Resistance to a nonselective 4-hydroxyphenylpyruvate dioxygenase-inhibiting herbicide via novel reduction-dehydration-glutathione conjugation in Amaranthus tuberculatus.

Metabolic resistance to 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides is a threat in controlling waterhemp (Amaranthus tuberculatus) in the USA. We investigated resistance mechanisms to syncarpic acid-3 (SA3), a nonselective, noncommercial HPPD-inhibiting herbicide metabolically robust to Phase I oxidation, in multiple-herbicide-resistant (MHR) waterhemp populations (SIR and NEB) and HPPD inhibitor-sensitive populations (ACR and SEN). Dose-response experiments with SA3 provided ED50 -based resistant : sensitive ratios of at least 18-fold. Metabolism experiments quantifying parent SA3 remaining in excised leaves during a time course indicated MHR populations displayed faster rates of SA3 metabolism compared to HPPD inhibitor-sensitive populations. SA3 metabolites were identified via LC-MS-based untargeted metabolomics in whole plants. A Phase I metabolite, likely generated by cytochrome P450-mediated alkyl hydroxylation, was detected but was not associated with resistance. A Phase I metabolite consistent with ketone reduction followed by water elimination was detected, creating a putative α,ß-unsaturated carbonyl resembling a Michael acceptor site. A Phase II glutathione-SA3 conjugate was associated with resistance. Our results revealed a novel reduction-dehydration-GSH conjugation detoxification mechanism. SA3 metabolism in MHR waterhemp is thus atypical compared to commercial HPPD-inhibiting herbicides. This previously uncharacterized detoxification mechanism presents a unique opportunity for future biorational design by blocking known sites of herbicide metabolism in weeds.

Derived Polymorphic Amplified Cleaved Sequence (dPACS): A Novel PCR-RFLP Procedure for Detecting Known Single Nucleotide and Deletion-Insertion Polymorphisms.

Most methods developed for detecting known single nucleotide polymorphisms (SNP) and deletion-insertion polymorphisms (DIP) are dependent on sequence conservation around the SNP/DIP and are therefore not suitable for application to heterogeneous organisms. Here we describe a novel, versatile and simple PCR-RFLP procedure baptised 'derived Polymorphic Amplified Cleaved Sequence' (dPACS) for genotyping individual samples. The notable advantage of the method is that it employs a pair of primers that cover the entire fragment to be amplified except for one or few diagnostic bases around the SNP/DIP being investigated. As such, it provides greater opportunities to introduce mismatches in one or both of the 35-55 bp primers for creating a restriction site that unambiguously differentiates wild from mutant sequences following PCR-RFLP and horizontal MetaPhorTM gel electrophoresis. Selection of effective restriction enzymes and primers is aided by the newly developed dPACS 1.0 software. The highly transferable dPACS procedure is exemplified here with the positive detection (in up to 24 grass and broadleaf species tested) of wild type proline106 of 5-enolpyruvylshikimate-3-phosphate synthase and its serine, threonine and alanine variants that confer resistance to glyphosate, and serine264 and isoleucine2041 which are key target-site determinants for weed sensitivities to some photosystem II and acetyl-CoA carboxylase inhibiting herbicides, respectively.

Key role for a glutathione transferase in multiple-herbicide resistance in grass weeds.

Multiple-herbicide resistance (MHR) in black-grass (Alopecurus myosuroides) and annual rye-grass (Lolium rigidum) is a global problem leading to a loss of chemical weed control in cereal crops. Although poorly understood, in common with multiple-drug resistance (MDR) in tumors, MHR is associated with an enhanced ability to detoxify xenobiotics. In humans, MDR is linked to the overexpression of a pi class glutathione transferase (GSTP1), which has both detoxification and signaling functions in promoting drug resistance. In both annual rye-grass and black-grass, MHR was also associated with the increased expression of an evolutionarily distinct plant phi (F) GSTF1 that had a restricted ability to detoxify herbicides. When the black-grass A. myosuroides (Am) AmGSTF1 was expressed in Arabidopsis thaliana, the transgenic plants acquired resistance to multiple herbicides and showed similar changes in their secondary, xenobiotic, and antioxidant metabolism to those determined in MHR weeds. Transcriptome array experiments showed that these changes in biochemistry were not due to changes in gene expression. Rather, AmGSTF1 exerted a direct regulatory control on metabolism that led to an accumulation of protective flavonoids. Further evidence for a key role for this protein in MHR was obtained by showing that the GSTP1- and MDR-inhibiting pharmacophore 4-chloro-7-nitro-benzoxadiazole was also active toward AmGSTF1 and helped restore herbicide control in MHR black-grass. These studies demonstrate a central role for specific GSTFs in MHR in weeds that has parallels with similar roles for unrelated GSTs in MDR in humans and shows their potential as targets for chemical intervention in resistant weed management.

