Sustainable production of 2,3,5,6-Tetramethylpyrazine at high titer in engineered Corynebacterium glutamicum.

The industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance, and commodity chemical. Here, we demonstrate TMP production (∼0.8 g L-1) in C. glutamicum type strain ATCC13032 via overexpression of acetolactate synthase and/or α-acetolactate decarboxylase from Lactococcus lactis in CGXII minimal medium supplemented with 40 g L-1 glucose. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v v-1) hydrolysates derived from ionic liquid-pretreated sorghum biomass. A key objective was to take the fully engineered strain developed in this study and interrogate medium parameters that influence the production of TMP, a critical post-strain engineering optimization. Design of experiments in a high-throughput plate format identified glucose, urea, and their ratio as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g L-1 TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol mol-1) from 80 g L-1 glucose and 11.9 g L-1 urea in shake flask batch cultivation. ONE-SENTENCE SUMMARY: Corynebacterium glutamicum was metabolically engineered to produce 2,3,5,6-tetramethylpyrazine followed by a design of experiments approach to optimize medium components for high-titer production.

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.

Connecting genes to whole plants in dilution effect of target-site ALS inhibitor resistance of Schoenoplectiella juncoides (Roxb.) Lye (Cyperaceae).

This study focuses on dilution effect of target-site resistance (TSR) to acetolactate synthase (ALS) inhibitors in Schoenoplectiella juncoides, which harbors two ALS genes, ALS1 and ALS2. We assessed gene expression, enzyme activity, and whole-plant resistance profiles across four S. juncoides lines: the susceptible line, the parental resistant lines with a homozygous mutation in either ALS1 or ALS2, and the bred progeny line with homozygous mutations in both ALS1 and ALS2. Gene expression and enzyme function showed a proportional relationship that the expression ratios of ALS1 to ALS2, approximately 70:30, were consistent with the functional ratio predicted by the double-sigmoidal plateau positions observed in enzyme assays. However, at the whole-plant level, resistance did not correlate to the putative abundance of susceptible enzyme, but the parental lines showed similar resistance to each other despite different enzyme-level resistances. This suggests a non-proportional mechanism in the reflection of physiological enzymatic profiles to whole-plant resistance profiles. These findings highlight the complexity of herbicide resistance and the need for further research to understand the mechanisms that influence resistance outcomes. Understanding these relationships is essential for developing strategies to manage herbicide resistance effectively.

Mechanism of multiple resistance to fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon in Avena fatua L. from China.

Avena fatua L. is one of the most damaging and malignant weeds in wheat fields in China. Fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon, which belong to Acetyl-CoA carboxylase- (ACCase), acetolactate synthase- (ALS), and photosystem II- (PS II) inhibitors, respectively, are commonly used in wheat fields and have a long history of use on A. fatua. An A. fatua population (R) resistant to fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon was collected from a wheat field in 2020. This study explored the mechanisms of target site resistance (TSR) and non-target site resistance (NTSR) in the multi-resistant A. fatua. Whole-plant bioassays showed that the R population had evolved high resistance to fenoxaprop-P-ethyl and moderate resistance to mesosulfuron-methyl and isoproturon. However, no mutations were detected in the ACCase, ALS, or psbA genes in the R population. In addition, the ACCase and ALS gene expression levels in the R group were significantly higher than those in the susceptible population (S) after treatment with fenoxaprop-P-ethyl or mesosulfuron-methyl. In vitro ACCase and ALS activity assays showed that ACCase and ALS from the R population were insensitive to fenoxaprop and mesosulfuron-methyl, respectively, with resistance indices 6.12-fold and 17.46-fold higher than those of the S population. Furthermore, pretreatment with P450 inhibitors significantly (P < 0.05) reversed the multi-resistant A. fatua's resistance to fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon. Sethoxydim, flucarbazone­sodium, chlortoluron, and cypyrafluone were effective in controlling multi-resistance A. fatua. Therefore, the overexpression of ACCase and ALS to synthesize sufficient herbicide-targeting proteins, along with P450-mediated metabolism, conferred resistance to fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon in the R population.

