Survey of ACCase and ALS resistance in winter annual grasses identifies target-site and nontarget-site imazamox resistance in Secale cereale.

BACKGROUND: Early detection of herbicide resistance in weeds is crucial for successful implementation of integrated weed management. We conducted a herbicide resistance survey of the winter annual grasses feral rye (Secale cereale), downy brome (Bromus tectorum), and jointed goatgrass (Aegilops cylindrica) from Colorado winter wheat production areas for resistance to imazamox and quizalofop. RESULTS: All samples were susceptible to quizalofop. All downy brome and jointed goatgrass samples were susceptible to imazamox. Out of 314 field collected samples, we identified three feral rye populations (named A, B, and C) that were imazamox resistant. Populations B and C had a target-site mechanism with mutations in the Ser653 residue of the acetolactate synthase (ALS) gene to Asn in B and to Thr in C. Both populations B and C had greatly reduced ALS in vitro enzyme inhibition by imazamox. ALS feral rye protein modeling showed that steric interactions induced by the amino acid substitutions at Ser653 impaired imazamox binding. Individuals from population A had no mutations in the ALS gene. The ALS enzyme from population A was equally sensitive to imazamox as to known susceptible feral rye populations. Imazamox was degraded two times faster in population A compared with a susceptible control. An oxidized imazamox metabolite formed faster in population A and this detoxification reaction was inhibited by malathion. CONCLUSION: Population A has a nontarget-site mechanism of enhanced imazamox metabolism that may be conferred by cytochrome P450 enzymes. This is the first report of both target-site and metabolism-based imazamox resistance in feral rye. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genomic-based epidemiology reveals independent origins and gene flow of glyphosate resistance in Bassia scoparia populations across North America.

Genomic-based epidemiology can provide insight into the origins and spread of herbicide resistance mechanisms in weeds. We used kochia (Bassia scoparia) populations resistant to the herbicide glyphosate from across western North America to test the alternative hypotheses that (i) a single EPSPS gene duplication event occurred initially in the Central Great Plains and then subsequently spread to all other geographical areas now exhibiting glyphosate-resistant kochia populations or that (ii) gene duplication occurred multiple times in independent events in a case of parallel evolution. We used qPCR markers previously developed for measuring the structure of the EPSPS tandem duplication to investigate whether all glyphosate-resistant individuals had the same EPSPS repeat structure. We also investigated population structure using simple sequence repeat markers to determine the relatedness of kochia populations from across the Central Great Plains, Northern Plains and the Pacific Northwest. We found that the original EPSPS duplication genotype was predominant in the Central Great Plains where glyphosate resistance was first reported. We identified two additional EPSPS duplication genotypes, one having geographical associations with the Northern Plains and the other with the Pacific Northwest. The EPSPS duplication genotype from the Pacific Northwest seems likely to represent a second, independent evolutionary origin of a resistance allele. We found evidence of gene flow across populations and a general lack of population structure. The results support at least two independent evolutionary origins of glyphosate resistance in kochia, followed by substantial and mostly geographically localized gene flow to spread the resistance alleles into diverse genetic backgrounds.

A needle in a seedstack: an improved method for detection of rare alleles in bulk seed testing through KASP.

BACKGROUND: Amaranthus palmeri is an aggressive and prolific weed species with major impact on agricultural yield and is a prohibited noxious weed across the Midwest. Morphological identification of A. palmeri from other Amaranthus species is extremely difficult in seeds, which has led to genetic testing for seed identification in commercial seed lots. RESULTS: We created an inexpensive and reliable genetic test based on novel, species-specific, single nucleotide polymorphisms (SNPs) from GBS (Genotyping by Sequencing) data. We report three SNP-based genetic tests for identifying A. palmeri alone or in a mixed pool of Amaranthus spp. Sensitivity ranged from 99.8 to 100%, specificity from 99.59 to 100%. Accuracy for all three tests is > 99.7%. All three are capable of reliably detecting one A. palmeri seed in a pool of 200 Amaranthus spp. seeds. The test was validated across 20 populations of A. palmeri, along with eight other Amaranthus species, the largest and most genetically diverse panel of Amaranthus samples to date. CONCLUSION: Our work represents a marked improvement over existing commercial assays resulting in an identification assay that is (i) accurate, (ii) robust, (iii) easy to interpret and (iv) applicable to both leaf tissue and pools of up to 200 seeds. Included is a data transformation method for calling of closely grouped competitive fluorescence assays. We also present a comprehensive GBS dataset from the largest geographic panel of Amaranthus populations sequenced. Our approach serves as a model for developing markers for other difficult to identify species. © 2021 Society of Chemical Industry.

