Non-target Site Resistance Research Articles

Transcriptome Profiling to Identify Genes Involved in Mesosulfuron-Methyl Resistance in Alopecurus aequalis.

Non-target-site resistance (NTSR) to herbicides is a worldwide concern for weed control. However, as the dominant NTSR mechanism in weeds, metabolic resistance is not yet well-characterized at the genetic level. For this study, we have identified a shortawn foxtail (Alopecurus aequalis Sobol.) population displaying both TSR and NTSR to mesosulfuron-methyl and fenoxaprop-P-ethyl, yet the molecular basis for this NTSR remains unclear. To investigate the mechanisms of metabolic resistance, an RNA-Seq transcriptome analysis was used to find candidate genes that may confer metabolic resistance to the herbicide mesosulfuron-methyl in this plant population. The RNA-Seq libraries generated 831,846,736 clean reads. The de novo transcriptome assembly yielded 95,479 unigenes (averaging 944 bp in length) that were assigned putative annotations. Among these, a total of 29,889 unigenes were assigned to 67 GO terms that contained three main categories, and 14,246 unigenes assigned to 32 predicted KEGG metabolic pathways. Global gene expression was measured using the reads generated from the untreated control (CK), water-only control (WCK), and mesosulfuron-methyl treatment (T) of R and susceptible (S). Contigs that showed expression differences between mesosulfuron-methyl-treated R and S biotypes, and between mesosulfuron-methyl-treated, water-treated and untreated R plants were selected for further quantitative real-time PCR (qRT-PCR) validation analyses. Seventeen contigs were consistently highly expressed in the resistant A. aequalis plants, including four cytochrome P450 monooxygenase (CytP450) genes, two glutathione S-transferase (GST) genes, two glucosyltransferase (GT) genes, two ATP-binding cassette (ABC) transporter genes, and seven additional contigs with functional annotations related to oxidation, hydrolysis, and plant stress physiology. These 17 contigs could serve as major candidate genes for contributing to metabolic mesosulfuron-methyl resistance; hence they deserve further functional study. This is the first large-scale transcriptome-sequencing study to identify NTSR genes in A. aequalis that uses the Illumina platform. This work demonstrates that NTSR is likely driven by the differences in the expression patterns of a set of genes. The assembled transcriptome data presented here provide a valuable resource for A. aequalis biology, and should facilitate the study of herbicide resistance at the molecular level in this and other weed species.

Unraveling the Transcriptional Basis of Temperature-Dependent Pinoxaden Resistance in Brachypodium hybridum.

Climate change endangers food security and our ability to feed the ever-increasing human population. Weeds are the most important biotic stress, reducing crop-plant productivity worldwide. Chemical control, the main approach for weed management, can be strongly affected by temperature. Previously, we have shown that temperature-dependent non-target site (NTS) resistance of Brachypodium hybridum is due to enhanced detoxification of acetyl-CoA carboxylase inhibitors. Here, we explored the transcriptional basis of this phenomenon. Plants were characterized for the transcriptional response to herbicide application, high-temperature and their combination, in an attempt to uncover the genetic basis of temperature-dependent pinoxaden resistance. Even though most of the variance among treatments was due to pinoxaden application (61%), plants were able to survive pinoxaden application only when grown under high-temperatures. Biological pathways and expression patterns of members of specific gene families, previously shown to be involved in NTS metabolic resistance to different herbicides, were examined. Cytochrome P450, glucosyl transferase and glutathione-S-transferase genes were found to be up-regulated in response to pinoxaden application under both control and high-temperature conditions. However, biological pathways related to oxidation and glucose conjugation were found to be significantly enriched only under the combination of pinoxaden application and high-temperature. Analysis of reactive oxygen species (ROS) was conducted at several time points after treatment using a probe detecting H2O2/peroxides. Comparison of ROS accumulation among treatments revealed a significant reduction in ROS quantities 24 h after pinoxaden application only under high-temperature conditions. These results may indicate significant activity of enzymatic ROS scavengers that can be correlated with the activation of herbicide-resistance mechanisms. This study shows that up-regulation of genes related to metabolic resistance is not sufficient to explain temperature-dependent pinoxaden resistance. We suggest that elevated activity of enzymatic processes at high-temperature may induce rapid and efficient pinoxaden metabolism leading to temperature-dependent herbicide resistance.

