Black-grass Populations Research Articles

The role of interspecific variability and herbicide pre-adaptation in the cinmethylin response of Alopecurus myosuroides.

Cinmethylin is an inhibitor of plant fatty acid biosynthesis, with in-plant activity caused by its binding to fatty acid thioesterases (FATs). The recent registration of cinmethylin for pre-emergence herbicidal use in the UK represents a new mode-of-action (MOA) for control of the grassweed blackgrass (Alopecurus myosuroides). To date there is little published information on the extent of blackgrass' inter-population variability in sensitivity to cinmethylin, nor on any potential effect of existing non-target-site resistance (NTSR) mechanisms on cinmethylin efficacy. Here we present a study of variability in cinmethylin sensitivity amongst 97 UK blackgrass populations. We demonstrate that under controlled conditions, a UK field-rate dose of 500 g ha-1 provides effective control of the tested populations. Nevertheless, we reveal significant inter-population variability at doses below this rate, with populations previously characterised as strongly NTSR displaying the lowest sensitivity to cinmethylin. Assessment of paired resistant 'R' and sensitive 'S' lines from standardised genetic backgrounds confirms that selection for NTSR to the acetyl-CoA-carboxylase inhibitor fenoxaprop, and the microtubule assembly inhibitor pendimethalin, simultaneously results in reduced sensitivity to cinmethylin at doses below 500 g ha-1. Whilst we find no resistance to the field-rate dose, we reveal that cinmethylin sensitivity can be further reduced through experimental selection with cinmethylin. Cinmethylin therefore represents a much-needed further MOA for blackgrass control, but needs to be carefully managed within a resistance monitoring and integrated weed management (IWM) framework to maximise the effective longevity of this compound. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

RNA and protein biomarkers for detecting enhanced metabolic resistance to herbicides mesosulfuron-methyl and fenoxaprop-ethyl in black-grass (Alopecurus myosuroides).

The evolution of non-target site resistance (NTSR) to herbicides leads to a significant reduction in herbicide control of agricultural weed species. Detecting NTSR in weed populations prior to herbicide treatment would provide valuable information for effective weed control. While not all NTSR mechanisms have been fully identified, enhanced metabolic resistance (EMR) is one of the better studied, conferring tolerance through increased herbicide detoxification. Confirming EMR towards specific herbicides conventionally involves detecting metabolites of the active herbicide molecule in planta, but this approach is time-consuming and requires access to well-equipped laboratories. In this study, we explored the potential of using molecular biomarkers to detect EMR before herbicide treatment in black-grass (Alopecurus myosuroides). We tested the reliability of selected biomarkers to predict EMR and survival after herbicide treatments in both reference and 27 field-derived black-grass populations collected from sites across the UK. The combined analysis of the constitutive expression of biomarkers and metabolism studies confirmed three proteins, namely, AmGSTF1, AmGSTU2 and AmOPR1, as differential biomarkers of EMR toward the herbicides fenoxaprop-ethyl and mesosulfuron in black-grass. Our findings demonstrate that there is potential to use molecular biomarkers to detect EMR toward specific herbicides in black-grass without reference to metabolism analysis. However, biomarker development must include testing at both transcript and protein levels in order to be reliable indicators of resistance. This work is a first step towards more robust resistance biomarker development, which could be expanded into other herbicide chemistries for on-farm testing and monitoring EMR in uncharacterised black-grass populations. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sequence Characterization of Extra-Chromosomal Circular DNA Content in Multiple Blackgrass (Alopecurus myosuroides) Populations.

Alopecurus myosuroides (blackgrass) is a problematic weed of Western European winter wheat, and its success is largely due to widespread multiple-herbicide resistance. Previous analysis of F2 seed families derived from two distinct blackgrass populations exhibiting equivalent non-target site resistance (NTSR) phenotypes shows resistance is polygenic and evolves from standing genetic variation. Using a CIDER-seq pipeline, we show that herbicide-resistant (HR) and herbicide-sensitive (HS) F3 plants from these F2 seed families as well as the parent populations they were derived from carry extra-chromosomal circular DNA (eccDNA). We identify the similarities and differences in the coding structures within and between resistant and sensitive populations. Although the numbers and size of detected eccDNAs varied between the populations, comparisons between the HR and HS blackgrass populations identified shared and unique coding content, predicted genes, and functional protein domains. These include genes related to herbicide detoxification such as Cytochrome P450s, ATP-binding cassette transporters, and glutathione transferases including AmGSTF1. eccDNA content was mapped to the A. myosuroides reference genome, revealing genomic regions at the distal end of chromosome 5 and the near center of chromosomes 1 and 7 as regions with a high number of mapped eccDNA gene density. Mapping to 15 known herbicide-resistant QTL regions showed that the eccDNA coding sequences matched twelve, with four QTL matching HS coding sequences; only one region contained HR coding sequences. These findings establish that, like other pernicious weeds, blackgrass has eccDNAs that contain homologs of chromosomal genes, and these may contribute genetic heterogeneity and evolutionary innovation to rapidly adapt to abiotic stresses, including herbicide treatment.

