from 68.6 ± 6.4% to 84.2 ± 5.1%, which was equivalent to that of the positive control (glyphosate). The potential structure-herbicidal activity relationship of these compounds was also discussed. To a certain extent, the results of this study will attract great interest for the potential practical application of promising fungal metabolites, α-pyrone derivatives, as ecofriendly herbicides.

Target-Site Mutations and Expression of ALS Gene Copies Vary According to Echinochloa Species.

The sustainability of rice cropping systems is jeopardized by the large number and variety of populations of polyploid Echinochloa spp. resistant to ALS inhibitors. Better knowledge of the Echinochloa species present in Italian rice fields and the study of ALS genes involved in target-site resistance could significantly contribute to a better understanding of resistance evolution and management. Using a CAPS-rbcL molecular marker, two species, E. crus-galli (L.) P. Beauv. and E. oryzicola (Vasinger) Vasing., were identified as the most common species in rice in Italy. Mutations involved in ALS inhibitor resistance in the different species were identified and associated with the ALS homoeologs. The relative expression of the ALS gene copies was evaluated. Molecular characterization led to the identification of three ALS genes in E. crus-galli and two in E. oryzicola. The two species also carried different point mutations conferring resistance: Ala122Asn in E. crus-galli and Trp574Leu in E. oryzicola. Mutations were carried in the same gene copy (ALS1), which was significantly more expressed than the other copies (ALS2 and ALS3) in both species. These results explain the high resistance level of these populations and why mutations in the other ALS copies are not involved in herbicide resistance.

Fate and adaptive plasticity of heterogeneous resistant population of Echinochloa colona in response to glyphosate.

Understanding the fate of heterogenous herbicide resistant weed populations in response to management practices can help towards overcoming the resistance issues. We selected one pair of susceptible (S) and resistant (R) phenotypes (2B21-R vs 2B21-S and 2B37-R vs 2B37-S) separately from two glyphosate resistant heterogeneous populations (2B21 and 2B37) of Echinochloa colona and their fate and adaptive plasticity were evaluated after glyphosate application. Our study revealed the glyphosate concentration required to cause a 50% plant mortality (LD50) was 1187, 200, 3064, and 192 g a. e. ha-1 for the four phenotypes 2B21-R, 2B21-S, 2B37-R, and 2B37-S respectively. Both S phenotypes accumulated more biomass than the R phenotypes at the lower application rates (34 and 67.5 g a. e. ha-1) of glyphosate. However, the R phenotypes generally produced more biomass at rates of glyphosate higher than 100 g a. e. ha-1 throughout the growth period. Plants from the R phenotypes of 2B21 and 2B37 generated 32% and 38% fewer spikesplant-1 than their respective S counterparts in the absence of glyphosate respectively. The spike and seed numbersplant-1 significantly higher in R than S phenotypes at increased rates of glyphosate and these relationships were significant. Our research suggests that glyphosate-resistant E. colona plants will be less fit than susceptible plants (from the same population) in the absence of glyphosate. But in the presence of glyphosate, the R plants may eventually dominate in the field. The use of glyphosate is widespread in field, would favour the selection towards resistant individuals.

An ABCC-type transporter endowing glyphosate resistance in plants.

Glyphosate is the most widely used herbicide in world agriculture and for general vegetation control in a wide range of situations. Global and often intensive glyphosate selection of very large weedy plant populations has resulted in widespread glyphosate resistance evolution in populations of many weed species. Here, working with a glyphosate-resistant (GR) Echinochloa colona population that evolved in a Western Australia agricultural field, we identified an ATP-binding cassette (ABC) transporter (EcABCC8) that is consistently up-regulated in GR plants. When expressed in transgenic rice, this EcABCC8 transporter endowed glyphosate resistance. Equally, rice, maize, and soybean overexpressing the EcABCC8 ortholog genes were made resistant to glyphosate. Conversely, CRISPR/Cas9-mediated knockout of the EcABCC8 ortholog gene OsABCC8 increased rice susceptibility to glyphosate. Subcellular localization analysis and quantification of glyphosate cellular levels in treated ABCC8 transgenic rice plants and isolated leaf protoplasts as well as structural modeling support that EcABCC8 is likely a plasma membrane-localized transporter extruding cytoplasmic glyphosate to the apoplast, lowering the cellular glyphosate level. This is a report of a membrane transporter effluxing glyphosate in a GR plant species, and its function is likely conserved in crop plant species.

