Acetolactate Synthase Herbicides Research Articles

Multiple resistance to EPSPS and ALS inhibitors in Palmer amaranth (Amaranthus palmeri) identified in Turkey

AbstractAmaranthus palmeri was first reported in Turkey in 2016, and an immediate heavy infestation of the weed was found in fruit orchards and summer crops such as maize, cotton, and sunflower. There have been farmers' complaints about the ineffective control of Palmer amaranth through the use of glyphosate and some sulfonylureas herbicides. Hence, this study aimed to determine the possible herbicide resistance evolution in Palmer amaranth against glyphosate and acetolactate synthase (ALS) herbicides. Seeds of 21 Palmer amaranth populations were collected from five provinces of Turkey where control problems with glyphosate and ALS inhibitors were reported in maize fields. Seeds of certain biotypes categorized as resistant or susceptible were grown to obtain the F2 generation. A single‐dose experiment determined the possible resistance to ALS inhibitors and glyphosate among the 21 populations. Of this, 18 populations were included in the subsequent dose–response experiments due to evident survival. Based on ED50 values from the dose–response experiment, SNU‐04 and ADN‐21 biotypes had the highest resistance index for glyphosate which was more than 7. The biotypes ADN‐21, OSM‐15, and DIR‐09 recorded the highest ED50 value with a resistance index of 9.21–10.35 after nicosulfuron application. Whereas, the biotypes SNU‐04, OSM‐15, and ADN‐21 were with the highest ED50 value and resistance index of 6.41–7.44, after the application of foramsulfuron + iodosulfuron methyl‐sodium. The increase in genomic 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) copy number has been observed in suspected cases that have been accepted as the molecular basis for the development of resistance against glyphosate. The sequence alignment results for the ALS gene contained Ala122Val and Pro197Arg mutations related to target‐site resistance against ALS herbicides.

Discovery and Mode-of-Action Characterization of a New Class of Acetolactate Synthase-Inhibiting Herbicides.

Herbicides are effective tools to manage weeds and enable food production and sustainable agriculture. Corteva Agriscience R&D has recently discovered new diphenyl-ether compounds displaying excellent postemergent efficacy on important weed species along with corn safety. Here, we describe the chemistry, biology, biochemistry, and computational modeling research that led to the discovery and elucidation of the primary mode of action for these compounds. The target protein was found to be acetolactate synthase (ALS), a key enzyme in the biosynthesis of branched chain amino acids (valine, leucine, and isoleucine). While weed resistance evolution to ALS herbicides is widespread, the molecular interaction of the diphenyl-ether compounds at the active site of the ALS enzyme differs significantly from that of some commercial ALS inhibitors. The unique biochemical profile of these molecules along with their excellent herbicidal activity and corn selectivity make them a noteworthy development in the pursuit of novel, safe, and sustainable weed control solutions.

Diversity and Spread of Acetolactate Synthase Allelic Variants at Position 574 Endowing Resistance in Amaranthus hybridus in Italy

Poor control of Amaranthus spp. with herbicides inhibiting acetolactate synthase (ALS) has been observed for several years in soybean fields in north-eastern Italy, but to date only a few ALS-resistant populations have been confirmed. An extensive sampling of putatively resistant Amaranthus accessions was completed in the Friuli Venezia Giulia region, across an arable land area of about 3000 km2. In total, 58 accessions were tested to confirm their resistance status, recognize the Amaranthus species, identify the mutant ALS alleles endowing the resistance and determine the efficacy of 3 pre-emergence herbicides. Most accessions resulted in cross-resistance to thifensulfuron-methyl and imazamox. Genomic DNA were extracted from single seeds with a newly developed protocol; an allele-specific PCR assay revealed the presence of the 574-leucine in 20 accessions, of the 574-methionine in 22, and of both alleles in 9 accessions. The two variants showed a different spatial distribution. All resistant populations were ascribed to A. hybridus. A. hybridus resistant to ALS herbicides is well-established in this Italian region and its resistance is due to two ALS mutant alleles. Metribuzin, clomazone and metobromuron can be used as alternative herbicides to be applied in pre-emergence and they should be integrated into the management strategies to limit the spread of resistance.