Recombinant glutathione transferases from flufenacet-resistant black-grass (Alopecurus myosuroides Huds.) form different flufenacet metabolites and differ in their interaction with pre- and post-emergence herbicides.

Black-grass (Alopecurus myosuroides Huds.) has become a problematic weed in cereals in Europe. Besides resistance to post-emergent herbicides becoming increasingly widespread, enhanced metabolism of inhibitors of the synthesis of very-long-chain fatty acids (VLCFAs), such as flufenacet, is evolving. Yet, cross-resistance patterns and evolution of this resistance remains poorly understood. The cDNA sequences of five glutathione transferases (GSTs) upregulated in flufenacet resistant black-grass were identified and used for recombinant protein expression. Moderate to slow detoxification of flufenacet was verified for all candidate GSTs expressed in E. coli, and the most active protein produced flufenacet-alcohol instead of a glutathione conjugate, in the presence of reduced glutathione (GSH). Moreover, cross-resistance to other VLCFA-inhibitors e.g., acetochlor and pyroxasulfone and the ACCase inhibitor fenoxaprop was verified in vitro. Various other herbicides of different modes of action including VLCFA-inhibitors were not detoxified by the candidate GSTs. As several in planta upregulated GSTs detoxified flufenacet in vitro, the shift in sensitivity observed in black-grass populations, is likely a result of an additive effect. The polygenic character and the relatively low turnover rate of the individual GSTs may explain the slow evolution of flufenacet resistance. In addition, flufenacet resistance was accompanied by cross-resistance with some, but not all, herbicides of the same mode of action, and furthermore to the ACCase inhibitor fenoxaprop-ethyl. Hence, not only the rotation of herbicide modes of action, but also of individual active ingredients is important for resistance management. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

ABC transporters linked to multiple herbicide resistance in blackgrass (Alopecurus myosuroides).

Enhanced detoxification is a prominent mechanism protecting plants from toxic xenobiotics and endows resistance to diverse herbicide chemistries in grass weeds such as blackgrass (Alopecurus myosuroides). The roles of enzyme families which impart enhanced metabolic resistance (EMR) to herbicides through hydroxylation (phase 1 metabolism) and/or conjugation with glutathione or sugars (phase 2) have been well established. However, the functional importance of herbicide metabolite compartmentalisation into the vacuole as promoted by active transport (phase 3), has received little attention as an EMR mechanism. ATP-binding cassette (ABC) transporters are known to be important in drug detoxification in fungi and mammals. In this study, we identified three distinct C-class ABCCs transporters namely AmABCC1, AmABCC2 and AmABCC3 in populations of blackgrass exhibiting EMR and resistance to multiple herbicides. Uptake studies with monochlorobimane in root cells, showed that the EMR blackgrass had an enhanced capacity to compartmentalize fluorescent glutathione-bimane conjugated metabolites in an energy-dependent manner. Subcellular localisation analysis using transient expression of GFP-tagged AmABCC2 assays in Nicotiana demonstrated that the transporter was a membrane bound protein associated with the tonoplast. At the transcript level, as compared with herbicide sensitive plants, AmABCC1 and AmABCC2 were positively correlated with EMR in herbicide resistant blackgrass being co-expressed with AmGSTU2a, a glutathione transferase (GST) involved in herbicide detoxification linked to resistance. As the glutathione conjugates generated by GSTs are classic ligands for ABC proteins, this co-expression suggested AmGSTU2a and the two ABCC transporters delivered the coupled rapid phase 2/3 detoxification observed in EMR. A role for the transporters in resistance was further confirmed in transgenic yeast by demonstrating that the expression of either AmABCC1 or AmABCC2, promoted enhanced tolerance to the sulfonylurea herbicide, mesosulfuron-methyl. Our results link the expression of ABCC transporters to enhanced metabolic resistance in blackgrass through their ability to transport herbicides, and their metabolites, into the vacuole.