Proteomic and biochemical assays of glutathione-related proteins in susceptible and multiple herbicide resistant Avena fatua L.

Extensive herbicide usage has led to the evolution of resistant weed populations that cause substantial crop yield losses and increase production costs. The multiple herbicide resistant (MHR) Avena fatua L. populations utilized in this study are resistant to members of all selective herbicide families, across five modes of action, available for A. fatua control in U.S. small grain production, and thus pose significant agronomic and economic threats. Resistance to ALS and ACCase inhibitors is not conferred by target site mutations, indicating that non-target site resistance mechanisms are involved. To investigate the potential involvement of glutathione-related enzymes in the MHR phenotype, we used a combination of proteomic, biochemical, and immunological approaches to compare their constitutive activities in herbicide susceptible (HS1 and HS2) and MHR (MHR3 and MHR4) A. fatua plants. Proteomic analysis identified three tau and one phi glutathione S-transferases (GSTs) present at higher levels in MHR compared to HS plants, while immunoassays revealed elevated levels of lambda, phi, and tau GSTs. GST specific activity towards 1-chloro-2,4-dinitrobenzene was 1.2-fold higher in MHR4 than in HS1 plants and 1.3- and 1.2-fold higher in MHR3 than in HS1 and HS2 plants, respectively. However, GST specific activities towards fenoxaprop-P-ethyl and imazamethabenz-methyl were not different between untreated MHR and HS plants. Dehydroascorbate reductase specific activity was 1.4-fold higher in MHR than HS plants. Pretreatment with the GST inhibitor NBD-Cl did not affect MHR sensitivity to fenoxaprop-P-ethyl application, while the herbicide safener and GST inducer mefenpyr reduced the efficacy of low doses of fenoxaprop-P-ethyl on MHR4 but not MHR3 plants. Mefenpyr treatment also partially reduced the efficacy of thiencarbazone-methyl or mesosulfuron-methyl on MHR3 or MHR4 plants, respectively. Overall, the GSTs described here are not directly involved in enhanced rates of fenoxaprop-P-ethyl or imazamethabenz-methyl metabolism in MHR A. fatua. Instead, we propose that the constitutively elevated GST proteins and related enzymes in MHR plants are representative of a larger, more global suite of abiotic stress-related changes.

Spatial Distribution of Acetolactate Synthase Resistance Mechanisms in Neighboring Populations of Silky Windgrass (Apera spica-venti)

Silky windgrass is a serious weed in central and northern Europe. Its importance has escalated in recent years because of its growing resistance to acetolactate synthase (ALS)-inhibiting herbicides. This study investigated the resistance level for three herbicide sites of action in eight silky windgrass populations, collected in fields neighboring a field where iodosulfuron sodium salt–resistant silky windgrass had previously been found. Target site resistance (TSR) and non–target site resistance (NTSR) mechanisms were identified, and a spatial gradient distribution hypothesis of ALS resistance was tested. Populations showed large variations in ED50values to iodosulfuron, with resistance indices (RIs) ranging from 0.1 to 372. No cross-resistance was found to other herbicide groups with the same site of action as iodosulfuron. In contrast, resistance was observed to the acetyl-CoA carboxylase inhibitor, fenoxaprop ethyl ester (RI from 0.7 to 776), while the activity of prosulfocarb, an inhibitor of long-chain fatty-acid synthesis, was unaffected. Iodosulfuron-resistant phenotypes were associated with NTSR, while fenoxaprop ethyl ester resistance was caused by both NTSR and TSR (Ile-1781-Leu mutation). A large-scale trend in the spatial distribution of resistance to ALS indicated a decreasing resistance with increased distance from an epicenter. After finer-scale analysis, less than 0.05% of the residual variation could be attributed to spatial autocorrelation. The spatial resistance pattern was not correlated with the dominant wind direction, while there was a correlation between the resistant phenotype and type of crop. This study underlines that NTSR mechanisms do not always confer broad resistance to different herbicide subclasses and site of action, hence the complex relationship to resistant phenotype. NTSR mechanisms, in particular detoxification, were present at different levels for the herbicides tested in the silky windgrass populations of this study. The factors contributing to the spatial distribution of resistance remain elusive.

