Mechanism Of Herbicide Resistance Research Articles

Resistance to Acetyl Coenzyme A Carboxylase (ACCase) Inhibitor in Lolium multiflorum: Effect of Multiple Target-Site Mutations

Italian ryegrass (Lolium multiflorum Lam.) is a persistent weed species that poses significant management challenges in key agricultural crops such as wheat, corn, cotton, and soybean. This study investigated the prevalence of resistance to ACCase inhibitor herbicides, specifically diclofop and pinoxaden, among field-collected Italian ryegrass populations. The survey revealed widespread resistance to diclofop and emerging cross-resistance to pinoxaden. To elucidate the physiological mechanism of ACCase herbicide resistance, we investigated mutations in the carboxyl-transferase (CT) domain of the ACCase enzyme, a critical region for herbicide sensitivity. Using dCAPS assays and CT domain sequencing, several known resistance-conferring mutations were detected in diclofop survivors, including I1781L, W2027C, I2041N, D2078G, and C2088R. Additionally, other mutations such as L1701M, E1874A, N1878H, G1946E/Q, V1992D, and E2039D were identified. To understand the functional role of these mutations in herbicide resistance, homology modeling was performed using AutoDock Vina for selected mutation combinations. The computational analysis revealed that all mutations and their combinations resulted in reduced binding affinity with diclofop and pinoxaden compared to the wild-type ACCase CT domain. Computational binding energy predictions indicated that the G1946E mutation and the L1701M + I1781L + E1874A + N1878H combination exhibited the lowest affinities for diclofop and pinoxaden, respectively. This study provides valuable insights into the molecular basis of ACCase inhibitor resistance in Italian ryegrass. However, further research is needed to validate the functional significance of each new substitution and its combinations in conferring herbicide resistance.

The mutation Asp-376-Glu in the ALS gene confers resistance to mesosulfuron-methyl in Beckmannia syzigachne

Understanding the mechanisms by which weeds develop herbicide resistance is crucial for managing resistance effectively and optimizing herbicide use. Beckmannia syzigachne, a harmful grass weed prevalent in wheat and rice-wheat rotation areas, poses a significant threat to crop productivity. A field herbicide resistance survey identified a resistant population with a new ALS mutation (Asp-376-Glu). The Glu-376-Asp population displayed varying resistance levels to seven ALS herbicides, verified using the dCAPS method. qRT-PCR analysis showed that no significant difference existed in the ALS gene expression between the Asp-376-Glu and S populations. P450 and GST inhibitors failed to reverse resistance to mesosulfuron-methyl, suggesting no involvement of P450- and GST-based metabolic resistance. Molecular docking indicated that the Asp-376-Glu mutation reduces the binding affinity between ALS-inhibitors and BsALS. The findings provide valuable insights into herbicide resistance mechanisms for weed resistance control.

Enhanced Herbicide Metabolism and Target Site Mutation Enabled the Multiple Resistance to Cyhalofop-butyl, Florpyrauxifen-benzyl, and Penoxsulam in Echinochloa crus-galli.

This study investigated the multiple herbicide resistance (MHR) mechanism of one Echinochloa crus-galli population that was resistant to florpyrauxifen-benzyl (FPB), cyhalofop-butyl (CHB), and penoxsulam (PEX). This population carried an Ala-122-Asn mutation in the acetolactate synthase (ALS) gene but no mutation in acetyl-CoA carboxylase (ACCase) and transport inhibitor response1 (TIR1) genes. The metabolism rate of PEX was 2-fold higher, and the production of florpyrauxifen-acid and cyhalofop-acid was lower in the resistant population. Malathion and 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) could reverse the resistance, suggesting that cytochrome P450 (CYP450) and glutathione S-transferase (GST) contribute to the enhanced metabolism. According to RNA-seq and qRT-PCR validation, two CYP450 genes (CYP71C42 and CYP71D55), one GST gene (GSTT2), two glycosyltransferase genes (rhamnosyltransferase 1 and IAAGLU), and two ABC transporter genes (ABCG1 and ABCG25) were induced by CHB, FPB, and PEX in the resistant population. This study revealed that the target mutant and enhanced metabolism were involved in the MHR mechanism in E. crus-galli.