The blackgrass genome reveals patterns of non-parallel evolution of polygenic herbicide resistance.

Globally, weedy plants are a major constraint to sustainable crop production. Much of the success of weeds rests with their ability to rapidly adapt in the face of human-mediated management of agroecosystems. Alopecurus myosuroides (blackgrass) is a widespread and impactful weed affecting agriculture in Europe. Here we report a chromosome-scale genome assembly of blackgrass and use this reference genome to explore the genomic/genetic basis of non-target site herbicide resistance (NTSR). Based on our analysis of F2 seed families derived from two distinct blackgrass populations with the same NTSR phenotype, we demonstrate that the trait is polygenic and evolves from standing genetic variation. We present evidence that selection for NTSR has signatures of both parallel and non-parallel evolution. There are parallel and non-parallel changes at the transcriptional level of several stress- and defence-responsive gene families. At the genomic level, however, the genetic loci underpinning NTSR are different (non-parallel) between seed families. We speculate that variation in the number, regulation and function of stress- and defence-related gene families enable weedy species to rapidly evolve NTSR via exaptation of genes within large multi-functional gene families. These results provide novel insights into the potential for, and nature of plant adaptation in rapidly changing environments.

Molecular Basis of Resistance to Mesosulfuron-Methyl in a Black-Grass (Alopecurus myosuroides Huds.) Population from China

Black-grass (Alopecurus myosuroides Huds.) is a common weed in Chinese wheat fields, and has become troublesome due to its evolution of herbicide resistance. One black-grass population (HN-14) collected from a wheat field where herbicides were applied was suspected to be resistant (R) to mesosulfuron-methyl. This study aims to establish a cross-resistance pattern and explore potential resistance mechanisms. The results of a whole-plant dose response assay showed that the resistant (R) population had a high of resistance to mesosulfuron-methyl (33-fold); meanwhile, no synergism of P450s activity inhibitor malathion was observed. The sequencing results revealed that ALS resistance mutation Trp-574-Leu occurred in R plants. The results of in vitro ALS enzyme activity assays also supported that the extractable ALS from R plants were 23.22-fold resistant to mesosulfuron-methyl. In the light of the “R” resistance rating system, HN-14 has evolved RRR and RR resistance to fenoxaprop-P-ethyl, clodinafop-propargyl, haloxyfop-methyl, and fluazifop-P-butyl and R? (resistance may be developing) to pinoxaden, however remains sensitive to imazethapyr, quizalofop-P-ethyl, tralkoxydim, and isoproturon. These results indicated that the mesosulfuron-methyl resistance in the black-grass population HN-14 was driven by a target-site mechanism rather than a nontarget (at least P450s-mediated) mechanism.

A novel naturally Phe206Tyr mutation confers tolerance to ALS-inhibiting herbicides in Alopecurus myosuroides

Herbicide-resistant weeds pose a serious threat to world food production. The rapid and widespread development of target-site based resistance limits the application of herbicides. Alopecurus myosuroides Huds. (blackgrass) has spread rapidly in winter wheat regions in China, and the field recommended dose of ALS herbicides no longer controls blackgrass populations in recent years. A highly resistant population TW18(R) was collected in 2018 from Shandong Province. Dose-response assays showed that the TW18 was resistant to mesosulfuron-methyl, flucarbazone-sodium, and imazethapyr, with resistance index values of 5.96, 6.1, and 4.09, respectively. DNA sequencing of the TW18 population revealed a Phe206Tyr (F206Y) mutation in the ALS, which was not yet reported. Blackgrass ALS gene with the F206Y mutation (R gene) was expressed in Arabidopsis and rice. Transgenic studies have shown that both Arabidopsis and rice expressing this R gene have resistance to imazethapyr. However, it did not confer resistance to tribenuron methyl and florasulam in transgenic Arabidopsis. This study showed that the F206Y substitution caused herbicide resistance in blackgrass. To our knowledge, this is the first-reported F206Y mutation of a weed species in the natural environment. Transgenic plants showed this functional site could be utilized to generate imazethapyr-resistant rice to control herbicide-resistant weed damage.

Non-target Site Herbicide Resistance Is Conferred by Two Distinct Mechanisms in Black-Grass (Alopecurus myosuroides).