MiR397/Laccase Gene Mediated Network Improves Tolerance to Fenoxaprop-P-ethyl in Beckmannia syzigachne and Oryza sativa.

Herbicide resistance can be either target-site or non-target-site based. The molecular mechanisms underlying non-target-site resistance (NTSR) are poorly understood, especially at the level of gene expression regulation. MicroRNAs (miRNAs) represent key post-transcriptional regulators of eukaryotic gene expression and play important roles in stress responses. In this study, the miR397 gene from Beckmannia syzigachne (referred to as bsy-miR397) was functionally characterized to determine its role in regulating fenoxaprop-P-ethyl resistance. We showed that (1) bsy-miR397 transcript level is constitutively higher in resistant than in sensitive B. syzigachne plants, whereas bsy-Laccase expression and activity show the opposite trend, and (2) bsy-miR397 suppresses the expression of bsy-Laccase in tobacco, indicating that it negatively regulates bsy-Laccase at the transcriptional level. We found evidences that miR397/laccase regulation might be involved in fenoxaprop-P-ethyl NTSR. First, the rice transgenic line overexpressing OXmiR397 showed improved fenoxaprop-P-ethyl tolerance. Second, following activation of bsy-Laccase gene expression by CuSO4 treatment, fenoxaprop resistance in B. syzigachne tended to decrease. Therefore, we suggest that bsy-miR397 might play a role in fenoxaprop-P-ethyl NTSR in B. syzigachne by down-regulating laccase expression, potentially leading to the enhanced expression of three oxidases/peroxidases genes to introduce an active moiety into herbicide molecules in Phase-2 metabolism. Bsy-miR397, bsy-Laccase, and other regulatory components might form a regulatory network to detoxify fenoxaprop-P-ethyl in B. syzigachne, supported by the differential expression of transcription factors and oxidases/peroxidases in the rice transgenic line overexpressing OXmiR397. This implies how down-regulation of a gene (laccase) can enhance NTSR. Our findings shed light on the daunting task of understanding and managing complex NTSR in weedy plant species.

Glyphosate resistance in Ambrosia trifida: Part 2. Rapid response physiology and non-target-site resistance.

The glyphosate-resistant rapid response (GR RR) resistance mechanism in Ambrosia trifida is not due to target-site resistance (TSR) mechanisms. This study explores the physiology of the rapid response and the possibility of reduced translocation and vacuolar sequestration as non-target-site resistance (NTSR) mechanisms. GR RR leaf discs accumulated hydrogen peroxide within minutes of glyphosate exposure, but only in mature leaf tissue. The rapid response required energy either as light or exogenous sucrose. The combination of phenylalanine and tyrosine inhibited the rapid response in a dose-dependent manner. Reduced glyphosate translocation was observed in GR RR, but only when associated with tissue death caused by the rapid response. Nuclear magnetic resonance studies indicated that glyphosate enters the cytoplasm and reaches chloroplasts, and it is not moved into the vacuole of GR RR, GR non-rapid response or glyphosate-susceptible A. trifida. The GR RR mechanism of resistance is not associated with vacuole sequestration of glyphosate, and the observed reduced translocation is likely a consequence of rapid tissue death. Rapid cell death was inhibited by exogenous application of aromatic amino acids phenylalanine and tyrosine. The mechanism by which these amino acids inhibit rapid cell death in the GR RR phenotype remains unknown, and it could involve glyphosate phytotoxicity or other agents generating reactive oxygen species. Implications of these findings are discussed. The GR RR mechanism is distinct from the currently described glyphosate TSR or NTSR mechanisms in other species. © 2017 Society of Chemical Industry.

