Target-site Mechanisms Research Articles

Metproxybicyclone, a Novel Carbocyclic Aryl-dione Acetyl-CoA Carboxylase-Inhibiting Herbicide for the Management of Sensitive and Resistant Grass Weeds.

Postemergence control of grass weeds has become problematic due to the evolution of resistance to 5-enolpyruvylshikimate-3-phosphate synthase, acetyl-CoA carboxylase (ACCase), and acetolactate synthase-inhibiting herbicides. Herein we describe the invention and synthesis journey toward metproxybicyclone, the first commercial carbocyclic aryl-dione ACCase-inhibiting herbicide for the cost-effective management of grass weeds in dicotyledonous crops and in preplant burndown applications. Glasshouse and field experiments have shown that metproxybicyclone is safe for use on soybean, cotton, and sugar beet, among other crops. It is effective on a variety of key grass weeds including Eleusine indica, Digitaria insularis, Sorghum halepense, and Echinochloa crus-galli. Importantly, metproxybicyclone was more efficacious at killing resistant grass weed populations than current ACCase herbicides. Metproxybicyclone controlled the main ACCase target-site and nontarget site resistant mechanisms in characterized Lolium multiflorum and E. indica populations under glasshouse conditions. Excellent control of a broad resistance-causing D2078G target-site mutant E. indica population was also observed under field conditions.

Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication.

Selective pressures on viruses provide opportunities to establish target site specificity and mechanisms of antivirals. Enterovirus (EV)-A71 with resistant mutations in the stem loop (SL) II internal ribosome entry site (IRES) (SLIIresist) were selected at low doses of the antiviral dimethylamiloride (DMA)-135. The EV-A71 mutants were resistant to DMA-135 at concentrations that inhibit replication of wild-type virus. EV-A71 IRES structures harboring resistant mutations induced efficient expression of Luciferase messenger RNA in the presence of noncytotoxic doses of DMA-135. Nuclear magnetic resonance indicates that the mutations change the structure of SLII at the binding site of DMA-135 and at the surface recognized by the host protein AU-rich element/poly(U)-binding/degradation factor 1 (AUF1). Biophysical studies of complexes formed between AUF1, DMA-135, and either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. This work demonstrates how viral evolution elucidates the (DMA-135)-RNA binding site specificity in cells and provides insights into the viral pathways inhibited by the antiviral.

Confirmation and differential metabolism associated with quinclorac resistance in smooth crabgrass (Digitaria ischaemum)

AbstractQuinclorac controls crabgrass (Digitaria spp.) in cool- and warm-season turfgrass species. Herbicide-resistant smooth crabgrass [Digitaria ischaemum (Schreb.) Schreb. ex Muhl.] biotypes have evolved due to recurrent usage of quinclorac. Two Mississippi populations (MSU1 and MSU2) of D. ischaemum were characterized using standard greenhouse dose–response screens to assess their resistance relative to known susceptible populations. Subsequent investigations explored mechanisms of resistance, including examining cyanide accumulation, glutathione S-transferase (GST) activity, and the potential involvement of cytochrome P450s in MSU1, MSU2, and a susceptible (SMT2). Resistant populations MSU1 and MSU2 required 80 and 5 times more quinclorac, respectively, to reach 50% biomass reduction than susceptible populations. The SMT2 biotype accumulated three times more cyanide than the resistant MSU1 and MSU2 populations. GST activity was elevated in resistant MSU1 and MSU2 populations. Furthermore, quinclorac concentrations in treated resistant populations were elevated when plants were pretreated with the P450 inhibitor malathion. These findings suggest a non–target site based mechanism of resistance involving the accumulation of cyanide. This may provide a scientific basis for understanding the occurrence of quinclorac-resistant D. ischaemum, although further research is needed to investigate potential target-site mechanisms of resistance.

Response characterization and target site mechanism study in glyphosate-resistant populations of Lolium multiflorum L. from Brazil

Italian ryegrass (Lolium multiflorum L.) is an invasive species widely spread in croplands worldwide. The intensive use of glyphosate has resulted in the selection of resistance to this herbicide in Italian ryegrass. This work characterized the response to glyphosate of Italian ryegrass populations from the South and Southwest regions of Paraná, Brazil. A total of 44 Italian ryegrass populations were collected in farming areas, and were classified for glyphosate resistance with 75% of populations resistant to gloyphosate. Of these, 3 resistant (VT05AR, MR20AR and RN01AR) and three susceptible (VT07AS, MR05AS and RN01AS) of these populations were selected to determine the resistance level and the involvement of the target site mechanisms for glyphosate resistance. Susceptible populations GR50 ranged from 165.66 to 218.17 g.e.a. ha−1 and resistant populations from 569.37 to 925.94, providing RI ranging from 2.88 and 4.70. No mutation in EPSPS was observed in the populations, however, in two (MR20AR and RN02AR) of the three resistant populations, an increase in the number of copies of the EPSPs gene (11 to 57×) was detected. The number of copies showed a positive correlation with the gene expression (R2 = 0.86) and with the GR50 of the populations (R2 = 0.81). The increase in EPSPS gene copies contributes to glyphosate resistance in Italian ryegrass populations from Brazil.