Non-target site resistance (NTSR) to herbicides in black-grass (Alopecurus myosuroides) results in enhanced tolerance to multiple chemistries and is widespread in Northern Europe. To help define the underpinning mechanisms of resistance, global transcriptome and biochemical analysis have been used to phenotype three NTSR black-grass populations. These comprised NTSR1 black-grass from the classic Peldon field population, which shows broad-ranging resistance to post-emergence herbicides; NTSR2 derived from herbicide-sensitive (HS) plants repeatedly selected for tolerance to pendimethalin; and NTSR3 selected from HS plants for resistance to fenoxaprop-P-ethyl. NTSR in weeds is commonly associated with enhanced herbicide metabolism catalyzed by glutathione transferases (GSTs) and cytochromes P450 (CYPs). As such, the NTSR populations were assessed for their ability to detoxify chlorotoluron, which is detoxified by CYPs and fenoxaprop-P-ethyl, which is acted on by GSTs. As compared with HS plants, enhanced metabolism toward both herbicides was determined in the NTSR1 and NTSR2 populations. In contrast, the NTSR3 plants showed no increased detoxification capacity, demonstrating that resistance in this population was not due to enhanced metabolism. All resistant populations showed increased levels of AmGSTF1, a protein functionally linked to NTSR and enhanced herbicide metabolism. Enhanced AmGSTF1 was associated with increased levels of the associated transcripts in the NTSR1 and NTSR2 plants, but not in NTSR3, suggestive of both pre- and post-transcriptional regulation. The related HS, NTSR2, and NTSR3 plants were subject to global transcriptome sequencing and weighted gene co-expression network analysis to identify modules of genes with coupled regulatory functions. In the NTSR2 plants, modules linked to detoxification were identified, with many similarities to the transcriptome of NTSR1 black-grass. Critical detoxification genes included members of the CYP81A family and tau and phi class GSTs. The NTSR2 transcriptome also showed network similarities to other (a)biotic stresses of plants and multidrug resistance in humans. In contrast, completely different gene networks were activated in the NTSR3 plants, showing similarity to the responses to cold, osmotic shock and fungal infection determined in cereals. Our results demonstrate that NTSR in black-grass can arise from at least two distinct mechanisms, each involving complex changes in gene regulatory networks.

Flufenacet activity is affected by GST inhibitors in blackgrass (Alopecurus myosuroides) populations with reduced flufenacet sensitivity and higher expression levels of GSTs

AbstractThe WSSA Group 15 (HRAC Group K3) herbicide flufenacet is a key compound in weed resistance management, primarily used for PRE control of grass weeds in winter cereal–based crop rotations in Europe. Although resistance to compounds of its mechanism of action (inhibition of the synthesis of very-long-chain fatty acids) generally evolves slowly, reduced flufenacet efficacy due to enhanced glutathione transferase (GST) activity has been described in several blackgrass (Alopecurus myosuroides Huds.) populations. The present study aimed to better understand of the mechanism of flufenacet detoxification in A. myosuroides. Therefore, we characterized four A. myosuroides populations with different levels of flufenacet sensitivity. Flufenacet degradation was significantly slowed down in a sensitive population and a population with reduced flufenacet sensitivity by the use of the GST inhibitors tridiphane and ethacrynic acid at sublethal rates. Finally, an RNA sequencing (RNA-seq) study with the four A. myosuroides populations was conducted. In total, six differentially expressed GSTs and nine transcription factors as well as a keto-acyl-CoA reductase involved in the biosynthesis of very-long-chain fatty acids were identified as candidate genes among a set of 319 significantly more highly expressed gene-associated contigs. Among a set of 218 contigs with significantly lower expression levels, receptor kinase activity was the most frequent annotation. In summary, the likely GST-mediated reduction in sensitivity evolves in A. myosuroides at a slow rate and can partially be reversed by an interaction between flufenacet and the GST inhibitors tridiphane and ethacrynic acid. This provides further evidence for enhanced GST activity as a key mechanism in flufenacet resistance in A. myosuroides and supports the hypothesis that the six differentially expressed GSTs detected in the present RNA-seq study are potentially involved in flufenacet resistance.

Enhanced metabolism causes reduced flufenacet sensitivity in black-grass (Alopecurus myosuroides Huds.) field populations.