A novel insight into the mode of action of glufosinate: how reactive oxygen species are formed.

Glufosinate targets glutamine synthetase (GS), but its fast herbicidal action is triggered by reactive oxygen species (ROS). The relationship between GS inhibition and ROS accumulation was investigated in Amaranthus palmeri. Glufosinate's fast action is light-dependent with no visual symptoms or ROS formation in the dark. Inhibition of GS leads to accumulation of ammonia and metabolites of the photorespiration pathway, such as glycolate and glyoxylate, as well as depletion of other intermediates such as glycine, serine, hydroxypyruvate, and glycerate. Exogenous supply of glycolate to glufosinate-treated plants enhanced herbicidal activity and dramatically increased hydrogen peroxide accumulation (possibly from peroxisomal glycolate oxidase activity). Glufosinate affected the balance between ROS generation and scavenging. The activity of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase increased after glufosinate treatment in an attempt to quench the nascent ROS burst. Low doses of atrazine and dinoseb were used to investigate the sources of ROS by manipulating photosynthetic electron transport and oxygen (O2) evolution. ROS formation depended on electron flow and O2 evolution in photosystem II (PSII). Inhibition of GS disrupted photorespiration, carbon assimilation, and linear electron flow in the light reactions. Consequently, the antioxidant machinery and the water-water cycle are overwhelmed in the presence of light and glufosinate. The O2 generated by the splitting of water in PSII becomes the acceptor of electrons, generating ROS. The cascade of events leads to lipid peroxidation and forms the basis for the fast action of glufosinate.

Trp2027Cys mutation evolves in Digitaria insularis with cross-resistance to ACCase inhibitors.

Sourgrass (Digitaria insularis) is one of the most problematic weeds in South America because glyphosate resistance is widespread across most crop production regions. Acetyl coenzyme A carboxylase (ACCase)-inhibiting herbicides have been intensively used to manage D. insularis, which substantially increased selection pressure for this class of herbicides. We confirmed resistance to ACCase herbicides in a D. insularis population from Brazil and characterized its molecular basis. Resistant plants showed high level of resistance to haloxyfop (resistance factor, RF = 613-fold), low level of resistance to pinoxaden (RF = 3.6-fold), and no resistance to clethodim. A target-site mutation, Trp2027Cys, was found in the ACCase sequence from resistant plants. A protein homology model shows that the Trp2027Cys mutation is near the herbicide-binding pocket formed between two ACCase chains, and is predicted to obstruct the access of aryloxyphenoxypropionates (FOP) herbicides to the binding site. A qPCR-based single nucleotide polymorphism genotyping method was validated to discriminate susceptible (wild-type Trp2027) and resistant (mutant Cys2027) alleles. All resistant plants were homozygous for the mutation and the assay could be used for early detection of resistance in D. insularis field samples with suspected resistance to ACCase inhibitors.

Physiological Factors Affecting Uptake and Translocation of Glufosinate.

Glufosinate is considered a contact herbicide because of its fast activity and limited translocation in plants. We used Palmer amaranth (Amaranthus palmeri S. Watson) as a model species to study plant-related factors affecting glufosinate uptake and translocation. Glufosinate uptake increased rapidly during the initial 24 h, achieving maximum uptake from this time on. The rate of uptake saturated with doses higher than 250 µM glufosinate, suggesting the involvement of a membrane transporter. When glufosinate concentrations were higher (>1 mM), uptake was a simple diffusion process in favor of a concentration gradient between the inside and the outside of the cells. Glufosinate uptake was inhibited by the presence of glutamine. The fast action of glufosinate did not limit its own translocation. Because glufosinate is highly water soluble, it translocates mostly through the apoplast or the xylem system. Consequently, old leaves tend to accumulate more herbicide than young meristematic leaves.