Nontarget site-based resistance to nicosulfuron and identification of candidate genes in Cucumis melo L. var. agrestis Naud. via RNA-Seq transcriptome analysis.

Herbicide resistance is a worldwide concern for weed control. Cucumis melo L. var. agrestis Naud. (C. melo) is an annual trailing vine weed that is commonly controlled by nicosulfuron, acetolactate synthase (ALS)-inhibiting herbicides. However, long-term use of this herbicide has led to the emergence of resistance and several nicosulfuron resistant populations of C. melo have been found. Here we identified a resistant (R) C. melo population exhibiting 7.31-fold resistance to nicosulfuron compared with a reference sensitive (S) population. ALS gene sequencing of the target site revealed no amino acid substitution in R plants, and no difference in enzyme activity, as shown by ALS activity assays in vitro. ALS gene expression was not significantly different before and after the application of nicosulfuron. Pretreatment with the cytochrome P450 monooxygenase (P450) inhibitor malathion reduced nicosulfuron resistance in the R population. RNA-Seq transcriptome analysis was used to identify candidate genes that may confer metabolic resistance to nicosulfuron. We selected genes with annotations related to detoxification functions. A total of 20 candidate genes (7 P450 genes, 1 glutathione S-transferase (GST) gene, 2 ATP-binding cassette (ABC) transporters, and 10 glycosyltransferase (GT)) were identified; 12 of them (7 P450s, 1 GST, 2 ABC transporters, and 2 GTs) were demonstrated significantly differential expression between R and S by quantitative real-time RT-PCR (qRT-PCR). Our findings revealed that the resistance mechanism in C. melo was nontarget-site based. Our results also provide a valuable resource for studying the molecular mechanisms of weed resistance.

Target gene overexpression and enhanced metabolism confer resistance to nicosulfuron in Eriochloa villosa (Thunb.).

Eriochloa villosa (Thunb.) Kunth is a troublesome weed widely distributed in maize (Zea mays L.) fields in Northeast China. Many populations of E. villosa have evolved resistance to nicosulfuron herbicides, which inhibit acetolactate synthase (ALS). The objectives of this research were to confirm that E. villosa is resistant to nicosulfuron and to investigate the basis of nicosulfuron resistance. Whole-plant dose-response studies revealed that the R population had not developed a high level of cross-resistance and exhibited greater resistant (25.62-fold) to nicosulfuron than that of the S population and had not yet developed a high level of cross-resistance. An in vitro ALS activity assay demonstrated that the I50 of nicosulfuron was 6.87-fold greater in the R population than the S population. However, based on ALS gene sequencing, the target ALS gene in the R population did not contain mutations. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that ALS gene expression between the R and S populations was significantly different after nicosulfuron application, but no differences were observed in the gene copy number. After the cytochrome P450 inhibitor malathion or the GST inhibitor NBD-Cl was applied, the resistant E. villosa population exhibited increased sensitivity to nicosulfuron. Based on the activities of GSTs and P450s, the activities of the R population were greater than those of the S population after nicosulfuron application. This is the first report that the resistance of E. villosa to ALS inhibitors results from increased target gene expression and increased metabolism. These findings provide a theoretical foundation for the effective control of herbicide-resistant E. villosa.

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.

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.

Unraveling the reasons for failure to control Amaranthus albus: insights into herbicide application at different growth stages, temperature effect, and herbicide resistance on a regional scale.

BACKGROUND: This study investigates factors contributing Amaranthus albus control failure in processing tomato fields in northern Israel. The study region is characterized by a significant climate gradient from east to west, providing the opportunity to investigate the effect of critical elements of the agricultural environment, e.g., temperature. Eight populations were collected from commercial fields in this region. Post-emergence herbicide efficacy of metribuzin, a photosystem II inhibitor, and rimsulfuron, an acetolactate synthase (ALS) inhibitor, was assessed through dose-response analyses at various growth stages. Temperature effects on control efficacy and resistance mechanisms were also explored. RESULTS: Standard metribuzin dose (X) was ineffective on A. albus plants with more than six true-leaves, whereas 2X dose proved effective. Rimsulfuron at 16X dose was ineffective on plants with more than four true-leaves. We report here the first case of target site resistance to ALS inhibitors in A. albus, due to point mutation in the ALS gene (Pro197 to Leu). Furthermore, our findings suggest potential involvement of CYT P450 enzymes in enhanced metabolizing of rimsulfuron. An overall decrease in dry weight was observed in response to both herbicides at 16/22 °C (P < 0.0001). Rimsulfuron was effective against only one population when applied at 28/34 °C. A possible fitness cost associated with target site-resistant biotypes was observed under low temperature conditions, leading to effective control. CONCLUSION: This regional-scale study highlights the challenges faced by growers, emphasizes the need for adapting management practices to the local climatic conditions and lays the groundwork for implementing location-specific weed management strategies in commercial fields. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Molecular and physiological characterization of AIP1, encoding the acetolactate synthase regulatory subunit in rice.