Black-grass (Alopecurus myosuroides Huds.) is a frequent grass weed that commonly occurs in winter wheat in temperate Europe. Evolving resistance to post-emergence herbicides, e.g. acetyl CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors requires more complex weed management strategies and ensuring good efficacy of pre-emergence treatments becomes increasingly important. Flufenacet, in particular, has become a key herbicide for the control of multiple-resistant A. myosuroides. However, in some of those populations, reduced flufenacet efficacy was already observed. In a screening of black-grass populations from several European countries, most populations were controlled with the registered field rate of flufenacet. However, differences in the level of flufenacet sensitivity were observed and correlated with glutathione S-transferase-mediated enhanced flufenacet metabolism. The efficacy of the pre-emergence herbicides pendimethalin, prosulfocarb, S-metolachlor and pethoxamid, was also significantly decreased in populations with reduced flufenacet sensitivity. The use of flufenacet in mixtures with diflufenican, particularly in combination with flurtamone or metribuzin, however, significantly improved efficacy in less susceptible black-grass populations. In several populations of different European origins, reduced efficacy of flufenacet was observed due to enhanced metabolism. Although differences between populations were relatively small, best weed management practices (e.g. application of full dose rates and herbicide mixtures and wide crop rotations) should be applied to reduce selection pressure and prevent flufenacet resistance from further evolving. This is particularly important as flufenacet is one of the few still-effective herbicides suitable for the control of multiple-resistant A. myosuroides genotypes in Europe, whereas alternative pre-emergence herbicides were less effective against multiple-resistant A. myosuroides populations. © 2019 Society of Chemical Industry.

Weed Suppressive Ability of Cover Crop Mixtures Compared to Repeated Stubble Tillage and Glyphosate Treatments

The utilization of an effective stubble management practice can reduce weed infestation before and in the following main crop. Different strategies can be used, incorporating mechanical, biological, and chemical measures. This study aims at estimating the effects of cover crop (CC) mixtures, various stubble tillage methods, and glyphosate treatments on black-grass, volunteer wheat and total weed infestation. Two experimental trials were conducted in Southwestern Germany including seven weed management treatments: flat soil tillage, deep soil tillage, ploughing, single glyphosate application, dual glyphosate application, and a CC mixture sown in a mulch-till and no-till system. An untreated control treatment without any processing was also included. Weed species were identified and counted once per month from October until December. The CC mixtures achieved a black-grass control efficacy of up to 100%, whereas stubble tillage and the single glyphosate treatment did not reduce the black-grass population, on the contrary it induced an increase of black-grass plants. The dual glyphosate application showed, similar to the CC treatments, best results for total weed and volunteer wheat reduction. The results demonstrated, that well developed CCs have a great ability for weed control and highlight that soil conservation systems do not have to rely on chemical weed control practices.

No Vegetative and Fecundity Fitness Cost Associated with Acetyl-Coenzyme A Carboxylase Non-target-site Resistance in a Black-Grass (Alopecurus myosuroides Huds) Population.

Attention should be devoted to weeds evolving herbicide resistance with non-target-site resistance (NTSR) mechanism due to their unpredictable resistance patterns. Quantification of fitness cost can be used in NTSR management strategies to determine the long-term fate of resistant plants in weed populations. To our knowledge, this is the first report evaluating potential fecundity and vegetative fitness of a NTSR black-grass (Alopecurus myosuroides Huds), the most important herbicide resistant weed in Europe, with controlled genetic background. The susceptible (S) and NTSR sub-populations were identified and isolated from a fenoxaprop-P-ethyl resistant population by a plant cloning technique. Using a target-neighborhood design, competitive responses of S and NTSR black-grass sub-populations to increasing density of winter wheat were quantified for 2 years in greenhouse and 1 year in field. Fitness traits including potential seed production, vegetative biomass and tiller number of both sub-populations significantly decreased with increasing density of winter wheat. More importantly, no statistically significant differences were found in fitness traits between S and NTSR sub-populations either grown alone (no competition) or in competition with winter wheat. According to the results, the NTSR black-grass is probably to persist in field even in the cessation of fenoxaprop-P-ethyl. So, effective herbicide resistant management strategies are strongly suggested to prevent and stop the spread of the NTSR black-grass, otherwise NTSR loci conferring resistance to a range of herbicides in black-grass will persist in the gene pool even in the absence of herbicide application. Consequently, herbicide as an effective tool for control of black-grass will gradually be lost in fields infested by NTSR black-grass.