A novel Phe‐206‐Leu mutation in acetolactate synthase confers resistance to penoxsulam in barnyardgrass (Echinochloa crus‐galli (L.) P. Beauv)

Barnyardgrass (Echinochloa crus-galli (L.) P. Beauv) has evolved resistance to the acetolactate synthase (ALS) inhibitor penoxsulam which is used to control weeds in rice fields in China. The present study is conducted to identify the target-site resistance (TSR) mechanisms conferring resistance in a penoxsulam-resistant population. The ALS sensitivity in vitro of the resistant population was sixfold lower to penoxsulam than that of the sensitive population. ALS sequencing revealed that no known mutation conferring ALS herbicide resistance was detected. However, a novel mutation Phe-206-Leu was identified in the ALS gene. Additionally, ALS gene expression level of the resistant population was lower than that of the sensitive population. Therefore, the penoxsulam resistance was not due to the overexpression of ALS gene. Molecular docking revealed that this mutation may change the interaction of the penoxsulam-ALS binding and weaken its mutual affinity by approximately 10%. Arabidopsis thaliana transformed with mutant ALS had fourfold greater resistance to penoxsulam and varied cross-resistance to other ALS herbicides than those transformed with sensitive ALS. Mutant and sensitive ALS proteins expressed by the baculovirus system exhibited different in vitro penoxsulam sensitivity levels. Mutant ALS had eightfold lower sensitivity to penoxsulam than sensitive ALS. This report provides clear evidence that the ALS mutation at position 206 (Phe-206-Leu) confers penoxsulam resistance in barnyardgrass. Phe-206 was confirmed to be the ninth amino acid residue related to ALS herbicide resistance in weeds. © 2022 Society of Chemical Industry.

CYP81A68 confers metabolic resistance to ALS and ACCase-inhibiting herbicides and its epigenetic regulation in Echinochloa crus-galli

Long-term and excessive herbicide use has led to some environmental concerns and especially, herbicide resistance evolution in weeds. Here, we confirmed acetolactate synthase (ALS) inhibiting herbicide penoxsulam resistance and cross resistance to acetyl-coenzyme carboxylase (ACCase) inhibiting herbicides (cyhalofop-butyl and metamifop) in a global weed Echinochloa crus-galli population resistant to these herbicides (R). Penoxsulam metabolism study indicated that degradation rate was significantly higher in R than susceptible E. crus-galli population (S). RNA-sequencing revealed that a cytochrome P450 (P450) gene, CYP81A68, expressed higher in R versus S. Rice seedlings overexpressing this CYP81A68 gene are resistant to penoxsulam, cyhalofop-butyl and metamifop, and penoxsulam resistance is due to enhanced metabolism via O-demethylation. Deletion analysis of the CYP81A68 gene promoter identified an efficient region, in which differential methylation of CpG islands occurred between R and S. Collectively, these results demonstrate that upregulation of E. crus-galli CYP81A68 gene endows generalist metabolic resistance to commonly used ALS- and ACCase-inhibiting herbicides in rice fields and epigenetic regulation may play a role in the resistance evolution. This research could contribute to strategies reducing herbicide environmental impacts by judicious selection of alternative herbicide and non-chemical control tactics.

Heterologous expression of CYP81A6 from rice (Oryza sativa) in Escherichia coli and structural analyses of bensulfuron‐methyl metabolites

AbstractHerbicides are often selective and kill only weeds without affecting crops. In rice, CYP81A6 is responsible for tolerance toward acetolactate synthase herbicides such as bensulfuron‐methyl (BSM). CYP81A6 is thought to metabolize BSM into a nontoxic metabolite, that is, O‐demethylated BSM. However, direct evidences for this reaction have not been reported, likely due to difficulties in the heterologous expression of plant P450s in bacterial expression systems. In this study, we established CYP81A6 expression in Escherichia coli via modification of its N‐terminal basic region coding sequence, optimization of cultivation temperature, and selection of E. coli strains. Heterologous production of CYP81A6 was evaluated by the conversion of BSM in autoinduction medium after the culture, compared to that with an empty vector control. The optimized heterologous production condition of rice CYP81A6 in E. coli using 48‐well plates involved replacement of the N‐terminal basic region with that of CYP2C3, the use of E. coli BL21(DE3) as an expression host, and culture at 34°C. Further, co‐expression of gene encoding glucose dehydrogenase enhanced the conversion of 0.5 mM BSM from 58.7 to 73.1% in the optimized condition. In the optimization process, we detected two metabolites, M1 and M2. Mass spectrometry and 1H nuclear magnetic resonance analyses indicated that M1 was O‐demethylated BSM. Although we could not isolate M2 because of its low abundance in the medium, mass spectrometry analysis suggested that it was the monooxygenated (+O) form of BSM. Overall, these results confirm that CYP81A6 catalyzes the formation of O‐demethylated BSM from BSM.