Enantioselective and Synergistic Herbicidal Activities of Common Amino Acids Against Amaranthus tricolor and Echinochloa crus-galli.

Amino acids have a wide range of biological activities, which usually rely on the stereoisomer presented. In this study, glycine and 21 common α-amino acids were investigated for their herbicidal property against Chinese amaranth (Amaranthus tricolor L.) and barnyard grass (Echinochloa crus-galli (L.) Beauv.). Both d- and l-isomers, as well as a racemic mixture, were tested and found that most compounds barely inhibited germination but moderately suppressed seedling growth. Various ratios of d:l-mixture were studied and synergy between enantiomers was found. For Chinese amaranth, the most toxic d:l-mixtures were at 3:7 (for glutamine), 8:2 (for methionine), and 5:5 (for tryptophan). For barnyard grass, rac-glutamine was more toxic than the pure forms; however, d-tryptophan exhibited greater activity than racemate and l-isomer, indicating the sign of enantioselective toxicity. The mode of action was unclear, but d-tryptophan caused bleaching of leaves, indicating pigment synthesis of the grass was inhibited. The results highlighted the enantioselective and synergistic toxicity of some amino acids, which relied upon plant species, chemical structures, and concentrations. Overall, our finding clarifies the effect of stereoisomers, and provides a chemical clue of amino acid herbicides, which may be useful in the development of herbicides from natural substances.

One-Step Encapsulation of ortho-Disulfides in Functionalized Zinc MOF. Enabling Metal-Organic Frameworks in Agriculture.

Application of natural products as new green agrochemicals with low average lifetime, low concentration doses, and safety is both complex and expensive due to chemical modification required to obtain desirable physicochemical properties. Transport, aqueous solubility, and bioavailability are some of the properties that have been improved using functionalized metal-organic frameworks based on zinc for the encapsulation of bioherbicides (ortho-disulfides). An in situ method has been applied to achieve encapsulation, which, in turn, led to an improvement in water solubility by more than 8 times after 2-hydroxypropyl-ß-cyclodextrin HP-ß-CD surface functionalization. High-resolution high-angle annular dark-field scanning transmission electron microscopy (HR HAADF-STEM) and integrated differential phase contrast (iDPC) imaging techniques were employed to verify the success of the encapsulation procedure and crystallinity of the sample. Inhibition studies on principal weeds that infect rice, corn, and potato crops gave results that exceed those obtained with the commercial herbicide Logran. This finding, along with a short synthesis period, i.e., 2 h at 25 °C, make the product an example of a new generation of natural-product-based herbicides with direct applications in agriculture.

Comparison of Efficacy and Detection of Clethodim and Glyphosate Applied with Dicamba and 2,4-D through Tank Mixture and Sequential Applications.

Greenhouse studies were planted at the R.R. Foil Plant Science Research Center in Starkville, MS. In the efficacy trial, pots were seeded with barnyardgrass (Echinochloa crus-galli), broadleaf signalgrass (Urochloa platyphylla), and giant foxtail (Setaria faberi). In the lab detection trial, only barnyardgrass was seeded. Both studies consisted of 16 treatments with four replications per treatment. The treatments consisted of clethodim, glyphosate, dicamba, and 2,4-D applied singularly and in combination with each other. Each herbicide combination was applied with three application methods: tank mixture, sequential applications where the synthetic auxin was applied first (auxin applied first), and sequential applications where glyphosate or clethodim was applied first (auxin applied second). The auxin applied second method had higher visual estimations of control ratings and lower biomass weights compared to the other two methods. The auxin applied second method had more glyphosate and clethodim detected with the use of liquid chromatography tandem mass spectrometry.

Design, Synthesis, and Herbicidal Activity of N-Benzyl-5-cyclopropyl-isoxazole-4-carboxamides.