Seed Germination and Seedling Emergence of Blackgrass (Alopecurus myosuroides) as Affected by Non–Target-Site Herbicide Resistance

Seedling emergence traits of susceptible (S) and resistant (R) blackgrass subpopulations isolated from a single non–target-site resistant (NTSR) population were studied in controlled conditions. The seedling emergence of the R subpopulation was lower and slower than that of the S subpopulation, especially at low temperature and deep burial. The burial depth inhibiting final emergence by 50% for the R subpopulation was significantly lower than that of the S subpopulation at low temperature. The present study revealed that under suboptimal conditions the NTSR loci conferring herbicide resistance were correlated with a fitness cost in relation to seedling emergence traits. The results suggest that deep soil cultivation and delayed sowing of autumn-sown crops can hamper germination of the R more than of the S subpopulation and thus potentially reduce the prevalence of the R subpopulation in the blackgrass population.

Transcriptome sequencing identifies novel persistent viruses in herbicide resistant wild-grasses

Herbicide resistance in wild grasses is widespread in the UK, with non-target site resistance (NTSR) to multiple chemistries being particularly problematic in weed control. As a complex trait, NTSR is driven by complex evolutionary pressures and the growing awareness of the role of the phytobiome in plant abiotic stress tolerance, led us to sequence the transcriptomes of herbicide resistant and susceptible populations of black-grass and annual rye-grass for the presence of endophytes. Black-grass (Alopecurus myosuroides; Am) populations, displaying no overt disease symptoms, contained three previously undescribed viruses belonging to the Partititiviridae (AMPV1 and AMPV2) and Rhabdoviridae (AMVV1) families. These infections were widespread in UK black-grass populations and evidence was obtained for similar viruses being present in annual rye grass (Lolium rigidum), perennial rye-grass (Lolium perenne) and meadow fescue (Festuca pratensis). In black-grass, while no direct causative link was established linking viral infection to herbicide resistance, transcriptome sequencing showed a high incidence of infection in the NTSR Peldon population. The widespread infection of these weeds by little characterised and persistent viruses and their potential evolutionary role in enhancing plant stress tolerance mechanisms including NTSR warrants further investigation.

Degradation and Metabolism of Fenoxaprop and Mesosulfuron + Iodosulfuron in Multiple Resistant Blackgrass (Alopecurus myosuroides)

Whole-plant glasshouse experiments were conducted to examine herbicide resistance in selected populations of blackgrass (Alopecurus myosuroides HUDS.). Three populations with either non-target-site resistance (NTSR) or target-site resistance (TSR) showed reduced sensitivity to the herbicides fenoxaprop and mesosulfuron + iodosulfuron. Degradation and metabolism of these herbicides were investigated using a liquid chromatography followed by tandem mass spectrometry. Fenoxaprop degraded in sensitive and resistant populations within 144 h after treatment without significant differences among populations. Fenoxaprop-P, the acid metabolite of fenoxaprop, was found in all populations. The dynamics of fenoxaprop-P differed significantly with an enhanced degradation of the substance in NTSR populations. The metabolite 6-chlorobenzoxazol-2(3H)-one could be detected in all populations 2 h after treatment and degraded almost completely within 144 h.

Resistance profile of herbicide-resistant Alopecurus myosuroides (black-grass) populations in Denmark

Alopecurus myosuroides Huds is one of the most important grass-weeds in North-western Europe and is also the most important herbicide-resistant weed species in European agricultural systems. Fifty-three Danish A. myosuroides populations, previously confirmed to be fenoxaprop-P resistant, were evaluated for five and two known mutation points within the ACCase and ALS genes, respectively. The resistance pattern of 28 out of the 53 populations was investigated to four herbicides using a seed bioassay technique. A whole plant dose response experiment was conducted on seven populations in 2012 and 2013 to evaluate the accuracy of the seed bioassay results. Two resistant populations from the UK and a susceptible population from Denmark were included as reference populations in all experiments. Of the 53 populations, nine (17%) populations were ACCase target site resistant (TSR), all with a mutation at the Ile-1781 position. No mutations conferring TSR to ALS inhibitors were detected in the 53 populations. The seed bioassay results showed that all populations had varying degrees of resistance to fenoxaprop-P. In contrast, all populations were susceptible to cycloxydim suggesting that non-target site resistance (NTSR) was present in all populations including the ones where TSR were found. The seed bioassay was not a suitable method for detecting resistance to the residual herbicides pendimethalin and prosulfocarb. The whole plant dose response experiment results confirmed the results of the seed bioassay for fenoxaprop-P and cycloxydim. Three and two out of seven populations were also resistant to flupyrsulfuron and pendimethalin, respectively, while all resistant populations were susceptible to mesosulfuron + iodosulfuron. The widespread occurrence of particularly NTSR is a severe challenge to the effective management of A. myosuroides. In Denmark this challenge is even more prominent due to few modes of action being available for A. myosuroides control mainly due to national regulation on groundwater protection.