Amaranthus palmeri S. Watson reproduction system: Implications for distribution and management strategies

AbstractAmaranthus palmeri S. Watson is one of the most troublesome weed species worldwide, and is considered one of the most devastating weeds in agricultural fields in the United States. Recently, A. palmeri populations have spread beyond their native range, into the Mediterranean region, and have been reported in several European countries. Resistance to 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) and acetolactate synthase (ALS) inhibitors have been found in European populations, exacerbating the management problems with this species in the Mediterranean region. While the biology, ecology, herbicide resistance mechanisms, and agricultural significance of A. palmeri have been studied, there remains a need to discuss how A. palmeri's unique reproductive traits influence its ability to adapt to various environments, especially as an invasive species spreading into new habitats. We review how the genetic and genomic characteristics of A. palmeri interact with aspects of its reproductive biology, including its breeding system, and alter its ability to hybridize and develop herbicide resistance. Finally, we discuss the breeding system of A. palmeri in the context of weed management, and explore different approaches such as irradiated pollens, genetic manipulations, and RNA interference (RNAi).

Gene flow and spontaneous mutations are responsible for imidazolinone herbicide-resistant weedy rice (Oryza sativa L.)

For more than two decades, weedy rice (Oryza sativa L.) has been controlled in rice fields by using imidazolinone (IMI) herbicide-resistant rice technology (Clearfield®). Outcrossing in weedy rice populations and spontaneous mutations are potential problems with herbicide-resistant crop management technologies, such as the IMI-resistant rice. The aim of this study was to characterize the mechanism of IMI herbicide resistance in weedy rice through dose-response bioassay study and evaluating amino acid substitutions in acetolactate synthase (ALS) protein. A total of 118 suspected IMI-resistant weedy rice samples, which survived in the field after an IMI herbicide application, were collected at harvest time from Türkiye in 2020 and 2021. Single-dose imazamox application experiment revealed that 38 plants survived herbicide treatment. The imazamox resistance of the surviving plants was confirmed by dose-response experiment. ALS gene region underwent a sanger DNA partial sequencing. No substitution was found in 10 samples, however, amino acid substitutions were found in 26 samples with S563N, one sample with S653T, and one sample with E630D. The S653N point is the same substitution point that serves as the origin of resistance for the Clearfield® rice varieties that are commonly cultivated in the region. It has been hypothesized that the gene flow from IMI-resistant rice may be the cause of resistance in the IMI resistant weedy rice samples with S653N. The other substitution, S653T, were considered spontaneous mutation to IMI resistance. Interestingly, the S653T mutation was detected for the first time in weedy rice. The mechanism of resistance of 10 resistant weedy rice was not confirmed in this study, however, it may be a non-target resistance or another mutation point in target site, but evidently, they did not acquire resistance by gene flow from IMI-resistant rice. It has been concluded that the effectiveness of IMI-resistant rice technology in controlling weedy rice has drastically decreased due to possible gene flow, spontaneous mutation and non-target resistance. In addition to cultural controls like clean seed, clean machinery and crop rotation, other herbicide-tolerant rice systems such as Provisia® and Roxy-RPS® rice are needed to create a diverse weedy rice management ensemble available for rice production and move towards sustainable rice farming.

Repeated evolution of herbicide resistance in Lolium multiflorum revealed by haplotype-resolved analysis of acetyl-CoA carboxylase.

Herbicide resistance in weeds is one of the greatest challenges in modern food production. The grass species Lolium multiflorum is an excellent model species to investigate evolution under similar selection pressure because populations have repeatedly evolved resistance to many herbicides, utilizing a multitude of mechanisms to neutralize herbicide damage. In this work, we investigated the gene that encodes acetyl-CoA carboxylase (ACCase), the target site of the most successful herbicide group available for grass weed control. We sampled L. multiflorum populations from agricultural fields with history of intense herbicide use, and studied their response to three ACCase-inhibiting herbicides. To elucidate the mechanisms of herbicide resistance and the genetic relationship among populations, we resolved the haplotypes of 97 resistant and susceptible individuals by sequencing ACCase amplicons using long-read DNA sequencing technologies. Our dose-response data indicated the existence of many, often unpredictable, resistance patterns to ACCase-inhibiting herbicides, where populations exhibited as much as 37-fold reduction in herbicide response. The majority of the populations exhibited resistance to all three herbicides studied. Phylogenetic and molecular genetic analyses revealed multiple evolutionary origins of resistance-endowing ACCase haplotypes, as well as widespread admixture in the region regardless of cropping system. The amplicons generated were diverse, with haplotypes exhibiting 26-110 polymorphisms. Polymorphisms included insertions and deletions 1-31 bp in length, none of which were associated with the resistance phenotype based on an association analysis. We also found evidence that some populations have multiple mechanisms of resistance. Our results highlight the astounding genetic diversity in L. multiflorum populations, and the potential for repeated evolution of herbicide resistance across the landscape that challenges weed management approaches and jeopardizes sustainable weed control practices. We provide an in-depth discussion of the evolutionary and practical implications of our results.