Based on the structures of isoxaflutole (IFT) and N-isobutyl-N-(4-chloro-benzyl)-4-chloro-2-pentenamide, a series of N-benzyl-5-cyclopropyl-isoxazole-4-carboxamides was designed by connecting their pharmacophores (i.e., a multitarget drug design strategy). A total of 27 N-benzyl-5-cyclopropyl-isoxazole-4-carboxamides were prepared from 5-cyclopropylisoxazole-4-carboxylic acid and substituted benzylamines, and their structures were confirmed by NMR and MS. Laboratory bioassays indicated that I-26 showed 100% inhibition against Portulaca oleracea and Abutilon theophrasti at a concentration of 10 mg/L, better than the positive control butachlor (50% inhibition for both weeds). A strong growth inhibition was observed, but a typical bleaching phenomenon of IFT could not be observed in the Petri dish assay. I-05 displayed excellent postemergence herbicidal activity against Echinochloa crusgalli and A. theophrasti at a rate of 150 g/ha, and bleaching symptoms were observed in the leaves of treated weeds. The bleaching effect of Chlamydomonas reinhardtii treated by I-05 could be reversed by adding homogentisate. Enzymatic bioassays indicated that I-05 could not inhibit 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, but II-05, an isoxazole ring-opening product of I-05, could inhibit HPPD activity with an EC50 value of 1.05 µM, similar to that of mesotrione (with an EC50 value of 1.35 µM). Detailed discussion about observed herbicidal symptoms is provided in the Results and Discussion section. This investigation provided a proof-of-concept foundation that a multitarget drug design strategy could be applied in agrochemical research.

Turpentine Derived Secondary Amines for Sustainable Crop Protection: Synthesis, Activity Evaluation and QSAR Study.

In this study, we will report on the synthesis and application of efficient botanical agrochemicals from turpentine for sustainable crop protection. Two series of turpentine derived secondary amines were synthesized and identified by FT-IR, 1H NMR, 13C NMR, and HRMS. The herbicidal activities against Echinochloa crus-galli were evaluated. The potential toxicity of the synthesized compounds was tested by MTT cytotoxicity analysis. The effect of structure of the synthesized secondary amines and corresponding Schiff base compounds on their activities was investigated by quantitative structure-activity relationship (QSAR) study. All target products were found to be low toxicity, with similar or higher herbicidal activities than commercial herbicides diuron and Glyphosate. Results of QSAR study showed that a best four-descriptor QSAR model with R2 of 0.880 and Rloo2 of 0.818 was obtained. The four descriptors most relevant to the herbicidal activities are the min valency of a N atom, the max total interaction for a C-H bond, the relative number of aromatic bonds, and the min partial charge (Qmin).

A novel mutation Asp-2078-Glu in ACCase confers resistance to ACCase herbicides in barnyardgrass (Echinochloa crus-galli).

Multiple-herbicide resistance (MHR) in barnyardgrass (Echinochloa crus-galli) is a threat to rice production. The Ala-205-Val mutation in acetolactate synthase (ALS) conferred resistance to several ALS inhibitors in the E. crus-galli population AXXZ-2; consequently, ALS-inhibitors were unable to control this noxious weed species. In the present study, the sensitivity to acetyl-coenzyme A carboxylase (ACCase) herbicides and other herbicides having different modes of action was evaluated to determine an effective strategy for chemical weed control. Compared with that of the reportedly sensitive population JLGY-3, the AXXZ-2 population showed differential resistance to three ACCase-inhibitors (cyhalofop-butyl, fenoxaprop-P-ethyl, and pinoxaden), in addition to quinclorac and pretilachlor. A novel substitution (Asp-2078-Glu) in ACCase was detected as the main target-site resistance mechanisms in the AXXZ-2 population. Structural modeling of the mutant ACCase protein predicted that Asp-2078-Glu confers resistance to three ACCase inhibitors by reducing the binding affinity between them and the ACCase protein. To the best of our knowledge, this is the first study to report that the novel Asp-2078-Glu mutation confers resistance to several ACCase inhibitors. Target-site mutations in ALS and ACCase were detected in this MHR population. Except for quinclorac, pretilachlor, ALS inhibitors, and the three ACCase inhibitors, a number of herbicides remain effective in controlling this MHR E. crus-galli population.

Sensitivity of yellow foxtail (Setaria glauca L.) and barnyardgrass (Echinochloa crus-galli L.) to aqueous extracts or dry biomass of cover crops.