Derived Polymorphic Amplified Cleaved Sequence (dPACS) Assay.

The derived polymorphic amplified cleaved sequence (dPACS) assay is a simple polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP)-based procedure for detecting known single-nucleotide polymorphisms (SNPs) and deletion-insertion polymorphisms (DIPs). It is relatively straightforward to carry out using basic and commonly available molecular biology kits. The method differs from other PCR-RFLP assays in that it employs 35-55 bp primer pairs that encompass the entire targeted DNA region except for a few diagnostic nucleotides being examined. In so doing, it allows for the introduction of nucleotide mismatches in one or both primers for differentiating wild from mutant sequences following polymerase chain reaction, restriction digestion and MetaPhor gel electrophoresis. Primer design and the selection of discriminating enzymes are achieved with the help of the dPACS 1.0 program. The method is exemplified here with the positive detection of serine 264-psbA, a key determinant for the effective binding of some photosystem II inhibitors to their target. A serine-to-glycine mutation at codon 264 of psbA causes resistance to serine-binding photosystem II herbicides in several grasses and broad-leaf weeds, including Amaranthus retroflexus, which is employed in this study.

Impact of a Novel W2027L Mutation and Non-Target Site Resistance on Acetyl-CoA Carboxylase-Inhibiting Herbicides in a French Lolium multiflorum Population.

Herbicides that inhibit acetyl-CoA carboxylase (ACCase) are among the few remaining options for the post-emergence control of Lolium species in small grain cereal crops. Here, we determined the mechanism of resistance to ACCase herbicides in a Lolium multiflorum population (HGR) from France. A combined biological and molecular approach detected a novel W2027L ACCase mutation that affects aryloxyphenoxypropionate (FOP) but not cyclohexanedione (DIM) or phenylpyraxoline (DEN) subclasses of ACCase herbicides. Both the wild-type tryptophan and mutant leucine 2027-ACCase alleles could be positively detected in a single DNA-based-derived polymorphic amplified cleaved sequence (dPACS) assay that contained the targeted PCR product and a cocktail of two discriminating restriction enzymes. Additionally, we identified three well-characterised I1781L, I2041T, and D2078G ACCase target site resistance mutations as well as non-target site resistance in HGR. The non-target site component endowed high levels of resistance to FOP herbicides whilst partially impacting on the efficacy of pinoxaden and cycloxydim. This study adequately assessed the contribution of the W2027L mutation and non-target site mechanism in conferring resistance to ACCase herbicides in HGR. It also highlights the versatility and robustness of the dPACS method to simultaneously identify different resistance-causing alleles at a single ACCase codon.

Fitness Cost Associated With Enhanced EPSPS Gene Copy Number and Glyphosate Resistance in an Amaranthus tuberculatus Population.

The evolution of resistance to pesticides in agricultural systems provides an opportunity to study the fitness costs and benefits of novel adaptive traits. Here, we studied a population of Amaranthus tuberculatus (common waterhemp), which has evolved resistance to glyphosate. The growth and fitness of seed families with contrasting levels of glyphosate resistance was assessed in the absence of glyphosate to determine their ability to compete for resources under intra- and interspecific competition. We identified a positive correlation between the level of glyphosate resistance and gene copy number for the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) glyphosate target, thus identifying gene amplification as the mechanism of resistance within the population. Resistant A. tuberculatus plants were found to have a lower competitive response when compared to the susceptible phenotypes with 2.76 glyphosate resistant plants being required to have an equal competitive effect as a single susceptible plant. A growth trade-off was associated with the gene amplification mechanism under intra-phenotypic competition where 20 extra gene copies were associated with a 26.5 % reduction in dry biomass. Interestingly, this growth trade-off was mitigated when assessed under interspecific competition from maize.