Resistance Mechanism to Metsulfuron-Methyl in Polypogon fugax.

Polypogon fugax is a common winter weed in China and other Asia countries. We have previously found a P. fugax biotype (R) resistant to acetyl co-enzyme A carboxylase (ACCase) herbicides also cannot be effectively controlled by some acetolactate synthase (ALS) herbicides. This study evaluated the level of resistance to four ALS herbicides (metsulfuron-methyl, chlorsulfuron, monosulfuron, pyribambenz isopropyl) in the R biotype and the associated resistance mechanism. The R biotype exhibited moderate level of resistance to metsulfuron-methyl (6.0-fold) compared with the sensitive biotype (S). Sequence analysis of ALS gene revealed that two ALS genes existed in P. fugax. However, no substitution associated with ALS resistance mechanism were found in ALS genes between the S and R biotypes. The activity of ALS enzyme isolated from the R biotype was inherently higher and less sensitive to metsulfuron-methyl than the S biotype. Glutathione S-transferases (GST) activity was also less sensitive to metsulfuron-methyl in the R than as the S biotypes. Malathion, a cytochrome P450 (CYP) monooxygenase inhibitor, had much greater synergistic effect with metsulfuron-methyl on the R than as the S plants, reducing the ED50 value (herbicide dose to inhibit growth by 50%) of metsulfuron-methyl by 23- and 6-fold, respectively, suggesting that CYP mediated enhanced metabolism might contribute to the resistance to ALS herbicides. These results suggest that metsulfuron-methyl resistance in the R biotype was associated with the up-regulated ALS enzymatic activity and the GST and CYP-mediated enhanced herbicide metabolism.

Metabolomic analysis of Schoenoplectus juncoides reveals common markers of acetolactate synthase inhibition among paddy weeds

Despite the increase in pressure for reducing the use of chemical pesticides in agriculture, herbicides remain one of the efficient tools for augmenting food production. Various herbicide-resistant weeds against most herbicidal modes of action (MoA) are emerging worldwide, and therefore, the necessity of developing herbicides with novel MoA is increasing. Toward this, rigid methods of determining MOA that can be applied for various weeds species are required. Despite the existence of weed species with resistance to acetolactate synthase (ALS) inhibitors, inhibition of ALS remains one of the most widely used herbicidal MoAs containing more than 50 commercial active ingredients. Here, we aimed to identify marker metabolites that are indicative of ALS inhibition. We performed non-targeted and targeted metabolomics using ALS inhibitor-treated Schoenoplectus juncoides. We identified internal metabolite markers for ALS inhibition, with excellent selectivity for ALS inhibitors and herbicides with different MOAs in various weed species. These markers will enable us to evaluate ALS inhibitory activity of chemicals in vivo in a wide variety of weed species.

Target-site mutation and enhanced metabolism confer resistance to thifensulfuron-methyl in a multiple-resistant redroot pigweed (Amaranthus retroflexus) population