Comprehensive insights into herbicide resistance mechanisms in weeds: a synergistic integration of transcriptomic and metabolomic analyses.

Omics techniques, including genomics, transcriptomics, proteomics, and metabolomics have smoothed the researcher's ability to generate hypotheses and discover various agronomically relevant functions and mechanisms, as well as their implications and associations. With a significant increase in the number of cases with resistance to multiple herbicide modes of action, studies on herbicide resistance are currently one of the predominant areas of research within the field of weed science. High-throughput technologies have already started revolutionizing the current molecular weed biology studies. The evolution of herbicide resistance in weeds (particularly via non-target site resistance mechanism) is a perfect example of a complex, multi-pathway integration-induced response. To date, functional genomics, including transcriptomic and metabolomic studies have been used separately in herbicide resistance research, however there is a substantial lack of integrated approach. Hence, despite the ability of omics technologies to provide significant insights into the molecular functioning of weeds, using a single omics can sometimes be misleading. This mini-review will aim to discuss the current progress of transcriptome-based and metabolome-based approaches in herbicide resistance research, along with their systematic integration.

Identification and Expression Analysis of EPSPS and BAR Families in Cotton.

Weeds seriously affect the yield and quality of crops. Because manual weeding is time-consuming and laborious, the use of herbicides becomes an effective way to solve the harm caused by weeds in fields. Both 5-enolpyruvyl shikimate-3-phosphate synthetase (EPSPS) and acetyltransferase genes (bialaphos resistance, BAR) are widely used to improve crop resistance to herbicides. However, cotton, as the most important natural fiber crop, is not tolerant to herbicides in China, and the EPSPS and BAR family genes have not yet been characterized in cotton. Therefore, we explore the genes of these two families to provide candidate genes for the study of herbicide resistance mechanisms. In this study, 8, 8, 4, and 5 EPSPS genes and 6, 6, 5, and 5 BAR genes were identified in allotetraploid Gossypium hirsutum and Gossypium barbadense, diploid Gossypium arboreum and Gossypium raimondii, respectively. Members of the EPSPS and BAR families were classified into three subgroups based on the distribution of phylogenetic trees, conserved motifs, and gene structures. In addition, the promoter sequences of EPSPS and BAR family members included growth and development, stress, and hormone-related cis-elements. Based on the expression analysis, the family members showed tissue-specific expression and differed significantly in response to abiotic stresses. Finally, qRT-PCR analysis revealed that the expression levels of GhEPSPS3, GhEPSPS4, and GhBAR1 were significantly upregulated after exogenous spraying of herbicides. Overall, we characterized the EPSPS and BAR gene families of cotton at the genome-wide level, which will provide a basis for further studying the functions of EPSPS and BAR genes during growth and development and herbicide stress.