Flooding deprives plants of oxygen and thereby causes severe stress by interfering with energy production, leading to growth retardation. Enzymes and metabolites may help protect plants from waterlogging and hypoxic environmental conditions. Acetolactate synthase (ALS) is a key enzyme in the biosynthesis of branched-chain amino acids (BCAAs), providing the building blocks for proteins and various secondary metabolites. Additionally, under energy-poor conditions, free BCAAs can be used as an alternative energy source by mitochondria through a catabolic enzyme chain reaction. In this study, we characterized ALS-INTERACTING PROTEIN 1 (OsAIP1), which encodes the regulatory subunit of ALS in rice (Oryza sativa). This gene was expressed in all parts of the rice plant, and its expression level was significantly higher in submerged and low-oxygen environments. Rice transformants overexpressing OsAIP1 showed a higher survival rate under hypoxic stress than did non-transgenic control plants under the same conditions. The OsAIP1-overexpressing plants accumulated increased levels of BCAAs, demonstrating that OsAIP1 is an important factor in the hypoxia resistance mechanism. These results suggest that ALS proteins are part of a defense mechanism that improves the tolerance of plants to low-oxygen environments.

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.

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.

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.

Introgression from local cultivars is a driver of agricultural adaptation in Argentinian weedy rice.

Weedy rice, a pervasive and troublesome weed found across the globe, has often evolved through fertilization of rice cultivars with little importance of crop-weed gene flow. In Argentina, weedy rice has been reported as an important constraint since the early 1970s, and, in the last few years, strains with herbicide-resistance are suspected to evolve. Despite their importance, the origin and genetic composition of Argentinian weedy rice as well its adaptation to agricultural environments has not been explored so far. To study this, we conducted genotyping-by-sequencing on samples of Argentinian weedy and cultivated rice and compared them with published data from weedy, cultivated and wild rice accessions distributed worldwide. In addition, we conducted a phenotypic characterization for weedy-related traits, a herbicide resistance screening and genotyped accessions for known mutations in the acetolactate synthase (ALS) gene, which confers herbicide resistance. Our results revealed large phenotypic variability in Argentinian weedy rice. Most strains were resistant to ALS-inhibiting herbicides with a high frequency of the ALS mutation (A122T) present in Argentinian rice cultivars. Argentinian cultivars belonged to the three major genetic groups of rice: japonica, indica and aus while weeds were mostly aus or aus-indica admixed, resembling weedy rice strains from the Southern Cone region. Phylogenetic analysis supports a single origin for aus-like South American weeds, likely as seed contaminants from the United States, and then admixture with local indica cultivars. Our findings demonstrate that crop to weed introgression can facilitate rapid adaptation to agriculture environments.

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

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

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

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

Induced mutation in the SiALS gene offers new weed management opportunities for sesame crop.