AbstractRedroot pigweed (Amaranthus retroflexus L.) is a troublesome dicot weed species widely distributed across China. A population of A. retroflexus that survived the recommended label rate of thifensulfuron-methyl was collected from the main soybean [Glycine max (L.) Merr.] production area in China. Whole-plant dose–response assays indicated that the resistant (R) population was highly resistant (61.80-fold) to thifensulfuron-methyl compared with the susceptible (S1 and S2) populations. In vitro acetolactate synthase (ALS) activity experiments showed that the thifensulfuron-methyl I50 value for the R population was 40.17 times higher than that for the S1 population. A preliminary malathion treatment study indicated that the R population might have cytochrome P450–mediated metabolic resistance. The R population exhibited a high level of cross-resistance to representative ALS herbicides (imazethapyr, flumetsulam, and bispyribac-sodium) and multiple resistance to the commonly used protoporphyrinogen oxidase (PPO)-inhibiting herbicides lactofen and fomesafen. Two common mutations, Trp-574-Leu in ALS and Arg-128-Gly in PPO2, were identified within the R population. This study identified possible enhanced metabolism of thifensulfuron-methyl coexisting with target-site mutations in both ALS and PPO2 in a multiple-resistant A. retroflexus population.

Mayweed chamomile (Anthemis cotula L.) biology and management—A review of an emerging global invader

AbstractThe globally invasive weed, mayweed chamomile (Anthemis cotula L.) is an annual, bushy, ill‐scented herb, originating in Eurasia. It is aggressively weedy in croplands, field‐side ditches, wet areas and along roadsides, especially in slightly acidic, nitrogen‐rich, clay‐loam soils. In addition to interfering with crop growth, the weed causes dermatitis and eye irritation in humans, skin rashes and sour mouth in domesticated mammals, and off‐flavours in cow's milk through forage contamination. Although A. cotula may be managed effectively with some broadleaf herbicides in cereal crops, it is much more difficult to manage in broadleaved crops and cover crops. In some regions, aggressive use of relatively few herbicides has selected for acetolactate synthase herbicide resistance in A. cotula. Therefore, A. cotula poses serious ecological and economic challenges in many regions. Despite this threat, the details of A. cotula biology and ecology in the context of environmental change remain relatively unknown. For example, the reason for the invasiveness of A. cotula remains uncertain, but is presumed to be due to abundant seed production, allelochemicals, mycorrhizal associations, minimal herbivory in invaded ranges, ability to attract generalist pollinators through an extended flowering period and adaptive capacity in a wide range of soil types and conditions. In this paper, we comprehensively review the literature and ongoing work on A. cotula and provide a research agenda to improve understanding and management of this species.

Evolving Multiple Resistance to EPSPS, GS, ALS, PSI, PPO, and Synthetic Auxin Herbicides in Dominican Republic Parthenium hysterophorus Populations. A Physiological and Biochemical Study

Two Parthenium hysterophorus populations resistant (R) and susceptible (S) harvested in banana crop from the Dominican Republic were studied. All S plants died when the herbicides were applied at field dose, except with paraquat. For the R population, the order of plant survival was as follows: glyphosate and paraquat > flazasulfuron > glufosinate > fomesafen > 2,4-D. The resistance factors obtained in the dose–response assays showed a high resistance to glyphosate, flazasulfuron, and fomesafen, medium resistance to glufosinate and 2,4-D, and a natural tolerance to paraquat (resistance factor (RF) = 1.0). The I50 values obtained in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetolactate synthase (ALS), and glutamine synthetase (GS) activity studies with glyphosate, flazasulfuron, and glufosinate, respectively, were greater in R than in S. The effect of fomesafen was measured by the Proto IX levels, obtaining five times more Proto IX in the S than in the R population. The resistance to 2,4-D in the R was determined by the lower accumulation of ethylene compared to the S population. The studies with 14C-paraquat conclude that the lower absorption and translocation in both the R and S populations would explain the natural tolerance of P. hysterophorus. This is the first case of multiple resistance to herbicides with different mechanisms of action confirmed in P. hysterophorus.

Detection and characterization of resistance to acetolactate synthase inhibiting herbicides in Anisantha and Bromus species in the United Kingdom.