Variability to flooding tolerance in barnyardgrass and early flooding benefits on weed management and rice grain yield

Barnyardgrass (Echinochloa crus-galli (L.) Beauv.) is one of the most problematic weed species in paddy fields. The recent large-scale evolution of herbicide resistance in this species requires the adoption of integrated methods of weed control. The present study aimed to determine the interaction of water depth, flooding irrigation onset, and pre-emergent herbicides on the control of barnyardgrass populations and to identify the variability and the putative genes involved in the regulation of flooding tolerance in this species. We identified tolerant, moderately tolerant, and flood-sensitive barnyardgrass populations through water depth-response experiments in a greenhouse. The relative expression of genes related to flooding tolerance were evaluated to confirm the flooding tolerance variability. A field experiment was conducted during the summer seasons of 2020/21 and 2021/22 to assess the interaction of the flooding onset, water depth, and pre-emergent herbicides on populations with different flood tolerances and herbicide resistance mechanisms. Water depths of 7.5 cm and 12.5 cm and plant height were selected for the diagnosis of flood tolerance. After 28 days under flooding, the 7.5 cm depth reduced plant height by an average of 26.82%. Of the 45 populations evaluated, 43 overcame the water depth of 7.5 cm, and four surpassed 12.5 cm. The relative expression of alcohol dehydrogenase 1 (ADH1), alcohol dehydrogenase 2 (ADH2), pyruvate decarboxylase (PDC1), aldehyde dehydrogenase (ALDH), and sucrose synthase 3 (SUS3) was between eight and sixteen times higher for the flood-tolerant population than for the sensitive one. In the field study, the anticipation of flooding to the two-leaves (V2) stage of rice resulted in an increase of up to 55.6% in the control of all populations evaluated compared with conventional irrigation at the four-leaves (V4) stage, including those resistant to herbicides. The grain yield was increased by approximately 11.4% with flooding onset in the V2 stage compared to the V4 stage. Barnyardgrass control and rice grain yield were not affected by water depths of 5 cm or 15 cm. The early flooding, in the V2 stage, increased rice grain yield and favored barnyardgrass control. However, variability in flood tolerance was identified in barnyardgrass populations, which may jeopardize the effect of early flooding for weed control in rice crops. The results we obtained add information on the basic knowledge of flooding tolerance in barnyardgrass and to the integrated weed management for dry direct-seeded rice.

Genomic adaptation of Burkholderia anthina to glyphosate uncovers a novel herbicide resistance mechanism.

Glyphosate (GS) specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that converts phosphoenolpyruvate (PEP) and shikimate-3-phosphate to EPSP in the shikimate pathway of bacteria and other organisms. The inhibition of the EPSP synthase depletes the cell of the EPSP-derived aromatic amino acids as well as of folate and quinones. A variety of mechanisms (e.g., EPSP synthase modification) has been described that confer GS resistance to bacteria. Here, we show that the Burkholderia anthina strain DSM 16086 quickly evolves GS resistance by the acquisition of mutations in the ppsR gene. ppsR codes for the pyruvate/ortho-Pi dikinase PpsR that physically interacts and regulates the activity of the PEP synthetase PpsA. The mutational inactivation of ppsR causes an increase in the cellular PEP concentration, thereby abolishing the inhibition of the EPSP synthase by GS that competes with PEP for binding to the enzyme. Since the overexpression of the Escherichia coli ppsA gene in Bacillus subtilis and E. coli did not increase GS resistance in these organisms, the mutational inactivation of the ppsR gene resulting in PpsA overactivity is a GS resistance mechanism that is probably unique to B. anthina.

Nontarget Site-Based Resistance to Fenoxaprop-P-ethyl and Candidate Genes Involved in Alopecurus japonicus

Nontarget-site resistance (NTSR) is a complex multigenic trait that is associated with the potential mechanisms of herbicide resistance which pose a serious threat to global crop protection. However, the NTSR mechanisms of Alopecurus japonicus, a malignant weed infesting wheat fields, are less characterized. In this study, we used RNA-sequencing transcriptome and enzyme activity detection to investigate the NTSR mechanisms and candidate genes involved in fenoxaprop-P-ethyl (FE) in a previously identified resistant population compared to the sensitive population of A. japonicus. Transcriptome analysis identified nine upregulated genes, which were constitutively overexpressed and upregulated by FE application in the resistant population, and the results were validated using quantitative real-time PCR. These genes including one cytochrome P450 monooxygenase (P450) gene (CYP75B4), one ATP-binding cassette (ABC) transporter gene (ABCG36), one laccase (LAC) gene (LAC15), one 9-cis-epoxycarotenoid dioxygenase (NCED) gene (NCED5), two purple acid phosphatase (PAP) genes (PAP4, PAP15), one sucrose phosphate synthase (SPS) gene (SPS3), one protein related to disease resistance gene (RGA3) and one immune protein gene (R1B-17). The activity assay of LAC, NCED, PAP and SPS revealed that the activities of these enzymes in the resistant population were significantly higher than those in the sensitive population at 0 h and after FE application at 12 h, 24 h and 72 h. Nevertheless, whether LAC, NCED, PAP and SPS genes were involved in herbicide metabolism needs to be further validated. Our results revealed that CYP, ABC transporter and LAC genes may participate in A. japonicus resistance. These genes identified in the present study provide new insights into the resistance mechanism of weeds in response to herbicide. Our study also implies the complexity of the NTSR mechanisms of weeds.