Weeds are the primary biotic constraint affecting sesame growth and production. Here, we applied EMS mutagenesis to an elite sesame cultivar and discovered a novel point mutation in the sesame SiALS gene conferring resistance to imidazolinone, a group of acetolactate-synthase (ALS)-inhibitors. The mutant line exhibited high resistance to imazamox, an ALS-inhibitor, with hybrid plants displaying an intermediate response. Field-based validation confirmed the mutant line's substantial resistance, leading to a significantly higher yield under imazamox treatment. Under pre-emergence application of imazapic, the mutant plants sustained growth, whereas wild-type and weed were effectively controlled. Field trials using s-metolachlor and imazapic combined resulted in weed-free plots compared to untreated controls. Consequently, this treatment showed a significantly greater yield (2280 vs. 880 Kg ha-1) than the commercial practice (s-metolachlor). Overall, our study unveils the potential of utilizing this point mutation in sesame breeding programs, offering new opportunities for integrated weed management strategies for sesame cultivation. Developing herbicide-resistant crop plants holds promise for supporting sustainable production and addressing the challenges of weed infestations in sesame farming.

Confirmation and characterization of the first case of acetolactate synthase (ALS)-inhibitor resistance in Japanese brome (Bromus japonicus) in the US.

BACKGROUND: Japanese brome (Bromus japonicus Thumb.) is one of the problematic annual weeds in winter wheat (Triticum aestivum L.) and is generally controlled by acetolactate synthase (ALS) inhibitors. Repeated use of the ALS inhibitor propoxycarbazone-Na resulted in the evolution of resistance to this herbicide in three B. japonicus populations, i.e., R1, R2, and R3 in Kansas (KS). However, the level of resistance and mechanism conferring resistance in these populations is unknown. The objectives of this research were to (i) evaluate the level of resistance to propoxycarbazone-Na in R1, R2, and R3 in comparison with a known susceptible population (S1), (ii) investigate the mechanism of resistance involved in conferring ALS-inhibitor resistance, and (iii) investigate the cross-resistance to other ALS inhibitors. RESULTS: Dose-response (0 to 16x; x = 44 g ai ha-1 of propoxycarbazone-Na) assay indicated 167, 125, and 667-fold resistance in R1, R2 and R3 populations, respectively, compared to S1 population. ALS gene sequencing confirmed the mutations resulting in amino acid substitutions, i.e., Pro-197-Thr (R3, R1)/Ser (R2, R1) bestowing resistance to these ALS inhibitors. Such amino acid substitutions also showed differential cross-resistance to sulfosulfuron, mesosulfuron-methyl, pyroxsulam, and imazamox among resistant populations. Pretreatment with malathion (a cytochrome P450 enzyme-inhibitor) followed by imazamox treatment suggested cross-resistance to this herbicide possibly via metabolism only in R3 population. CONCLUSION: Overall, these results confirm the first case of target-site based resistance to ALS inhibitors in B. japonicus in the US, highlighting the need for exploring herbicides with alternative modes of action to enhance weed control in winter wheat. © 2024 Society of Chemical Industry.

Development of a rapid detection assay for acetolactate synthase inhibitors resistance in three Amaranthus weed species through loop-mediated isothermal amplification.

BACKGROUND: The early detection of herbicide resistance in weeds is a key factor to avoid herbicide waste and improve agriculture sustainability. The present study aimed to develop and validate an allele-specific loop-mediated isothermal amplification (AS-LAMP) assay for the quick on-site detection of the resistance-endowing point mutation Trp-574-Leu in the acetolactate synthase (ALS) gene in three widely diffused Amaranthus weed species: Amaranthus retroflexus, Amaranthus hybridus and Amaranthus tuberculatus. RESULTS: The AS-LAMP protocol was developed on wild-type and ALS-mutant plants of the three species and revealed that the amplification approach with only the primer set specific for the mutant allele (574-Leu) was the most promising. The validation and estimation of the AS-LAMP performance evaluated by comparing the results with those of the molecular marker (cleaved amplified polymorphic sequences) indicated that, although the sensitivity and specificity were relatively high in all species (overall 100 and > 65%, respectively), precision was high for A. hybridus L. and A. retroflexus L. (75 and 79%, respectively), but quite low for A. tuberculatus (Moq.) J. D. Sauer (59%). The LAMP assay was also effective on crude genomic DNA extraction, allowing the quick detection of mutant plants in field situation (on site resistance detection). CONCLUSION: The proposed AS-LAMP method has proven to be a promising technique for rapid detection of resistance as a result of Trp-574-Leu on the two monoecious weedy Amaranthus species but resulted less effective in the genetically variable dioecious species A. tuberculatus. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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

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