A novel TIPT double mutation in EPSPS conferring glyphosate resistance in tetraploid Bidens subalternans.

BACKGROUND: Bidens subalternans (greater beggarticks) is a tetraploid and troublesome weed infesting annual crops in most tropical regions of the world. A glyphosate-resistant (GR) B. subalternans biotype was detected in a soybean field from Paraguay. A series of physiological and molecular analyses were conducted to elucidate its resistance mechanisms. RESULTS: The GR biotype had a high level of resistance (> 15-fold LD50 ), relative to a glyphosate-susceptible (GS) biotype. Shikimate accumulation was up to ten-fold greater for GS compared with GR. We found no differences in sensitivity when plants were treated and kept under lower (10/4 °C) or higher temperatures (25/20 °C). GS and GR had the same relative EPSPS gene copy number, and similar glyphosate absorption and translocation rates. Neither biotype metabolized glyphosate. A double amino acid substitution (TIPT - Thr102Ile and Pro106Thr) was found in only one EPSPS allele from one of the two EPSPS homoeologs present in tetraploid GR B. subalternans. CONCLUSION: This is the first report of a TIPT double mutation conferring high levels of glyphosate resistance in a weed species. The presence of both wild-type and TIPT mutant EPSPS on the polyploid genome of GR B. subalternans may offset a potential fitness cost, requiring additional research to confirm the absence of deleterious effects. © 2019 Society of Chemical Industry.

Physiological and molecular analysis of glyphosate resistance in non-rapid response Ambrosia trifida from Wisconsin.

BACKGROUND: We previously identified a glyphosate-resistant A. trifida phenotype from Wisconsin USA that showed a non-rapid response to glyphosate. The mechanism of glyphosate resistance in this phenotype has yet to be elucidated. We conducted experiments to investigate non-target-site resistance and target-site resistance mechanisms. The roles of glyphosate absorption, translocation, and metabolism in resistance of this phenotype have not been reported previously, nor have EPSPS protein abundance or mutations to the full-length sequence of EPSPS. RESULTS: Whole-plant dose-response results confirmed a 6.5-level of glyphosate resistance for the resistant (R) phenotype compared to a susceptible (S) phenotype. Absorption and translocation of 14 C-glyphosate were similar between R and S phenotypes over 72 h. Glyphosate and AMPA concentrations in leaf tissue did not differ between R and S phenotypes over 96 h. In vivo shikimate leaf disc assays confirmed that glyphosate EC50 values were 4.6- to 5.4-fold greater for the R than S phenotype. Shikimate accumulation was similar between phenotypes at high glyphosate concentrations (>1000 µM), suggesting that glyphosate entered chloroplasts and inhibited EPSPS. This finding was supported by results showing that EPSPS copy number and EPSPS protein abundance did not differ between R and S phenotypes, nor did EPSPS sequence at Gly101, Thr102, and Pro106 positions. Comparison of full-length EPSPS sequences found five nonsynonymous polymorphisms that differed between R and S phenotypes. However, their locations were distant from the glyphosate target site and, therefore, not likely to affect enzyme-glyphosate interaction. CONCLUSION: The results suggest that a novel mechanism confers glyphosate resistance in this A. trifida phenotype. © 2019 Society of Chemical Industry.

The Draft Genome of Kochia scoparia and the Mechanism of Glyphosate Resistance via Transposon-Mediated EPSPS Tandem Gene Duplication.

Increased copy number of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene confers resistance to glyphosate, the world's most-used herbicide. There are typically three to eight EPSPS copies arranged in tandem in glyphosate-resistant populations of the weed kochia (Kochia scoparia). Here, we report a draft genome assembly from a glyphosate-susceptible kochia individual. Additionally, we assembled the EPSPS locus from a glyphosate-resistant kochia plant by sequencing select bacterial artificial chromosomes from a kochia bacterial artificial chromosome library. Comparing the resistant and susceptible EPSPS locus allowed us to reconstruct the history of duplication in the structurally complex EPSPS locus and uncover the genes that are coduplicated with EPSPS, several of which have a corresponding change in transcription. The comparison between the susceptible and resistant assemblies revealed two dominant repeat types. Additionally, we discovered a mobile genetic element with a FHY3/FAR1-like gene predicted in its sequence that is associated with the duplicated EPSPS gene copies in the resistant line. We present a hypothetical model based on unequal crossing over that implicates this mobile element as responsible for the origin of the EPSPS gene duplication event and the evolution of herbicide resistance in this system. These findings add to our understanding of stress resistance evolution and provide an example of rapid resistance evolution to high levels of environmental stress.