BACKGROUND: The common weeds Echinochloa crus-galli L. and Setaria glauca L. were studied for their sensitivity to aqueous extracts or dry biomass of the following cover crops (CCs): Brassicaceae (Sinapis alba, Raphanus sativus var. Oleiformis, Camellina sativa), Fagopyrum esculentum and Guizotia abyssinica. RESULTS: Treating E. crus-galli with aqueous extracts of mixed CCs or individual brassica CC significantly reduced germination. Treating S. glauca with aqueous extracts of C. sativa or G. abyssinicia reduced germination. Aqueous extracts of all CCs significantly reduced radicle length of E. crus-galli and S. glauca, with C. sativa and mixed CCs showing the greatest effect. Aqueous extracts of nearly all CCs delayed start and middle germination of E. crus-galli and S. glauca, with S. alba and R. sativus showing the strongest effects. Aqueous extracts of Brassicaceae leaf and flower significantly reduced germination, coleoptile length, radicle length and seedling biomass of E. crus-galli and S. glauca. Brassicaceae leaves and flowers contained higher phenolics than other tissues. Adding 4 or 8% S. alba and R. sativus dry powder to soil significantly reduced growth of E. crus-galli and S. glauca; even concentrations of 1% measurably slowed growth of E. crus-galli. CONCLUSIONS: Brassicaceae may be allelopathic to S. glauca and E. crus-galli. Aqueous extracts of leaves and flowers showed greater phytotoxic activity than other tissues and also contained more phenolics. Therefore Brassicaceae CCs may be most effective against S. glauca and E. crus-galli if incorporated into soil during their flowering stage. © 2020 Society of Chemical Industry.

Response of glyphosate-resistant and susceptible biotypes of Echinochloa colona to low doses of glyphosate in different soil moisture conditions.

To evaluate the hormetic effect of glyphosate on Echinochloa colona, two pot studies were done in the screenhouse at the Gatton Campus, the University of Queensland, Australia. Glyphosate was sprayed at the 3-4 leaf stage using different doses [(0, 5, 10, 20, 40, 80 and 800 g a.e. ha-1) and (0, 2.5, 5, 10, 20 and 800 g a.e. ha-1)] in the first and second study, respectively. In the second study, two soil moistures (adequately-watered and water-stressed), and two E. colona biotypes, glyphosate-resistant and glyphosate-susceptible, were included. In both studies, plants that were treated with glyphosate at 2.5-40 g ha-1 grew taller and produced more leaves, tillers, inflorescences and seeds than the control treatment. In the first study, 5 g ha-1 glyphosate resulted in the maximum aboveground biomass (increase of 34% to 118%) compared with the control treatment. In the second study, the adequately-watered and glyphosate low dose treatments caused an increase in all the measured growth parameters for both biotypes. For example, total dry biomass was increased by 64% and 54% at 5 g ha-1 in the adequately-watered treatments for the resistant and susceptible biotypes, respectively, compared with the control treatment. All measured traits tended to decrease with increasing water stress and the stimulative growth of low doses of glyphosate could not compensate for the water stress effect. The results of both studies showed a hormetic effect of low doses of glyphosate on E. colona biotypes and such growth stimulation was significant in the range of 5 to 10 g ha-1 glyphosate. Water availability was found to be effective in modulating the stimulatory outcomes of glyphosate-induced hormesis. No significant difference was observed between the resistant and susceptible biotypes for hormesis phenomenon. The study showed the importance of precise herbicide application for suppressing weed growth and herbicide resistance evolution.

Effects of sorghum residue in presence of pre-emergence herbicides on emergence and biomass of Echinochloa colona and Chloris virgata.