A derived Polymorphic Amplified Cleaved Sequence assay for detecting the Δ210 PPX2L codon deletion conferring target-site resistance to protoporphyrinogen oxidase-inhibiting herbicides.

BACKGROUND: Resistance to protoporphyrinogen oxidase (PPO)-inhibiting herbicides in Amaranthus rudis from corn/soybean production systems in the USA appears to be mainly due to a codon deletion at position 210 of the target PPX2L gene. In this study, we have developed a simple and cost-effective derived Polymorphic Amplified Cleaved Sequenced (dPACS) marker for detecting this resistance-causing deletion in A. rudis and other relevant weed species. RESULTS: Ninety-six plants from 16 diverse fomesafen-sensitive and resistant A. rudis populations from Illinois and Iowa were used to establish the dPACS procedure. The assay requires forced mismatches in both the forward and reverse PCR primers and uses the restriction enzyme XcmI for the positive identification of wild type glycine residue at PPX2L codon position 210. The data from the dPACS method, using either leaf tissues or seeds as starting material, were completely correlated with direct Sanger sequencing results for samples that gave readable nucleotide peaks around codon 210 of PPX2L. Furthermore, the assay was directly transferable to all four other Amaranthus species tested, and to Ambrosia artemisiifolia using species-specific primers. CONCLUSION: The proposed assay will allow the rapid detection of the Δ210 codon deletion in the PPX2L gene and the timely development of management strategies for tackling growing resistance to PPO-inhibiting herbicides in A. rudis and other broadleaf weed species. © 2019 Society of Chemical Industry.

A holistic approach in herbicide resistance research and management: from resistance detection to sustainable weed control.

Agricultural weeds can adapt rapidly to human activities as exemplified by the evolution of resistance to herbicides. Despite its multi-faceted nature, herbicide resistance has rarely been researched in a holistic manner. A novel approach combining timely resistance confirmation, investigation of resistance mechanisms, alternative control solutions and population modelling was adopted for the sustainable management of the Amaranthus palmeri weed in soybean production systems in Argentina. Here, we show that resistance to glyphosate in the studied population from Cordoba province was mainly due to a P106S target-site mutation in the 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) gene, with minor contributions from EPSPS gene duplication/overexpression. Alternative herbicides, such as fomesafen, effectively controlled the glyphosate-resistant plants. Model simulations revealed the tendency of a solo herbicidal input to primarily select for a single resistance mechanism and suggested that residual herbicides, alongside chemical diversity, were important for the sustainable use of these herbicides. We also discuss the value of an interdisciplinary approach for improved understanding of evolving weeds.

Evolution of Target-Site Resistance to Glyphosate in an Amaranthus palmeri Population from Argentina and Its Expression at Different Plant Growth Temperatures.

The mechanism and expression of resistance to glyphosate at different plant growing temperatures was investigated in an Amaranthus palmeri population (VM1) from a soybean field in Vicuña Mackenna, Cordoba, Argentina. Resistance was not due to reduced glyphosate translocation to the meristem or to EPSPS duplication, as reported for most US samples. In contrast, a proline 106 to serine target-site mutation acting additively with EPSPS over-expression (1.8-fold increase) was respectively a major and minor contributor to glyphosate resistance in VM1. Resistance indices based on LD50 values generated using progenies from a cross between 52 PS106 VM1 individuals were estimated at 7.1 for homozygous SS106 and 4.3 for heterozygous PS106 compared with homozygous wild PP106 plants grown at a medium temperature of 24 °C day/18 °C night. A larger proportion of wild and mutant progenies survived a single commonly employed glyphosate rate when maintained at 30 °C day/26 °C night compared with 20 °C day/16 night in a subsequent experiment. Interestingly, the P106S mutation was not identified in any of the 920 plants analysed from 115 US populations, thereby potentially reflecting the difference in A. palmeri control practices in Argentina and USA.

Mechanism of resistance to mesotrione in an Amaranthus tuberculatus population from Nebraska, USA.