Evolution of Herbicide Resistant Weeds in Agro-ecological Systems

Weed management through herbicides continues to be the most efficient and cost-effective component of integrated weed management (IWM) in crop production systems. However, the current production systems are characterised by intensive, inappropriate use of herbicides along with misapplication which creates selection pressure and leads to the rapid evolution of herbicide resistance in weeds. Currently, there are 511 unique cases of herbicide-resistant weed species globally, involving 266 species and these comprise 153 and 113 dicots and monocots respectively. To date, herbicide resistance has been reported from 96 crops in more than 71 countries. Furthermore, weeds have evolved resistance to 21 of the 31 known sites of herbicide action in different crops. Understanding the mechanisms of herbicide resistance is therefore essential which when coupled with the ecological and management factors that affect herbicide resistance would lead to the development of appropriate, profitable, and sustainable weed management strategies. The review is aimed at the development of herbicide resistance, mechanisms of herbicide resistance, factors that influence the rate of resistance development and management of herbicide resistance by growers.

Reduced coenzyme Q synthesis confers non-target site resistance to the herbicide thaxtomin A

Herbicide resistance in weeds is a growing threat to global crop production. Non-target site resistance is problematic because a single resistance allele can confer tolerance to many herbicides (cross resistance), and it is often a polygenic trait so it can be difficult to identify the molecular mechanisms involved. Most characterized molecular mechanisms of non-target site resistance are caused by gain-of-function mutations in genes from a few key gene families-the mechanisms of resistance caused by loss-of-function mutations remain unclear. In this study, we first show that the mechanism of non-target site resistance to the herbicide thaxtomin A conferred by loss-of-function of the gene PAM16 is conserved in Marchantia polymorpha, validating its use as a model species with which to study non-target site resistance. To identify mechanisms of non-target site resistance caused by loss-of-function mutations, we generated 107 UV-B mutagenized M. polymorpha spores and screened for resistance to the herbicide thaxtomin A. We isolated 13 thaxtomin A-resistant mutants and found that 3 mutants carried candidate resistance-conferring SNPs in the MpRTN4IP1L gene. Mprtn4ip1l mutants are defective in coenzyme Q biosynthesis and accumulate higher levels of reactive oxygen species (ROS) than wild-type plants. Mutants are weakly resistant to thaxtomin A and cross resistant to isoxaben, suggesting that loss of MpRTN4IP1L function confers non-target site resistance. Mutants are also defective in thaxtomin A metabolism. We conclude that loss of MpRTN4IP1L function is a novel mechanism of non-target site herbicide resistance and propose that other mutations that increase ROS levels or decrease thaxtomin A metabolism could contribute to thaxtomin A resistance in the field.

Determination of Paraquat in Arabidopsis Tissues and Protoplasts by UHPLC-MS/MS.

Paraquat is a cost-effective herbicide, widely used in many countries, that can induce severe oxidative stress in photosynthetic tissues. Studying plant herbicide resistance or antioxidant stress mechanisms requires determining the cellular paraquat level when plants are treated by paraquat. The traditional isotopic labeling method has the potential risk to cause problems to both human health and the environment. For radioisotope manipulation, special operation spaces and strict environmental inspection are also required. In addition, the radiolabeled paraquat is increasingly hard to buy due to the extended production cycle. Here, we describe a nonradioactive method to determine the paraquat level in a small number of Arabidopsis tissues or protoplasts, using a high resolution ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry (MS)/MS method. This method is highly selective and sensitive, and more environmentally compatible and technically feasible than the isotope detection method.