First confirmation and characterization of target and non-target site resistance to glyphosate in Palmer amaranth (Amaranthus palmeri) from Mexico

Following the introduction of glyphosate-resistant (GR)-cotton crops in Mexico, farmers have relied upon glyphosate as being the only herbicide for in-season weed control. Continuous use of glyphosate within the same year and over multiple successive years has resulted in the selection of glyphosate resistance in Palmer amaranth (Amarantus palmeri). Dose-response assays confirmed resistance in seven different accessions. The resistance ratio based on GR50 values (50% growth reduction) varied between 12 and 83. At 1000 μM glyphosate, shikimic acid accumulation in the S-accession was 30- to 2-fold higher at compared to R-accessions. At 96 h after treatment, 35–44% and 61% of applied 14C-glyphosate was taken up by leaves of plants from R- and S-accessions, respectively. At this time, a significantly higher proportion of the glyphosate absorbed remained in the treated leaf of R-plants (55–69%) compared to S-plants (36%). Glyphosate metabolism was low and did not differ between resistant and susceptible plants. Glyphosate was differentially metabolized to AMPA and glyoxylate in plants of R- and S-accessions, although it was low in both accessions (<10%). There were differences in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme activity by 50% (I50) between R- and S-accessions. However, no significant differences were found in the basal EPSPS activity (μmol inorganic phosphate μg−1 total soluble protein min−1) between R- and S-accessions. A point mutation Pro-106-Ser was evidenced in three accessions. The results confirmed the resistance of Palmer amaranth accessions to glyphosate collected from GR-cotton crops from Mexico. This is the first study demonstrating glyphosate-resistance in Palmer amaranth from Mexico.

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.

Non-target Site Mechanisms of Resistance to Herbicides

ABSTRACTEvolution of resistance in weeds to herbicides is threatening world agricultural production. Weed management has become more complicated with the development of non-target site resistance (NTSR) to herbicides in weeds. The NTSR mechanisms can be caused by herbicide metabolism, altered patterns of translocation, and herbicide absorption/penetration. Metabolism of herbicides consists of an activation phase and then a conjugation phase, though in some cases no activation phase is needed. The activation phase mainly involves the addition of functional groups to herbicide molecules using enzymes such as cytochrome P-450. Transcriptome-wide gene expression has shown that genes which encode for several cytochrome P-450s are upregulated in weeds resistant to ACCase-inhibitor and ALS-inhibitor herbicides. In the conjugated phase, several studies have shown that two important types of enzyme, glutathione S-transferases and glucosyltransferases, play crucial roles in conferring resistance to herbicides. An altered pattern of translocation can also play a crucial role in conferring NTSR to weeds. With glyphosate and paraquat, it has been shown that altered patterns of translocation are due to sequestration of the herbicide into vacuoles. However, some other little known mechanisms such as “hyper-sensitive” reactions, impaired translocation, and root exudation can affect the patterns of herbicide translocation within resistant weeds. The molecular understanding of NTSR mechanisms is still in its infancy. Recent developments in techniques such as high-throughput DNA/RNA sequencing technologies will soon allow further insights into the NTSR mechanisms in weeds. This information is essential for developing strategies to overcome weeds with the NTSR mechanisms.

A “Stressed” Alfalfa-Based Cropping System Leads to the Selection of Quizalofop-Resistant Italian Ryegrass (Lolium perennessp.multiflorum)

Italian ryegrass populations investigated in this study were harvested in an alfalfa-based cropping system. In that system, the agronomic practices and chemical weed management, based on the use of aryloxyphenoxy-propionates herbicides (i.e., quizalofop ethyl ester), were optimized to obtain a dual seed–forage production. Five of seven populations tested were confirmed resistant to quizalofop ethyl ester with resistance indexes ranging from 4.5 to &gt;209. Both target- and nontarget-site resistance mechanisms were most likely involved. Three allelic variants were detected (Ile-1781–Leu, Trp-2027–Cys, and Ile-2041–Asn) in four resistant populations, whereas no known mutations were found in one resistant population. The herbicide treatment on Italian ryegrass plants at different phenological stages suggested that to control regrowth, it is necessary to use two to fives times the herbicide dose suitable for younger plants. This situation is encountered in fields when Italian ryegrass plants need to be controlled to maximize the alfalfa seed production, and it is comparable to using a sublethal herbicide dose, leading to the selection of herbicide-resistant biotypes. In such a situation, the cropping system is not sustainable, and integrated weed management should be implemented to deplete the soil weed seed bank and prevent new weed seed production.