Reactive oxygen species trigger the fast action of glufosinate.

MAIN CONCLUSION: Glufosinate is primarily toxic to plants due to a massive light-dependent generation of reactive oxygen species rather than ammonia accumulation or carbon assimilation inhibition. Glutamine synthetase (GS) plays a key role in plant nitrogen metabolism and photorespiration. Glufosinate (C5H12NO4P) targets GS and causes catastrophic consequences leading to rapid plant cell death, and the causes for phytoxicity have been attributed to ammonia accumulation and carbon assimilation restriction. This study aimed to examine the biochemical and physiological consequences of GS inhibition to identify the actual cause for rapid phytotoxicity. Monocotyledonous and dicotyledonous species with different forms of carbon assimilation (C3 versus C4) were selected as model plants. Glufosinate sensitivity was proportional to the uptake of herbicide between species. Herbicide uptake also correlated with the level of GS inhibition and ammonia accumulation in planta even with all species having the same levels of enzyme sensitivity in vitro. Depletion of both glutamine and glutamate occurred in glufosinate-treated leaves; however, amino acid starvation would be expected to cause a slow plant response. Ammonia accumulation in response to GS inhibition, often reported as the driver of glufosinate phytotoxicity, occurred in all species, but did not correlate with either reductions in carbon assimilation or cell death. This is supported by the fact that plants can accumulate high levels of ammonia but show low inhibition of carbon assimilation and absence of phytotoxicity. Glufosinate-treated plants showed a massive light-dependent generation of reactive oxygen species, followed by malondialdehyde accumulation. Consequently, we propose that glufosinate is toxic to plants not because of ammonia accumulation nor carbon assimilation inhibition, but the production of reactive oxygen species driving the catastrophic lipid peroxidation of the cell membranes and rapid cell death.

The power and potential of genomics in weed biology and management.

There have been previous calls for, and efforts focused on, realizing the power and potential of weed genomics for better understanding of weeds. Sustained advances in genome sequencing and assembly technologies now make it possible for individual research groups to generate reference genomes for multiple weed species at reasonable costs. Here, we present the outcomes from several meetings, discussions, and workshops focused on establishing an International Weed Genomics Consortium (IWGC) for a coordinated international effort in weed genomics. We review the 'state of the art' in genomics and weed genomics, including technologies, applications, and on-going weed genome projects. We also report the outcomes from a workshop and a global survey of the weed science community to identify priority species, key biological questions, and weed management applications that can be addressed through greater availability of, and access to, genomic resources. Major focus areas include the evolution of herbicide resistance and weedy traits, the development of molecular diagnostics, and the identification of novel targets and approaches for weed management. There is increasing interest in, and need for, weed genomics, and the establishment of the IWGC will provide the necessary global platform for communication and coordination of weed genomics research. © 2018 Society of Chemical Industry.

Cross-resistance to dicamba, 2,4-D, and fluroxypyr in Kochia scoparia is endowed by a mutation in an AUX/IAA gene.

The understanding and mitigation of the appearance of herbicide-resistant weeds have come to the forefront of study in the past decade, as the number of weed species that are resistant to one or more herbicide modes of action is on the increase. Historically, weed resistance to auxin herbicides has been rare, but examples, such as Kochia scoparia L. Schrad (kochia), have appeared, posing a challenge to conventional agricultural practices. Reports of dicamba-resistant kochia populations began in the early 1990s in areas where auxin herbicides were heavily utilized for weed control in corn and wheat cropping systems, and some biotypes are resistant to other auxin herbicides as well. We have further characterized the auxin responses of one previously reported dicamba-resistant biotype isolated from western Nebraska and found that it is additionally cross-resistant to other auxin herbicides, including 2,4-dichlorophenoxyacetic acid (2,4-D) and fluroxypyr. We have utilized transcriptome sequencing and comparison to identify a 2-nt base change in this biotype, which results in a glycine to asparagine amino acid change within a highly conserved region of an AUX/indole-3-acetic acid (IAA) protein, KsIAA16. Through yeast two-hybrid analysis, characterization of F2 segregation, and heterologous expression and characterization of the gene in Arabidopsis thaliana, we show that that the single dominant KsIAA16R resistance allele is the causal basis for dicamba resistance in this population. Furthermore, we report the development of a molecular marker to identify this allele in populations and facilitate inheritance studies. We also report that the resistance allele confers a fitness penalty in greenhouse studies.