Amaranthus tuberculatus is a troublesome weed in corn and soybean production systems in Midwestern USA, due in part to its ability to evolve multiple resistance to key herbicides including 4-hydroxyphenylpyruvate dioxygenase (HPPD). Here we have investigated the mechanism of resistance to mesotrione, an important chemical for managing broadleaf weeds in corn, in a multiple herbicide resistant population (NEB) from Nebraska. NEB showed a 2.4-fold and 45-fold resistance increase to mesotrione compared to a standard sensitive population (SEN) in pre-emergence and post-emergence dose-response pot tests, respectively. Sequencing of the whole HPPD gene from 12 each of sensitive and resistant plants did not detect any target-site mutations that could be associated with post-emergence resistance to mesotrione in NEB. Resistance was not due to HPPD gene duplication or over-expression before or after herbicide treatment, as revealed by qPCR. Additionally, no difference in mesotrione uptake was detected between NEB and SEN. In contrast, higher levels of mesotrione metabolism via 4-hydroxylation of the dione ring were observed in NEB compared to the sensitive population. Overall, the NEB population was characterised by lower levels of parent mesotrione exported to other parts of the plant, either as a consequence of metabolism in the treated leaves and/or impaired translocation of the herbicide. This study demonstrates another case of non-target-site based resistance to an important class of herbicides in an A. tuberculatus population. The knowledge generated here will help design strategies for managing multiple herbicide resistance in this problematic weed species.

A novel W1999S mutation and non-target site resistance impact on acetyl-CoA carboxylase inhibiting herbicides to varying degrees in a UK Lolium multiflorum population.

BACKGROUND: Acetyl-CoA carboxylase (ACCase) inhibiting herbicides are important products for the post-emergence control of grass weed species in small grain cereal crops. However, the appearance of resistance to ACCase herbicides over time has resulted in limited options for effective weed control of key species such as Lolium spp. In this study, we have used an integrated biological and molecular biology approach to investigate the mechanism of resistance to ACCase herbicides in a Lolium multiflorum Lam. from the UK (UK21). METHODOLOGY/PRINCIPAL FINDINGS: The study revealed a novel tryptophan to serine mutation at ACCase codon position 1999 impacting on ACCase inhibiting herbicides to varying degrees. The W1999S mutation confers dominant resistance to pinoxaden and partially recessive resistance to cycloxydim and sethoxydim. On the other hand, plants containing the W1999S mutation were sensitive to clethodim and tepraloxydim. Additionally population UK21 is characterised by other resistance mechanisms, very likely non non-target site based, affecting several aryloxyphenoxyproprionate (FOP) herbicides but not the practical field rate of pinoxaden. The positive identification of wild type tryptophan and mutant serine alleles at ACCase position 1999 could be readily achieved with an original DNA based derived cleaved amplified polymorphic sequence (dCAPS) assay that uses the same PCR product but two different enzymes for positively identifying the wild type tryptophan and mutant serine alleles identified here. CONCLUSION/SIGNIFICANCE: This paper highlights intrinsic differences between ACCase inhibiting herbicides that could be exploited for controlling ryegrass populations such as UK21 characterised by compound-specific target site and non-target site resistance.

Role of a novel I1781T mutation and other mechanisms in conferring resistance to acetyl-CoA carboxylase inhibiting herbicides in a black-grass population.

BACKGROUND: Knowledge 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 FINDINGS: Comparison 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/SIGNIFICANCE: This 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.

Broad resistance to ACCase inhibiting herbicides in a ryegrass population is due only to a cysteine to arginine mutation in the target enzyme.

BACKGROUND: The design of sustainable weed management strategies requires a good understanding of the mechanisms by which weeds evolve resistance to herbicides. Here we have conducted a study on the mechanism of resistance to ACCase inhibiting herbicides in a Lolium multiflorum population (RG3) from the UK. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of plant phenotypes and genotypes showed that all the RG3 plants (72%) that contained the cysteine to arginine mutation at ACCase codon position 2088 were resistant to ACCase inhibiting herbicides. Whole plant dose response tests on predetermined wild and mutant 2088 genotypes from RG3 and a standard sensitive population indicated that the C2088R mutation is the only factor conferring resistance to all ten ACCase herbicides tested. The associated resistance indices ranged from 13 for clethodim to over 358 for diclofop-methyl. Clethodim, the most potent herbicide was significantly affected even when applied on small mutant plants at the peri-emergence and one leaf stages. CONCLUSION/SIGNIFICANCE: This study establishes the clear and unambiguous importance of the C2088R target site mutation in conferring broad resistance to ten commonly used ACCase inhibiting herbicides. It also demonstrates that low levels "creeping", multigenic, non target site resistance, is not always selected before single gene target site resistance appears in grass weed populations subjected to herbicide selection pressure.