Establishing a herbicide-metabolizing enzyme library inBeckmannia syzigachneto identify genes associated with metabolic resistance

Non-target site resistance (NTSR) to herbicides is an increasing concern for weed control. Metabolic herbicide resistance is an important mechanism for NTSR. However, little is known about metabolic resistance at the genetic level. In this study, we have identified three fenoxaprop-P-ethyl-resistant American sloughgrass (Beckmannia syzigachne Steud.) populations, in which the molecular basis for NTSR remains unclear. To reveal the mechanisms of metabolic resistance, the genes likely to be involved in herbicide metabolism (e.g. for cytochrome P450s, esterases, hydrolases, oxidases, peroxidases, glutathione S-transferases, glycosyltransferases, and transporter proteins) were isolated using transcriptome sequencing, in combination with RT-PCR (reverse transcription-PCR) and RACE (rapid amplification of cDNA ends). Consequently, we established a herbicide-metabolizing enzyme library containing at least 332 genes, and each of these genes was cloned and the sequence and the expression level compared between the fenoxaprop-P-ethyl-resistant and susceptible populations. Fifteen metabolic enzyme genes were found to be possibly involved in fenoxaprop-P-ethyl resistance. In addition, we found five metabolizing enzyme genes that have a different gene sequence in plants of susceptible versus resistant B. syzigachne populations. These genes may be major candidates for herbicide metabolic resistance. This established metabolic enzyme library represents an important step forward towards a better understanding of herbicide metabolism and metabolic resistance in this and possibly other closely related weed species. This new information may help to understand weed metabolic resistance and to develop novel strategies of weed management.

Cross‐resistance patterns to acetyl coenzyme A carboxylase (ACCase) inhibitors associated with different ACCase mutations in Beckmannia syzigachne

SummaryBeckmannia syzigachne (American sloughgrass) is a competitive grass weed found in China. Fenoxaprop‐P‐ethyl is widely used for control of this species in China. Resistance to fenoxaprop‐P‐ethyl in B. syzigachne has been reported to be conferred by an isoleucine(Ile)‐1781‐leucine(Leu) substitution in the gene encoding the herbicide target, acetyl‐CoA carboxylase (ACCase). In this study, three mutations were detected by derived cleaved amplified polymorphic sequence (dCAPS) method in fenoxaprop‐P‐ethyl‐resistant B. syzigachne populations: Ile‐1781‐Leu in population JCWL‐R, Ile‐2041‐Asn in JCJT‐R and Gly‐2096‐Ala in JYJD‐R. The data indicated they were genetically homogeneous (homozygous mutant) at the ACCase locus. The use of cytochrome P450 inhibitors was shown to slightly reduce the GR50 value of fenoxaprop‐P‐ethyl‐resistant populations, from which we inferred a combination of target‐site resistance (TSR) and non‐target‐site resistance (NTSR) was involved in fenoxaprop‐P‐ethyl‐resistance. We characterised the cross‐resistance patterns to ACCase inhibitors in B. syzigachne. The plants in the JCWL‐R population were highly resistant to all tested APPs (aryloxyphen‐oxypropionates), sethoxydim and pinoxaden, and moderately resistant to clethodim. The plants in the JCJT‐R population were highly resistant to fluazifop‐P‐butyl, clodinafop‐propargyl, cyhalofop‐butyl, metamifop and pinoxaden; moderately resistant to haloxyfop‐R‐methyl, quizalofop‐P‐ethyl and sethoxydim; and sensitive to clethodim. The plants in the JYJD‐R population were highly resistant to clodinafop‐propargyl, metamifop and pinoxaden; moderately resistant to haloxyfop‐R‐methyl, cyhalofop‐butyl, quizalofop‐P‐ethyl, fluazifop‐P‐butyl and sethoxydim; and sensitive to clethodim. If resistance to ACCase inhibitors is present in B. syzigachne populations in the field, then our results indicate that clethodim should be used. While we demonstrated the cross‐resistance patterns of TSR resulting from three mutations in B. syzigachne, we also demonstrated that NTSR plays a role in resistance, which will complicate weed management.