Metabolism of 2,4-dichlorophenoxyacetic acid contributes to resistance in a common waterhemp (Amaranthus tuberculatus) population.

BACKGROUND: Synthetic auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) have been widely used for selective control of broadleaf weeds since the mid-1940s. In 2009, an Amaranthus tuberculatus (common waterhemp) population with 10-fold resistance to 2,4-D was found in Nebraska, USA. The 2,4-D resistance mechanism was examined by conducting [14 C] 2,4-D absorption, translocation and metabolism experiments. RESULTS: No differences were found in 2,4-D absorption or translocation between resistant and susceptible A. tuberculatus plants. Resistant plants metabolized [14 C] 2,4-D more rapidly than did susceptible plants. The half-life of [14 C] 2,4-D in susceptible plants was 105 h, compared with 22 h in resistant plants. Pretreatment with the cytochrome P450 inhibitor malathion inhibited [14 C] 2,4-D metabolism in resistant plants and reduced the 2,4-D dose required for 50% growth inhibition (GR50 ) of resistant plants by 7-fold to 27 g ha-1 , similar to the GR50 for susceptible plants in the absence of malathion. CONCLUSION: Our results demonstrate that rapid 2,4-D metabolism is a contributing factor to resistance in A. tuberculatus, potentially mediated by cytochrome P450. Metabolism-based resistance to 2,4-D could pose a serious challenge for A. tuberculatus control because of the potential for cross-resistance to other herbicides. © 2017 Society of Chemical Industry.

Increased chalcone synthase (CHS) expression is associated with dicamba resistance in Kochia scoparia.

BACKGROUND: Resistance to the synthetic auxin herbicide dicamba is increasingly problematic in Kochia scoparia. The resistance mechanism in an inbred dicamba-resistant K. scoparia line (9425R) was investigated using physiological and transcriptomics (RNA-Seq) approaches. RESULTS: No differences were found in dicamba absorption or metabolism between 9425R and a dicamba-susceptible line, but 9425R was found to have significantly reduced dicamba translocation. Known auxin-responsive genes ACC synthase (ACS) and indole-3-acetic acid amino synthetase (GH3) were transcriptionally induced following dicamba treatment in dicamba-susceptible K. scoparia but not in 9425R. Chalcone synthase (CHS), the gene regulating synthesis of the flavonols quertecin and kaemperfol, was found to have twofold higher transcription in 9425R both without and 12 h after dicamba treatment. Increased CHS transcription co-segregated with dicamba resistance in a forward genetics screen using an F2 population. CONCLUSION: Prior work has shown that the flavonols quertecin and kaemperfol compete with auxin for intercellular movement and vascular loading via ATP-binding cassette subfamily B (ABCB) membrane transporters. The results of this study support a model in which constitutively increased CHS expression in the meristem produces more flavonols that would compete with dicamba for intercellular transport by ABCB transporters, resulting in reduced dicamba translocation. © 2017 Society of Chemical Industry.

Glyphosate resistance in Ambrosia trifida: Part 1. Novel rapid cell death response to glyphosate.