Anisantha and Bromus spp. are widespread and difficult to control, potentially due to the evolution of herbicide resistance. In this study, UK populations of four brome species have been tested for the early development of resistance to acetolactate synthase (ALS)-inhibiting herbicides commonly used in their control. Glasshouse assays confirmed reduced sensitivity to ALS-inhibiting herbicides in single populations of A. diandra, B. commutatus and B. secalinus, and in three populations of A. sterilis. By contrast, all 60 brome populations tested were sensitive to the ACCase-inhibiting herbicide propaquizafop and glyphosate. Dose-response with two ALS herbicides showed broad-ranging resistance in the A. diandra, A. sterilis and B. commutatus populations. In the B. commutatus population, this was associated with a point mutation in the ALS enzyme conferring target site resistance (TSR). Additionally, resistant populations of A. sterilis and B. commutatus populations contained enhanced amounts of an orthologue of the glutathione transferase phi (F) class 1 (GSTF1) protein, a functional biomarker of nontarget site resistance (NTSR) in Alopecurus myosuroides. There was further evidence of NTSR as these plants also demonstrated an enhanced capacity to detoxify herbicides. This study confirms the evolution of resistance to ALS inhibiting herbicides in brome species in the UK by mechanisms consistent with the evolution of both TSR and NTSR. These findings point to the need for increased vigilance in detecting and mitigating against the evolution of herbicide resistance in brome species in Northern Europe. © 2020 Society of Chemical Industry.

Multiple mechanisms are involved in new imazamox-resistant varieties of durum and soft wheat

Weed control in wheat is one of the major goals of farmers in their efforts toward obtaining the highest crop yields for human foods. Several studies (dose-response, enzyme activity, absorption-translocation and metabolism) were conducted to characterize the resistance level of two new wheat cultivars called Rafalín (Triticum aestivum) and Antoñín (T. durum) that were obtained by conventional breeding based on Clearfield® technology; they are resistant (R) to imazamox compared to their sensitive (S) counterparts (Gazul and Simeto, respectively). The R-cultivars were 93.7-fold (Rafalín) and 43.7-fold (Antoñín) more resistant than their respective S-cultivars. The acetolactate synthase (ALS) enzyme activity revealed high resistance to imidazolinone (IMI) herbicides in R-cultivars, but no cross-resistance to other ALS herbicides was found. The Ser653Asn mutation that confers resistance to IMI herbicides was identified in the imi1 and imi2 genes of Rafalín and only in the imi1 gene of Antoñín. The 14C-imazamox absorption did not differ between the R- and S-cultivars. Imazamox was metabolized by Cyt-P450 into imazamox-hydroxyl and imazamox-glucoside in the R-cultivars, altering their translocation patterns. The differential sensitivity to imazamox between R-cultivars was due to the number of resistance genes that carry each genotype. The R-cultivars Rafalín and Antoñín could be excellent weed control tools.

Annual Bluegrass Herbicide Resistance Caused by ALS Gene Mutation in Korea

Acetolactate synthase (ALS)‐inhibiting herbicide is widely used for control of annual bluegrass (Poa annua L.) infestation in zoysiagrass (Zoysia spp.) fairways. Field experiments were conducted 2015 and 2016 to investigate ALS herbicide resistance of annual bluegrass at the Ansung Benest Golf Club in Korea, where application of trifloxysulfuron (0.008 g m−2) resulted in poor control for 2 yr. Control of annual bluegrass was significantly less (<34%) in flazasulfuron, foramsulfuron, and trifloxysulfuron treatments than in asulam treatment (83%). In dose‐response experiments, the resistant annual bluegrass survived (>80%) 40 d after treatment in flazasulfuron, foramsulfuron, and trifloxysulfuron treatments, which were applied 10 times at labeled rates. DNA sequencing results indicated that the resistance of annual bluegrass was associated with the tryptophan to leucine target‐site mutation at ALS codon position 574. These results confirm the risk of annual bluegrass herbicide resistance at golf courses in Korea and the need for new effective herbicides that may be useful in delaying or avoiding herbicide resistance.

Commercial Safened ALS‐herbicide Formulation Effects on ALS‐sensitive Field Corn Hybrids