Integrated transcriptomic and metabolomic analyses of the molecular mechanisms of two highland barley genotypes with pyroxsulam responses.

Highland barley is one of the few crops that can be grown at high elevations, making it a key resource within the Tibet Plateau. Weeds are a significant threat to highland barley production, and new herbicides and tolerant barley varieties are needed to control this ever-growing problem. A better understanding of existing herbicide resistance mechanisms is therefore needed. In this study, transcriptomic and metabolomic analyses were used to identify molecular and physiological changes in two highland barley genotypes with differing sensitivities to the herbicide pyroxsulam. We identified several stress-responsive metabolites, including flavonoids and antioxidants, which accumulated to significantly higher levels in the pyroxsulam-resistant genotype. Additionally, we found key genes in both the flavonoid biosynthesis pathway and the antioxidant system that were up-regulated in pyroxsulam-resistant barley. This work significantly expands on the current understanding of the molecular mechanisms underlying differing pyroxsulam tolerance among barley genotypes and provides several new avenues to explore for breeding or engineering tolerant barley.

Mutations of Asn321 and Glu322 Improve Resistance of 4-Hydroxyphenylpyruvate Dioxygenase SpHPPDm to Topramezone.

As a highly efficient 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide, topramezone is an ideal target for herbicide-resistant genetic engineering. In this study, two mutants, K-19 (N321Y) and K-63 (Q166R/E322V), with topramezone resistance increased by 205.3 and 58.5%, respectively, were screened from the random mutation library of SpHPPDm, a topramezone-resistant HPPD mutant that we previously obtained. Sites N321 and E322 were identified as key sites for increased topramezone resistance by single-site mutation analysis. A mutant KB-145 (N321Y/E322K) was further obtained by saturation mutation at sites N321 and E322. The topramezone resistance of KB-145 increased by 955.3% compared to mutant SpHPPDm. In conclusion, this study identifies two new sites that significantly affect the topramezone resistance of SpHPPDm, which provides new insights into the molecular mechanism of herbicide resistance of HPPD, and the acquired mutants have great application potential in the construction of herbicide-resistant crops.

Diversity of Herbicide-Resistance Mechanisms of Avena fatua L. to Acetyl-CoA Carboxylase-Inhibiting Herbicides in the Bajio, Mexico.

Herbicide resistance is an evolutionary process that affects entire agricultural regions’ yield and productivity. The high number of farms and the diversity of weed management can generate hot selection spots throughout the regions. Resistant biotypes can present a diversity of mechanisms of resistance and resistance factors depending on selective conditions inside the farm; this situation is similar to predictions by the geographic mosaic theory of coevolution. In Mexico, the agricultural region of the Bajio has been affected by herbicide resistance for 25 years. To date, Avena fatua L. is one of the most abundant and problematic weed species. The objective of this study was to determine the mechanism of resistance of biotypes with failures in weed control in 70 wheat and barley crop fields in the Bajio, Mexico. The results showed that 70% of farms have biotypes with target site resistance (TSR). The most common mutations were Trp–1999–Cys, Asp–2078–Gly, Ile–2041–Asn, and some of such mutations confer cross-resistance to ACCase-inhibiting herbicides. Metabolomic fingerprinting showed four different metabolic expression patterns. The results confirmed that in the Bajio, there exist multiple selection sites for both resistance mechanisms, which proves that this area can be considered as a geographic mosaic of resistance.

Full chloroplast sequencing using genome skimming for novel plant DNA barcode discovery in Amaranths

DNA barcoding has been established as an efficient, sensitive and reliable methodology for plant identification. However, in spite of efforts to find a universal DNA plant barcode, some taxa are not sufficiently resolved by typical plant barcoding genes like matK or rbcL. We have used a technique known as genome skimming, which relies on the empiric low coverage sequencing of a full plant genome, resulting in high coverage of the high copy genome fractions such as chloroplast and rDNA. Phylogenetic studies show that these regions are a reservoir of variability which could be further exploited for DNA barcode discovery. We sequenced eight amaranth species using Illumina Next Generation Sequencing technology to test the feasibility of this technique. Amaranths were chosen due to their increasing impact as invasive species bearing multiple herbicide resistance mechanisms. Our results showed that complete chloroplast genomes could be assembled for all of the eight species tested. We obtained an average of 47 million reads for each one of the amaranth nuclear genomes, which range in size between 400-700Mb approximately. These reads provide an average theoretical coverage of 10-15X for each nuclear genome, but resulted in an average chloroplast genome coverage in the range of 500-8000X due to multiple chloroplast genome copies per cell. Alignment of the eight chloroplast genomes shows variability in the single copy regions (Fig. 1), especially on intergenic sections (Fig. 2). Additional preliminary analyses also show variation among different populations of the same species, demonstrating the importance of studying both inter and intraspecific diversity to design reliable and accurate DNA barcodes that can be used in species identification.