In conservation agriculture systems, farmers gain many advantages from retaining crop residue on the soil surface, but crop residue retention in these systems may intervene with the activity of pre-emergence herbicides. A pot study was conducted to evaluate the effect of different rates of pre-emergence herbicides [imazethapyr (100 and 150 g a. i. ha-1), isoxaflutole (100 and 200 g a. i. ha-1), metolachlor (1.5 and 2.25 kg a. i. ha-1), pendimethalin (2.25 and 3.38 kg a. i. ha-1) and prosulfocarb + metolachlor (2.5 and 3.75 kg a. i. ha-1)] on seedling emergence and biomass of Echinochloa colona and Chloris virgata when applied in the presence of sorghum residue at rates equivalent to (0, 3 and 6 t ha-1). When seeds of E. colona and C. virgata were not covered with sorghum residue, the seedling emergence and biomass of both weeds was inhibited by 93-100% and 56-100%, respectively, with the application (both rates) of isoxaflutole, metolachlor, pendimethalin and prosulfocarb + metolachlor. Using sorghum residue resulted in lower herbicide efficacy on both weeds. At 3 t ha-1 sorghum residue, E. colona emergence and biomass reduced by 38-100% and 30-100%, respectively, with application of isoxaflutole, metolachlor and pendimethalin (both rates) in comparison with the no-herbicide treatment. Similarly, the emergence and biomass of C. virgata was also reduced by 92-100% and 25-100%, respectively. The results of this study suggest that crop residue may influence efficacy of commonly used pre-emergence herbicides and that the amount of crop residue on the soil surface should be adjusted according to the nature of the pre-emergence herbicides to achieve adequate weed control.

Ethylene Biosynthesis Inhibition Combined with Cyanide Degradation Confer Resistance to Quinclorac in Echinochloa crus-galli var. mitis.

Echinochloa crus-galli var. mitis has rarely been reported for herbicide resistance, and no case of quinclorac resistance has been reported so far. Synthetic auxin-type herbicide quinclorac is used extensively to control rice weeds worldwide. A long history of using quinclorac in Chinese rice fields escalated the resistance in E. crus-galli var. mitis against this herbicide. Bioassays in Petri plates and pots exhibited four biotypes that evolved into resistance to quinclorac ranking as JS01-R > AH01-R > JS02-R > JX01-R from three provinces of China. Ethylene production in these biotypes was negatively correlated with resistance level and positively correlated with growth inhibition. Determination of the related ethylene response pathway exhibited resistance in biotypes that recorded a decline in 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase oxidase activities, and less inducible ACS and ACO genes expressions than the susceptible biotype, suggesting that there was a positive correlation between quinclorac resistance and ethylene biosynthesis inhibition. Cyanides produced during the ethylene biosynthesis pathway mainly degraded by the activity of ß-cyanoalanine synthase (ß-CAS). Resistant biotypes exhibited higher ß-CAS activity than the susceptible ones. Nucleotide changes were found in the EcCAS gene of resistant biotypes as compared to sensitive ones that caused three amino acid substitutions (Asn-105-Lys, Gln-195-Glu, and Gly-298-Val), resulting in alteration of enzyme structure, increased binding residues in the active site with its cofactor, and decreased binding free energy; hence, its activity was higher in resistant biotypes. Moreover, these mutations increased the structural stability of the enzyme. In view of the positive correlation between ethylene biosynthesis inhibition and cyanide degradation with resistance level, it is concluded that the alteration in ethylene response pathway or at least variation in ACC synthase and ACC oxidase enzyme activities-due to less relative expression of ACS and ACO genes and enhanced ß-CAS activity, as well as mutation and increased relative expression of EcCAS gene-can be considered as a probable mechanism of quinclorac resistance in E. crus-galli var. mitis.

Synthesis and Evaluation of Halogenated 5-(2-Hydroxyphenyl)pyrazoles as Pseudilin Analogues Targeting the Enzyme IspD in the Methylerythritol Phosphate Pathway.

This work reports halogenated 5-(2-hydroxyphenyl)pyrazoles as pseudilin analogues with the potential to target the enzyme IspD in the methylerythritol phosphate (MEP) pathway. Such analogues were designed using the bioisosteric replacement of the pseudilin core structure and synthesized via an efficient three-step route. With AtIspD-based screening and pre- and post-emergence herbicidal tests, these compounds were demonstrated to have considerable activities against AtIspD, with IC50 up to 3.27 µM, and against model plants rape and barnyard grass, with moderate to excellent activities. At a rate of 150 g/ha in the greenhouse test, three compounds exhibited higher or comparable herbicidal activities than pseudilin. Molecular docking of representative compounds into the allosteric site of AtIspD revealed a binding mode similar to that of pseudilin. The established bioisosterism and synthesis method in this work may serve as an important tool for the development of new herbicides and antimicrobials targeting IspD in the MEP pathway.