Core Ideas Corn hybrid sensitivity to ALS herbicides is an issue. Herbicide safeners may not provide complete crop safety. Growers who want to use safened ALS herbicides on sensitive corn hybrids must be aware of yield loss potential. Research was conducted in 2014 and 2015 to determine if two common commercial acetolactate synthase (ALS) inhibitor herbicide formulations, that contain the crop safener isoxadifen, could be used on field corn (Zea mays L.) hybrids with reported ALS sensitivity. Two ALS‐sensitive field corn hybrids (DEKALB DKC 62‐08 and DKC 64‐69) were treated 18 to 21 days after planting with nicosulfuron + rimsulfuron + isoxadifen or thiencarbazone + tembotrione + isoxadifen at 1× and 2× labeled rates. No interactions were observed between herbicide treatment and field corn hybrid. When averaged over herbicide treatment, DKC 62‐08 produced less aboveground biomass and was shorter in height than DKC 64‐69. However, no difference in grain yield between hybrids was observed. When averaged over corn hybrid, only the 2× rate of tembotrione + thiencarbazone methyl + isoxadifen caused a significant reduction (19%) in aboveground biomass. Nicosulfuron + rimsulfuron + isoxadifen and tembotrione + thiencarbazone + isoxadifen, at both 1× and 2× rates, reduced plant height up to 3 inches 26 days after treatment (DAT). However, only the 2× rates of these safened herbicides caused significant height reductions 62 DAT. Both herbicide formulations at 1× and 2× labeled rates caused corn yield losses of 11 to 16 bu/acre. These data suggest that these particular commercial herbicide formulations can cause crop injury and subsequent yield loss when used on ALS‐sensitive field corn hybrids.

Non-target-site resistance to ALS-inhibiting herbicides in a Sagittaria trifolia L. population

Sagittaria trifolia L. is one of the most competitive weeds in rice fields in northeastern China. The continuous use of acetolactate synthase (ALS)-inhibitors has led to the evolution of herbicide resistant S. trifolia. A subpopulation BC1, which was derived from the L1 population, was analyzed using DNA sequencing and ALS enzyme activity assays and levels of resistance to five ALS-inhibiting herbicides was determined. DNA sequencing and ALS enzyme assays revealed no amino acid substitutions and no significant differences in enzyme sensitivity between susceptible and resistant populations. Whole-plant dose-response experiments showed that the BC1 population exhibited different levels of resistance (resistance ratios ranging from 2.14 to 51.53) to five ALS herbicides, and the addition of malathion (P450 inhibitor) to bensulfuron-methyl, penoxsulam and bispyribac-sodium strongly reduced the dry weight accumulation of the BC1 population compared with the effects of the three herbicides alone. The results of the present study demonstrated that the BC1 population has evolved non-target-site resistance to ALS-inhibiting herbicides.

Bensulfuron-methyl resistant Sagittaria trifolia L.: Multiple resistance, cross-resistance and molecular basis of resistance to acetolactate synthase-inhibiting herbicides

Acetolactate synthase (ALS)-inhibiting herbicides play an important role in controlling broad-leaved weeds. Populations of Sagittaria trifolia L. showed resistance to ALS-inhibiting sulfonylurea herbicides (e.g. bensulfuron-methyl) in paddy fields in the northeast of China. In our study, whole-plant bioassays were performed on eight suspected resistant S. trifolia populations that showed high levels of resistance to bensulfuron-methyl, with resistance indices from 31.06 to 120.35. The results of ALS-activity assays were consistent with the observed whole-plant dose-response data. This confirmed that resistant populations displayed significantly higher ALS activity than the sensitive population due to prevention of normal enzyme-herbicide interaction. The mutations Pro-197-Ser, Pro-197-His, Pro-197-Thr and Pro-197-Leu were identified in the ALS gene of resistant populations. Pro-197-His and Pro-197-Thr mutations conferring resistance to bensulfuron-methyl are reported for the first time in S. trifolia. All resistant populations were resistant to sulfonylurea (SU) herbicides, but not to imidazolinone (IMI) herbicides. HLJ-5 and JL-3 populations were resistant to bispyribac-sodium of the pyrimidinyl-thiobenozoate (PTB) class of ALS herbicides, JL-2 to penoxsulam of triazolopyrimidine (TP) class and JL-1 to pyribenzoxim, also of PTB class. The eight S. trifolia populations were susceptible to other herbicide modes of action tested.

Multiple Mechanisms Increase Levels of Resistance in Rapistrum rugosum to ALS Herbicides.