Target and non-target site mechanisms of fungicide resistance and their implications for the management of crop pathogens.

Fungicides are indispensable for high-quality crops, but the rapid emergence and evolution of fungicide resistance have become the most important issues in modern agriculture. Hence, the sustainability and profitability of agricultural production have been challenged due to the limited number of fungicide chemical classes. Resistance to site-specific fungicides has principally been linked to target and non-target site mechanisms. These mechanisms change the structure or expression level, affecting fungicide efficacy and resulting in different and varying resistance levels. This review provides background information about fungicide resistance mechanisms and their implications for developing anti-resistance strategies in plant pathogens. Here, our purpose was to review changes at the target and non-target sites of quinone outside inhibitor fungicides (QoIs), methyl-benzimidazole fungicides (MBCs), demethylation inhibitor fungicides (DMIs), and succinate dehydrogenase inhibitor fungicides (SDHIs) and to evaluate if they may also be associated with a fitness cost on crop pathogen populations. The current knowledge suggests that understanding fungicide resistance mechanisms can facilitate resistance monitoring and assist in developing anti-resistance strategies and new fungicide molecules to help solve this issue. This article is protected by copyright. All rights reserved.

ACCase gene mutations and P450-mediated metabolism contribute to cyhalofop-butyl resistance in Eleusine indica biotypes from direct-seeding paddy fields

Eleusine indica causes problems in direct-seeding rice fields across Jiangsu Province in China. Long-term application of chemical herbicides has led to the widespread evolution of resistance in E. indica. In this study, we surveyed the resistance level of cyhalofop-butyl (CyB) in 19 field-collected E. indica biotypes, and characterized its underlying resistance mechanisms. All 19 biotypes evolved moderate- to high-level resistance to CyB (from 5.8- to 171.1-fold). 18 biotypes had a target-site mechanism with Trp-1999-Ser, Trp-2027-Cys, or Asp-2078-Gly mutations, respectively. One biotype (JSSQ-1) was identified to have metabolic resistance, in which malathion pretreatment significantly reduced the CyB resistance, and cyhalofop acid was degraded 1.7- to 2.5-times faster in this biotype compared with a susceptible control. Furthermore, the JSSQ-1 biotype showed multiple resistance to acetyl-CoA carboxylase (ACCase) inhibitor metamifop (RI = 4.6) and fenoxaprop-p-ethyl (RI = 5.1), acetolactate synthase (ALS) inhibitor imazethapyr (RI = 4.1), and hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor mesotrione (RI = 3.5). In addition, 11 out of 19 E. indica biotypes exhibited multiple resistance to glyphosate. This research has identified the widespread occurrence of CyB resistance in E. indica, attributed to target-site mutations or enhanced metabolism. Moreover, certain biotypes have exhibited resistance to multiple herbicides or even cross-resistance. Consequently, there is an urgent need to implement diverse weed management practices to effectively combat the proliferation of this weed in rice fields.

Asp376Glu Mutation and Enhanced Metabolism Controlling the Resistance to ALS-Inhibiting Herbicides in Ixophorus unisetus (J. Presl) Schltdl from the Bajio, Mexico

A study was carried out to determine the ALS (acetolactate synthase)-inhibitor herbicide resistance in the Mexican grass Ixophorus unisetus, a troublesome weed in corn crops in Mexico. First, the resistance was confirmed in field screening assays. Eight populations that survived nicosulfuron treatment at a field rate of 40 g ai ha−1 were labeled as putative-resistant. Dose–response trials demonstrated a high resistance in the eight populations (GR50 from 140.33 to 245.46 g ai ha−1). The synergism of malathion plus nicosulfuron demonstrated that the non-target-site resistance (NTSR) mechanism based on cytochrome P450 (Cyt-P450) was involved in five populations of I. unisetus. Molecular studies revealed that a single-nucleotide change occurs in the amino acid at position 376 (from GAT to GAG), which codifies from Asp-376 to Glu-376. This is the first time that Asp-376-Glu has been reported in this species. Assays in vitro and in vivo demonstrated I. unisetus cross-resistance to flucarbazone, penoxsulam, bispyribac-Na, and imazamox. No multiple resistance was found in two resistant populations exposed to different herbicides. Our results indicate that the lack of good control over Mexican grass in corn with ALS inhibitors is due to target-site mutation and NTSR mechanisms (Cyt-P450-mediated metabolism). A strategy should be established in Mexican fields to continue controlling this weed, including mechanical control practices and a good combination of the available pre- and post-emergence herbicides.