Development of target-site resistance (TSR) in Alopecurus myosuroides in Germany between 2004 and 2012

Alopecurus myosuroides is one of the most important grass weeds on arable land in Germany. For many years, it has developed herbicide-resistant populations. ACCase- and ALS-inhibiting herbicides, in particular, have lost significant efficacy. Two different mechanisms are commonly known to confer resistance to herbicides: target-site resistance (TSR) and non-target-site resistance (NTSR). In Germany, only approximately 5 to 10% of the herbicide-resistant black-grass populations have been reported to show TSR so far. To quantify the status and the distribution of TSR in Germany, 653 black-grass samples were collected between 2004 and 2012 all over the country. The samples were tested by bioassays and genetic analysis. Averaged over all tested samples, ACCase-TSR increased from 5.0% in 2004 to 54.3% in 2011. In 2012, the proportion of ACCase-TSR was lower than the previous years (38.5%). Similarly, ALS-TSR rose from 0.8% in 2007 to 13.9% in 2012. Regions with a high density of resistant black-grass samples were identified in the north and south of Germany. The data suggest a rapidly working selection of herbicide-resistant populations which may be due to current agricultural practices and the increasing use of herbicides acting on a single site.

Role of a Novel I1781T Mutation and Other Mechanisms in Conferring Resistance to Acetyl-CoA Carboxylase Inhibiting Herbicides in a Black-Grass Population

BackgroundKnowledge of the mechanisms of herbicide resistance is important for designing long term sustainable weed management strategies. Here, we have used an integrated biology and molecular approach to investigate the mechanisms of resistance to acetyl-CoA carboxylase inhibiting herbicides in a UK black-grass population (BG2).Methodology/Principal FindingsComparison between BG2 phenotypes using single discriminant rates of herbicides and genotypes based on ACCase gene sequencing showed that the I1781L, a novel I1781T, but not the W2027C mutations, were associated with resistance to cycloxydim. All plants were killed with clethodim and a few individuals containing the I1781L mutation were partially resistant to tepraloxydim. Whole plant dose response assays demonstrated that a single copy of the mutant T1781 allele conferred fourfold resistance levels to cycloxydim and clodinafop-propargyl. In contrast, the impact of the I1781T mutation was low (Rf = 1.6) and non-significant on pinoxaden. BG2 was also characterised by high levels of resistance, very likely non-target site based, to the two cereal selective herbicides clodinafop-propargyl and pinoxaden and not to the poorly metabolisable cyclohexanedione herbicides. Analysis of 480 plants from 40 cycloxydim resistant black grass populations from the UK using two very effective and high throughput dCAPS assays established for detecting any amino acid changes at the 1781 ACCase codon and for positively identifying the threonine residue, showed that the occurrence of the T1781 is extremely rare compared to the L1781 allele.Conclusion/SignificanceThis study revealed a novel mutation at ACCase codon position 1781 and adequately assessed target site and non-target site mechanisms in conferring resistance to several ACCase herbicides in a black-grass population. It highlights that over time the level of suspected non-target site resistance to some cereal selective ACCase herbicides have in some instances surpassed that of target site resistance, including the one endowed by the most commonly encountered I1781L mutation.

Comparison of different black-grass populations ( Alopecurus myosuroides Huds.) in their susceptibility to herbicides under field conditions