Rapistrum rugosum (turnip weed) is a common weed of wheat fields in Iran, which is most often controlled by tribenuron-methyl (TM), a sulfonylurea (SU) belonging to the acetolactate synthase (ALS) inhibiting herbicides group. Several cases of unexplained control failure of R. rugosum by TM have been seen, especially in Golestan province-Iran. Hence, there is lack of research in evaluation of the level of resistance of the R. rugosum populations to TM, using whole plant dose-response and enzyme assays, then investigating some potential resistance mechanisms Results revealed that the resistance factor (RF) for resistant (R) populations was 2.5–6.6 fold higher than susceptible (S) plant. Neither foliar retention, nor 14C-TM absorption and translocation were the mechanisms responsible for resistance in turnip weed. Metabolism of TM was the second resistant mechanism in two populations (Ag-R5 and G-1), in which three metabolites were found. The concentration of TM for 50% inhibition of ALS enzyme activity in vitro showed a high level of resistance to the herbicide (RFs were from 28 to 38) and cross-resistance to sulfonyl-aminocarbonyl-triazolinone (SCT), pyrimidinyl-thiobenzoate (PTB) and triazolopyrimidine (TP), with no cross-resistance to imidazolinone (IMI). Substitution Pro 197 to Ser 197 provided resistance to four of five ALS-inhibiting herbicides including SU, TP, PTB, and SCT with no resistance to IMI. These results documented the first case of R. rugosum resistant population worldwide and demonstrated that both RST and NRST mechanisms are involved to the resistance level to TM.

Molecular basis of multiple resistance to ACCase- and ALS-inhibiting herbicides in Alopecurus japonicus from China

Fenoxaprop-P-ethyl-resistant Alopecurus japonicus has become a recurring problem in winter wheat fields in eastern China. Growers have resorted to using mesosulfuron-methyl, an acetolactate synthase (ALS)-inhibiting herbicide, to control this weed. A single A. japonicus population (AH-15) resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl was found in Anhui Province, China. The results of whole-plant dose–response experiments showed that AH-15 has evolved high-level resistance to fenoxaprop-P-ethyl (95.96-fold) and mesosulfuron-methyl (39.87-fold). It was shown via molecular analysis that resistance to both fenoxaprop-P-ethyl and mesosulfuron-methyl was due to an amino acid substitution of Ile1781 to Leu in acetyl-CoA carboxylase (ACCase) and a substitution of Trp 574 to Leu in ALS, respectively. Whole-plant bioassays indicated that the AH-15 population was resistant to the ACCase herbicides clodinafop-propargyl, clethodim, sethoxydim and pinoxaden as well as the ALS herbicides pyroxsulam, flucarbazone-Na and imazethapyr, but susceptible to the ACCase herbicide haloxyfop-R-methyl. This work reports for the first time that A. japonicus has developed resistance to ACCase- and ALS-inhibiting herbicides due to target site mutations in the ACCase and ALS genes.

Common Cocklebur (<i>Xanthium strumarium</i>) Response to Nicosulfuron

The response of two populations (CC1, 43.59°N & 20.40°E; CC2, 44.46°N & 20.17°E) of common cocklebur (Xanthium strumarium L.) to nicosulfuron was investigated both in field experiments and in the laboratory. Population CC1 had no history of treatment with any herbicide, while population CC2 was treated with ALS inhibitor herbicides for six consecutive years. In the field, plants were treated post-emergence with nicosulfuron (0, 10, 20, 40, 60 and 80 g ai ha-1) at four true leaves. Visual injury estimation and vegetative parameters (plant height, fresh weight, leaf area) were recorded about month after herbicide application. The acetolactate synthase (ALS) enzyme activity in response to herbicide concentrations of 0, 0.01, 0.1, 1, 10, 100 μM was determined in vitro. GR50 values for vegetative parameters and I50 values for ALS activity were slightly greater for the CC2 than for the CC1population, but the results confirmed that neither population was susceptible to nicosulfuron. Namely, based on results for fresh weight, the population CC1 was about 3.9 and 2.6-fold more susceptible to nicosulfuron than population CC2 in two consecutive years, but differences were not so prominent for other parameters (plant height, leaf area and ALS activity), ranging from 1.18 to 1.8-fold. The differences between population CC1 and CC2 could be attributed to inter-population variability in susceptibility to nicosulfuron or could be the consequence of repeated application of ALS herbicides to the CC2 population during the six previous years. Future investigations are necessary in order to clarify this dilemma.