Nontarget-site resistance due to rapid physiological response in 2,4-D resistant Conyza sumatrensis: reduced 2,4-D translocation and auxin-induced gene expression.

Resistance to 2,4-Dichlorophenoxyacetic acid (2,4-D) has been reported in several weed species since the 1950s; however, a biotype of Conyza sumatrensis showing a novel physiology of the rapid response minutes after herbicide application was reported in 2017. The objective of this research was to investigate the mechanisms of resistance and identify transcripts associated with the rapid physiological response of C. sumatrensis to 2,4-D herbicide. Differences were found in 2,4-D absorption between the resistant and susceptible biotypes. Herbicide translocation was reduced in the resistant biotype compared to the susceptible. In resistant plants 98.8% of [14 C] 2,4-D was found in the treated leaf, whereas ≈13% translocated to other plant parts in the susceptible biotype at 96 h after treatment. Resistant plants did not metabolize [14 C] 2,4-D and had only intact [14 C] 2,4-D at 96 h after application, whereas susceptible plants metabolized [14 C] 2,4-D into four detected metabolites, consistent with reversible conjugation metabolites found in other 2,4-D sensitive plant species. Pre-treatment with the cytochrome P450 inhibitor malathion did not enhance 2,4-D sensitivity in either biotype. Following treatment with 2,4-D, resistant plants showed increased expression of transcripts within plant defense response and hypersensitivity pathways, whereas both sensitive and resistant plants showed increased expression of auxin-response transcripts. Our results demonstrate that reduced 2,4-D translocation contributes to resistance in the C. sumatrensis biotype. The reduction in 2,4-D transport is likely to be a consequence of the rapid physiological response to 2,4-D in resistant C. sumatrensis. Resistant plants had increased expression of auxin-responsive transcripts, indicating that a target-site mechanism is unlikely. © 2023 Society of Chemical Industry.

Target-site and non–target site mechanisms of pronamide resistance in annual bluegrass (Poa annua) populations from Mississippi golf courses

AbstractThe mitotic-inhibiting herbicide pronamide controls susceptible annual bluegrass (Poa annua L.) pre- and postemergence, but in some resistant populations, postemergence activity is compromised, hypothetically due to a target-site mutation, lack of root uptake, or an unknown resistance mechanism. Three suspected pronamide-resistant (LH-R, SC-R, and SL-R) and two pronamide-susceptible (BS-S and HH-S) populations were collected from Mississippi golf courses. Dose–response experiments were conducted to confirm and quantify pronamide resistance, as well as resistance to flazasulfuron and simazine. Target sites known to confer resistance to mitotic-inhibiting herbicides were sequenced, as were target sites for herbicides inhibiting acetolactate synthase (ALS) and photosystem II (PSII). Pronamide absorption and translocation were investigated following foliar and soil applications. Dose–response experiments confirmed pronamide resistance of LH-R, SC-R, and SL-R populations, as well as instances of multiple resistance to ALS- and PSII-inhibiting herbicides. Sequencing of the α-tubulin gene confirmed the presence of a mutation that substituted isoleucine for threonine at position 239 (Thr-239-Ile) in LH-R, SC-R, SL-R, and BS-S populations. Foliar application experiments failed to identify differences in pronamide absorption and translocation between the five populations, regardless of harvest time. All populations had limited basipetal translocation—only 3% to 13% of the absorbed pronamide—across harvest times. Soil application experiments revealed that pronamide translocation was similar between SC-R, SL-R, and both susceptible populations across harvest times. The LH-R population translocated less soil-applied pronamide than susceptible populations at 24, 72, and 168 h after treatment, suggesting that reduced acropetal translocation may contribute to pronamide resistance. This study reports three new pronamide-resistant populations, two of which are resistant to two modes of action (MOAs), and one of which is resistant to three MOAs. Results suggest that both target site– and translocation-based mechanisms may be associated with pronamide resistance. Further research is needed to confirm the link between pronamide resistance and the Thr-239-Ile mutation of the α-tubulin gene.