Occurrence, genetic control and evolution of non-target-site based resistance to herbicides inhibiting acetolactate synthase (ALS) in the dicot weed Papaver rhoeas

Non-target-site resistance (NTSR) to herbicides is a major issue for the chemical control of weeds. Whilst predominant in grass weeds, NTSR remains largely uninvestigated in dicot weeds. We investigated the occurrence, inheritance and genetic control of NTSR to acetolactate synthase (ALS) inhibitors in Papaver rhoeas (corn poppy) using progenies from plants with potential NTSR to the imidazolinone herbicide imazamox. NTSR to imazamox was inherited from parents over two successive generations. NTSR to tritosulfuron (a sulfonylurea) was observed in F1 generations and inherited in F2 generations. NTSR to florasulam (a triazolopyrimidine) emerged in F2 generations. Our findings suggest NTSR was polygenic and gradually built-up by accumulation over generations of loci with moderate individual effects in single plants. We also demonstrated that ALS alleles conferring herbicide resistance can co-exist with NTSR loci in P. rhoeas plants. Previous research focussed on TSR in P. rhoeas, which most likely caused underestimation of NTSR significance in this species. This may also apply to other dicot species. From our data, resistance to ALS inhibitors in P. rhoeas appears complex, and involves well-known mutant ALS alleles and a set of unknown NTSR loci that confer resistance to ALS inhibitors from different chemical families.

Nontarget-Site Resistance to ALS Inhibitors in Waterhemp (Amaranthus tuberculatus)

A waterhemp population (MCR) previously characterized as resistant to 4-hydroxyphenylpyruvate dioxygenase and photosystem II inhibitors demonstrated both moderate and high levels of resistance to acetolactate synthase (ALS) inhibitors. Plants from the MCR population exhibiting high resistance to ALS inhibitors contained the commonly found Trp574Leu ALS amino acid substitution, whereas plants with only moderate resistance did not have this substitution. A subpopulation (JG11) was derived from the MCR population in which the moderate-resistance trait was isolated from the Trp574Leu mutation. Results from DNA sequencing and ALS enzyme assays demonstrated that resistance to ALS inhibitors in the JG11 population was not due to an altered site of action. This nontarget-site ALS-inhibitor resistance was characterized with whole-plant dose–response experiments using herbicides from each of the five commercialized families of ALS-inhibiting herbicides. Resistance ratios ranging from 3 to 90 were obtained from the seven herbicides evaluated. Nontarget-site resistance to ALS has been rarely documented in eudicot weeds, and adds to the growing list of resistance traits evolved in waterhemp.

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.

Molecular Mechanisms of Herbicide Resistance

Resistance to herbicides occurs in weeds as the result of evolutionary adaptation (Jasieniuk et al. 1996). Basically, two types of mechanisms are involved in resistance (Beckie and Tardif 2012; Délye 2013). Target-site resistance (TSR) is caused by changes in the tridimensional structure of the herbicide target protein that decrease herbicide binding, or by increased activity (e.g., due to increased expression or increased intrinsic activity) of the target protein. Nontarget-site resistance (NTSR) is endowed by any mechanism not belonging to TSR, e.g., reduction in herbicide uptake or translocation in the plant, or enhanced herbicide detoxification (reviewed in Délye 2013; Yuan et al. 2007).

RNA-Seq analysis of rye-grass transcriptomic response to an herbicide inhibiting acetolactate-synthase identifies transcripts linked to non-target-site-based resistance.