Multiple resistance to ACCase- and ALS-inhibiting herbicides in black-grass (Alopecurus myosuroides Huds.) in China

Two black-grass (Alopecurus myosuroides Huds.) populations (R2105 and R1027) that were suspected to be resistant to clodinafop-propargyl, an acetyl-CoAcarboxylase (ACCase) inhibitor, were found in winter wheat fields in China. Research was carried out to investigate whether resistance to clodinafop-propargyl was present and the molecular mechanism of herbicide resistance in these two populations. Dose–response assays confirmed high level resistance to clodinafop-propargyl in both R2105 and R1027 populations, with resistance indexes 25.1 and 22.1. ACCase gene sequence comparison revealed three amino acid mutations (Trp-1999-Leu, Ile-2041-Asn, or Asp-2078-Gly) in R2105 population and Ile-2041-Asn mutation in R1027 population. Sensitivity to other herbicides assays indicated that R2105 and R1027 populations were cross resistant to fenoxaprop-P-ethyl and multiple resistant to pyroxsulam and mesosulfuron-methyl. The ALS gene sequence analysis revealed that all resistant individuals in R2105 and R1027 populations had the Trp-574-Leu mutation. Applying malathion, significantly decreased the rate of metabolism of clodinafop-propargyl in both R2105 and R1027 populations. This is the first report of multiple resistance to ACCase- and ALS-inhibiting herbicides conferred by target-site mutations and enhanced metabolism in black-grass in China.

A nonnative Palmer amaranth (Amaranthus palmeri) population in the Republic of South Africa is resistant to herbicides with different sites of action

AbstractPalmer amaranth (Amaranthus palmeriS. Watson) is not native to Africa. Based on the presence and persistence ofA. palmeripopulations, its invasive status in southern Africa is classified as “naturalized.” Globally,A. palmeriis one of the most troublesome weed species in several crops, including soybean [Glycine max(L.) Merr.], maize (Zea maysL.), and cotton (Gossypium hirsutumL.). Certain populations ofA. palmeriin various countries were reported to be resistant to herbicides with different sites of action (SOAs). Two biotypes ofA. palmeriin the United States reportedly each have resistance to herbicides representing five different SOAs, and between them a total of eight different SOAs are involved. Resistance mechanisms in these biotypes involve target-site and/or non–target site resistance. Here we characterize a specificA. palmeripopulation that was found in the Douglas district in South Africa and showed resistance to various herbicide SOAs. Initially, thisA. palmeripopulation was discovered in a glyphosate-tolerant cotton field, where it survived glyphosate treatment. Subsequently, greenhouse experiments were conducted to characterize thisA. palmeripopulation for potential resistance to herbicides of additional SOAs, and molecular analyses were conducted to reveal the mechanisms of herbicide resistance. Results indicated resistance to chlorimuron-ethyl and glyphosate in this population, while <90% control (decreased sensitivity) was observed at the label rate for mesotrione, atrazine, saflufenacil, andS-metolachlor. However, glufosinate, tembotrione, acifluorfen, dicamba, 2,4-D, metribuzin, acetochlor, isoxaflutole, diflufenican, and pyroxasulfone were effective at controlling this population. This profiling of herbicide sensitivity has allowed development of programs to control and potentially minimize the spread of this weed. In addition, molecular analysis ofEPSPSrevealed the role of higher copy number as a mechanism for glyphosate resistance in this population and a Ser-653-Asn target-site mutation likely conferring resistance to the acetolactate synthase–inhibitor chlorimuron-ethyl. No known target-site mutations were identified for the protoporphyrinogen oxidase–inhibitor group.