Multiple Pesticide Resistance in Rust-Red Flour Beetle (Tribolium castaneum, Herbst 1797) from Northern Nigeria Is Probably Driven by Metabolic Mechanisms

There is a severe lack of information about molecular mechanisms of pesticide resistance in the rust-red flour beetle, a major pest destroying grains and flour across Nigeria, hindering evidence-based control. Here, we identified to the species level three populations of the red flour beetle from Kano, Nigeria, as Tribolium castaneum (Herbst 1797) and investigated the mechanism driving their insecticide resistance. The IRAC susceptibility bioassays established cypermethrin resistance, with LC50s of 4.35–5.46 mg/mL in the three populations, NNFM, R/Zaki and Yankaba. DDT and malathion resistance were observed in NNFM, with LC50s of 15.32 mg/mL and 3.71 mg/mL, respectively. High susceptibility was observed towards dichlorvos in all three populations with LC50s of 0.17–0.35 mg/mL. The synergist bioassay with piperonylbutoxide significantly restored cypermethrin susceptibility, with mortality increasing almost threefold, from 24.8% obtained with 1.5 mg/mL of cypermethrin to 63.3% in the synergised group (p = 0.013), suggesting a preeminent role of P450s. The two major knockdown resistance (kdr) mutations, T929I and L1014F, in the IIS4 and IIS6 fragments of the voltage-gated sodium channel were not detected in both cypermethrin-alive and cypermethrin-dead beetles, suggesting a lesser role of target-site insensitivity mechanisms. These findings highlight the need to explore alternative control tools for this pest and/or utilise synergists, such as piperonyl butoxide, as additional chemistries in pesticide formulations to improve their efficacy.

Different Non-Target Site Mechanisms Endow Different Glyphosate Susceptibility in Avena Species from Spain

In recent decades, cereal agriculture across main producing areas in Spain has progressively adopted direct sowing, associated with an increased use of herbicides in pre-sowing. Weedy species from genus Avena have been observed after the application of glyphosate in wheat. Here, Avena fatua (two accessions), Avena byzantina and Avena sterilis subsp. sterilis, one accession each, were taxonomically characterized by a biometric study. Dose-response trials confirmed that one A. fatua accession evolved to resistance, because it was four times more resistant (R) than the others, ascribed as susceptible (S). In addition, based on LD50, A. byzantina and A. sterilis had low susceptibility to glyphosate, with 604 and 458 g ae ha−1. Shikimic acid accumulation was able to discriminate between high susceptible (A. fatua (S)), low susceptible (A. byzantina and A. sterilis), and low resistant (A. fatua (R)) populations. On the other hand, the study revealed that A. fatua (R), A. byzantina and A. sterilis had low foliar uptake and decreased movement of glyphosate. In addition, the metabolism study showed less metabolite accumulation in A. byzantina and A. fatua (S). However, at 96 h after glyphosate application, A. fatua (R) and A. sterilis were able to convert more than 30% of glyphosate to aminomethylphosphonic acid (AMPA) and sarcosine. Enzyme basal activity and I50 values predicted high affinity between the herbicide and the target enzyme for all accessions, thus indicating that a target-site (TS) mechanism is probably not involved in the differences in glyphosate susceptibility. In closing, non-target site (NTS) mechanisms could participate both in A. fatua R to glyphosate, and low susceptibility in A. byzantina and A. sterilis from Spain.

High-Resolution Genetic Mapping Combined with Transcriptome Profiling Reveals That Both Target-Site Resistance and Increased Detoxification Confer Resistance to the Pyrethroid Bifenthrin in the Spider Mite Tetranychus urticae.

Pyrethroids are widely applied insecticides in agriculture, but their frequent use has provoked many cases of resistance, in which mutations in the voltage-gated sodium channel (VGSC), the pyrethroid target-site, were shown to play a major role. However, for the spider mite Tetranychus urticae, it has also been shown that increased detoxification contributes to resistance against the pyrethroid bifenthrin. Here, we performed QTL-mapping to identify the genomic loci underlying bifenthrin resistance in T. urticae. Two loci on chromosome 1 were identified, with the VGSC gene being located near the second QTL and harboring the well-known L1024V mutation. In addition, the presence of an L925M mutation in the VGSC of a highly bifenthrin-resistant strain and its loss in its derived, susceptible, inbred line indicated the importance of target-site mutations in bifenthrin resistance. Further, RNAseq experiments revealed that genes encoding detoxification enzymes, including carboxyl/choline esterases (CCEs), cytochrome P450 monooxygenases and UDP-glycosyl transferases (UGTs), were overexpressed in resistant strains. Toxicity bioassays with bifenthrin (ester pyrethroid) and etofenprox (non-ester pyrethroid) also indicated a possible role for CCEs in bifenthrin resistance. A selection of CCEs and UGTs were therefore functionally expressed, and CCEinc18 was shown to metabolize bifenthrin, while teturUGT10 could glycosylate bifenthrin-alcohol. To conclude, our findings suggest that both target-site and metabolic mechanisms underlie bifenthrin resistance in T. urticae, and these might synergize high levels of resistance.