Non-target-site resistance (NTSR) to herbicides that disrupts agricultural weed control is a worldwide concern for food security. NTSR is considered a polygenic adaptive trait driven by differential gene regulation in resistant plants. Little is known about its genetic determinism, which precludes NTSR diagnosis and evolutionary studies. We used Illumina RNA-sequencing to investigate transcriptomic differences between plants from the global major weed rye-grass sensitive or resistant to the acetolactate-synthase (ALS) inhibiting herbicide pyroxsulam. Plants were collected before and along a time-course after herbicide application. De novo transcriptome assembly yielded a resource (LOLbase) including 92,381 contigs representing potentially active transcripts that were assigned putative annotations. Early effects of ALS inhibition consistent with the literature were observed in resistant and sensitive plants, proving LOLbase data were relevant to study herbicide response. Comparison of resistant and sensitive plants identified 30 candidate NTSR contigs. Further validation using 212 plants resistant or sensitive to pyroxsulam and/or to the ALS inhibitors iodosulfuron+mesosulfuron confirmed four contigs (two cytochromes P450, one glycosyl-transferase and one glutathione-S-transferase) were NTSR markers which combined expression levels could reliably identify resistant plants. This work confirmed that NTSR is driven by differential gene expression and involves different mechanisms. It provided tools and foundation for subsequent NTSR investigations.

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.

Evolution of herbicide resistance mechanisms in grass weeds

Herbicide resistant weeds are becoming increasingly common, threatening global food security. Here, we present BrIFAR: a new model system for the functional study of mechanisms of herbicide resistance in grass weeds. We have developed a large collection of Brachypodium accessions, the BrI collection, representing a wide range of habitats. Wide screening of the responses of the accessions to four major herbicide groups (PSII, ACCase, ALS/AHAS and EPSPS inhibitors) identified 28 herbicide-resistance candidate accessions. Target-site resistance to PSII inhibitors was found in accessions collected from habitats with a known history of herbicide applications. An amino acid substitution in the psbA gene (serine264 to glycine) conferred resistance and also significantly affected the flowering and shoot dry weight of the resistant accession, as compared to the sensitive accession. Non-target site resistance to ACCase inhibitors was found in accessions collected from habitats with a history of herbicide application and from a nature reserve. In-vitro enzyme activity tests and responses following pre-treatment with malathion (a cytochrome-P450 inhibitor) indicated sensitivity at the enzyme level, and give strong support to diclofop-methyl and pinoxaden enhanced detoxification as NTS resistance mechanism. BrIFAR can promote better understanding of the evolution of mechanisms of herbicide resistance and aid the implementation of integrative management approaches for sustainable agriculture.

Impact of non-target-site-resistance on herbicidal activity of imazamox on black¬grass ( Alopecurus myosuroides Huds.) in comparison to other ALS-graminicides

A black-grass (Alopecurus myosuroides Huds.) resistance-monitoring conducted by BASF in 2010 - 2012 revealed a high number of accessions with resistance against imazamox. However, application of imazamox- based products in a winter crop was limited to winter beans in France and United Kingdom only until the introduction of the Clearfield ® -production system in autumn 2012 in winter oilseed rape. It is therefore assumed that the resistance mechanisms were probably selected by the frequent use of ACCase- and ALS- inhibitors in winter crop rotations during the last 2 decades. Resistance level for each product-biotype combination was calculated according the R-classification system (S, R?, RR, RRR) by directly comparing the product performance on a biotype versus untreated control. Majority of resistant biotypes did not show a target-site mutation at the known codon Pro197 or Trp574. In order to better evaluate the impact of Non- Target-Site-Resistance (NTSR) on the activity of BEYOND (imazamox), ATLANTIS WG (mesosulfuron+iodosulfuron) and ABAK (pyroxsulam), biotypes who have shown an ALS-target-site mutation were removed from further analysis. At the dose rate of 35 g ai/ha BEYOND provided good activity on susceptible biotypes of black-grass almost matching up with ATLANTIS WG and ABAK. However, activity of BEYOND declined stronger on biotypes classified as R? or RR for that product, while ATLANTIS WG and ABAK hardly showed any decline in control on this group of biotypes when applied at the recommended dose rate. It is assumed that the underlying NTSR- mechanism is not effective enough yet to confer resistance to ATLANTIS WG and ABAK, but on BEYOND. In contrast, biotypes classified as R? for ATLANTIS WG did show a stronger impact on the activity of BEYOND and ABAK then of ATLANTIS WG. These differences in control level probably do translate into differences in selection pressure as well.