In autumn 2009 and 2010, black-grass samples from different regions in Germany were sown into a field near Munster. The sandy soil of the field was free of black-grass seeds until the sowing date. The aim of the trial was to compare the fitness and resistance value of different black-grass populations under outdoor conditions. Particularly the impact of pre-emergence herbicides was of interest. Different efficiency factors of CADOU SC ® (flufenacet) against the tested black-grass populations were observed. The disparities in the level of control were even greater under the use of STOMP AQUA ® (pendimethalin), BOXER ® (prosulfocarb) and ARELON TOP ® (isoproturon). A hundred percent control of all black-grass populations in both seasons was only achieved by the use of KERB FLO ® (propyzamide). It was visible that the black-grass populations that were difficult to control had a powerful constitution, even better than that of those populations that turned out to be highly susceptible to the herbicide treatments. The fall results in both seasons of the experiment (2009/2010 and 2010/2011) were consistent. This was also confirmed for the efficacy results for foliar herbicides in spring. Subject to field history, there was a wide range in sensitivity to the tested herbicides. Black-grass populations from coastal regions were poorly controlled by FOCUS ULTRA ® (cycloxydim) or SELECT 240 EC ® (clethodim) + PARA SOMMER ® (paraffin oil), but were highly susceptible to MOTIVELL ® (nicosulfuron). Contrary results were observed with reference to the blackgrass populations from the inland. These populations were derived from fields where the crop rotation contains of 66 % cereals and 33 % maize. The populations from the coast were sampled in crop rotations dominated by cereals or cereals followed by oil seed rape. Poor efficacy of ATLANTIS OD ® (mesosulfuron + iodosulfuron) was observed in black-grass populations from the coastal regions as well as from the inland. Keywords: Crop rotation, fitness, flufenacet, mesosulfuron, propyzamide, prosulfocarb, quizalofop-p Vergleich verschiedener Ackerfuchsschwanz-Populationen ( Alopecurus myosuroides Huds.) in ihrer Empfindlichkeit gegenuber Herbiziden unter Freilandbedingungen Im Herbst der Jahre 2009 und 2010 wurden Samen unterschiedlicher Ackerfuchsschwanz-Populationen auf einem Feld in der Nahe von Munster ausgesat. Bis zur Saat war der Boden, ein Sandboden, frei von Ackerfuchsschwanz-Samen. Ziel des Versuches war es, die okologische Fitness und den Resistenzgrad unterschiedlicher Ackerfuchsschwanz-Populationen unter Freilandbedingungen miteinander zu vergleichen. Hierbei ging es besonders um die Wirksamkeit von Bodenherbiziden. Es wurde deutlich, dass die Wirkung von CADOU SC ® (Flufenacet) gegen die gepruften Ackerfuchsschwanz-Populationen unterschiedlich war. Deutlich groser waren die Unterschiede im Bekampfungserfolg bei Einsatz von STOMP AQUA ® (Pendimethalin), BOXER ® (Prosulfocarb) und ARELON TOP ® (Isoproturon). Eine hundertprozentige Bekampfung aller Ackerfuchsschwanz-Herkunfte konnte in beiden Versuchsjahren nur durch den Einsatz von KERB FLO ® (Propyzamid) erreicht werden. Es war zu beobachten, dass die schwer bekampfbaren Ackerfuchsschwanz-Herkunfte uber eine gute Konstitution verfugten. Diese war besser als die Konstitution der Herkunfte, die sehr empfindlich auf die Herbizidbehandlungen reagierten. Die Ergebnisse aus den Herbstbehandlungen der Versuchsjahre 2009/2010 und 2010/2011 waren konsistent. Dies gilt auch fur die Bekampfungserfolge, die nach den Behandlungen im Fruhjahr erhoben wurden. In Abhangigkeit von der Schlaghistorie war die Empfindlichkeit der unterschiedlichen Ackerfuchsschwanz-Herkunfte sehr unterschiedlich. Ackerfuchsschwanz-Populationen aus den Kustenregionen wurden mit FOCUS ULTRA ® (Cycloxydim) oder SELECT 240 EC ® (Clethodim) + PARA SOMMER ® (Paraffinol) schlecht bekampft, waren aber empfindlich gegenuber MOTIVELL ® (Nicosulfuron). Gegenteilig war die Reaktion der Ackerfuchsschwanz-Populationen aus dem Binnenland. Diese Populationen entwickelten sich in Fruchtfolgen, die zu 66 % aus Getreide und zu 33 % aus Mais bestehen. Die Populationen der Kustenregionen sind durch Getreide- bzw. Getreide-Raps-Fruchtfolgen gepragt. Eine unzureichende Wirkung von ATLANTIS OD ® (Mesosulfuron + Iodosulfuron) konnte sowohl bei den Populationen aus den Kustenregionen wie aus dem Binnenland beobachtet werden. Stichworter: Cycloxydim, Fitness, Flufenacet, Freiland, Fruchtfolge, Mesosulfuron, Propyzamid