BACKGROUND: Glyphosate-resistant (GR) Ambrosia trifida is now present in the midwestern United States and in southwestern Ontario, Canada. Two distinct GR phenotypes are known, including a rapid response (GR RR) phenotype, which exhibits cell death within hours after treatment, and a non-rapid response (GR NRR) phenotype. The mechanisms of resistance in both GR RR and GR NRR remain unknown. Here, we present a description of the RR phenotype and an investigation of target-site mechanisms on multiple A. trifida accessions. RESULTS: Glyphosate resistance was confirmed in several accessions, and whole-plant levels of resistance ranged from 2.3- to 7.5-fold compared with glyphosate-susceptible (GS) accessions. The two GR phenotypes displayed similar levels of resistance, despite having dramatically different phenotypic responses to glyphosate. Glyphosate resistance was not associated with mutations in EPSPS sequence, increased EPSPS copy number, EPSPS quantity, or EPSPS activity. CONCLUSION: These encompassing results suggest that resistance to glyphosate in these GR RR A. trifida accessions is not conferred by a target-site resistance mechanism. © 2017 Society of Chemical Industry.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

EPSPS Gene Copy Number and Whole-Plant Glyphosate Resistance Level in Kochia scoparia.

Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism of resistance involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Agricultural fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska, and Montana. Glyphosate resistance was also confirmed in K. scoparia accessions collected from wheat-fallow fields in Montana. All GR samples had increased EPSPS gene copy number, with median population values up to 11 from sugarbeet fields and up to 13 in Montana wheat-fallow fields. The results indicate that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number.

Gene amplification of 5-enol-pyruvylshikimate-3-phosphate synthase in glyphosate-resistant Kochia scoparia.

MAIN CONCLUSION: Field-evolved resistance to the herbicide glyphosate is due to amplification of one of two EPSPS alleles, increasing transcription and protein with no splice variants or effects on other pathway genes. The widely used herbicide glyphosate inhibits the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Globally, the intensive use of glyphosate for weed control has selected for glyphosate resistance in 31 weed species. Populations of suspected glyphosate-resistant Kochia scoparia were collected from fields located in the US central Great Plains. Glyphosate dose response verified glyphosate resistance in nine populations. The mechanism of resistance to glyphosate was investigated using targeted sequencing, quantitative PCR, immunoblotting, and whole transcriptome de novo sequencing to characterize the sequence and expression of EPSPS. Sequence analysis showed no mutation of the EPSPS Pro106 codon in glyphosate-resistant K. scoparia, whereas EPSPS genomic copy number and transcript abundance were elevated three- to ten-fold in resistant individuals relative to susceptible individuals. Glyphosate-resistant individuals with increased relative EPSPS copy numbers had consistently lower shikimate accumulation in leaf disks treated with 100 µM glyphosate and EPSPS protein levels were higher in glyphosate-resistant individuals with increased gene copy number compared to glyphosate-susceptible individuals. RNA sequence analysis revealed seven nucleotide positions with two different expressed alleles in glyphosate-susceptible reads. However, one nucleotide at the seven positions was predominant in glyphosate-resistant sequences, suggesting that only one of two EPSPS alleles was amplified in glyphosate-resistant individuals. No alternatively spliced EPSPS transcripts were detected. Expression of five other genes in the chorismate pathway was unaffected in glyphosate-resistant individuals with increased EPSPS expression. These results indicate increased EPSPS expression is a mechanism for glyphosate resistance in these K. scoparia populations.

Tandem amplification of a chromosomal segment harboring 5-enolpyruvylshikimate-3-phosphate synthase locus confers glyphosate resistance in Kochia scoparia.

Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.

Characterization of glyphosate resistance in Amaranthus tuberculatus populations.

The evolution of glyphosate-resistant weeds has recently increased dramatically. Six suspected glyphosate-resistant Amaranthus tuberculatus populations were studied to confirm resistance and determine the resistance mechanism. Resistance was confirmed in greenhouse for all six populations with glyphosate resistance factors (R/S) between 5.2 and 7.5. No difference in glyphosate absorption or translocation was observed between resistant and susceptible individuals. No mutation at amino acid positions G101, T102, or P106 was detected in the EPSPS gene coding sequence, the target enzyme of glyphosate. Analysis of EPSPS gene copy number revealed that all glyphosate-resistant populations possessed increased EPSPS gene copy number, and this correlated with increased expression at both RNA and protein levels. EPSPS Vmax and Kcat values were more than doubled in resistant plants, indicating higher levels of catalytically active expressed EPSPS protein. EPSPS gene amplification is the main mechanism contributing to glyphosate resistance in the A. tuberculatus populations analyzed.