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

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. 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. 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.

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

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

Insecticide resistant Anopheles gambiae have enhanced longevity but reduced reproductive fitness and a longer first gonotrophic cycle

Widespread insecticide resistance in African malaria vectors raises concerns over the potential to compromise malaria vector control interventions. Understanding the evolution of resistance mechanisms, and whether the selective disadvantages are large enough to be useful in resistance management or designing suitable control strategies is crucial. This study assessed whether insecticide resistance to pyrethroids has an effect on the gonotrophic cycle and reproductive potential of malaria vector Anopheles gambiae. Comparative tests were performed with pyrethroid-resistant and susceptible colonies of Anopheles gambiae colonized from the same geographical area, and the reference Kisumu strain was used as a control. Adult females aged 3 days old were given a blood meal and kept separately for individual egg-laying. The number of days taken to lay eggs post-blood-feeding was recorded to determine the length of the gonotrophic cycle. To measure adult longevity and reproduction potential, newly emerged males and females of equal numbers were aspirated into a cage and females allowed to blood feed daily. The number of eggs laid and the surviving mosquitoes were recorded daily to determine fecundity, net reproduction rate, intrinsic growth rate and adult longevity. Overall, the resistant females had a significantly longer (1.8 days) gonotrophic cycle than susceptible females (F2, 13 = 9. 836, P < 0.01). The proportion of resistant females that laid eggs was lower 31.30% (94/300) compared to 54% (162/300) in the susceptible colony and 65.7% (197/300) in the Kisumu strain. The mean number of eggs laid per female was significantly lower in the resistant colony (88.02 ± 20) compared to the susceptible colony (104.9 ± .28.8) and the Kisumu strain (97.6 ± 34.8). The adult longevity was significantly higher for resistant (39.7 ± 1.6 days) compared to susceptible (29.9 ± 1.7 days) and the Kisumu strain was (29.6 ± 1.1 days) (F2,8 = 45.05, P < 0.0001). Resistant colony exhibited a lower fecundity (4.3 eggs/females/day) and net reproductive rate (2.6 offsprings/female/generation) compared to the susceptible colony (8.6 eggs/female/day; 4.7 offsprings/female/generation respectively) and Kisumu strain (9.7 eggs/female/day; 4.1 offsprings/female/generation respectively). The study suggests high fitness cost on reproductive parameters of pyrethroid-resistant mosquitoes particularly on the duration of gonotrophic cycle, fecundity and net reproductive rate. These fitness costs are likely associated with maintaining both target site and metabolic mechanisms of resistance to pyrethroids. Despite these costs, resistant mosquitoes had longer longevity. These results give insights to understanding the fitness cost of insecticide resistance and thus are critical when predicting the epidemiological impact of insecticide resistance.

Combined metabolic and target-site resistance mechanisms confer fipronil and deltamethrin resistance in field-collected German cockroaches (Blattodea: Ectobiidae)

Despite insecticide resistance issues, pyrethroids and fipronil have continued to be used extensively to control the German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae) for more than two decades. We evaluated the physiological insecticide resistance in five German cockroach populations collected from 2018 to 2020 and measured the extent of metabolic detoxification and target-site insensitivity resistance mechanisms. Topically applied doses of the 3 x LD95 of deltamethrin, fipronil, DDT, or dieldrin of a susceptible strain (UCR, Diagnostic Dose) failed to cause >23% mortality, and the 10 x LD95 of deltamethrin or fipronil failed to cause >53% mortality. All field-collected strains possessed a combination of metabolic and target-site insensitivity mechanisms that cause reduced susceptibility. Elevated activities of esterase and glutathione S-transferase were measured, and the synergists piperonyl butoxide or S,S,S-tributyl phosphorotrithioate increased topical mortality up to 100% for deltamethrin and 93% for fipronil 10 x LD95. The target-site mutations L993F of the para-homologous sodium channel and A302S of the GABA-gated chloride channel associated with pyrethroid and fipronil resistance, respectively, were found at ~80–100% frequency in field populations. Pyrethroid and fipronil spray formulations also were ineffective in a choice box assay against field-collected strains suggesting that these treatments would fail to control cockroaches under field conditions.

Pinoxaden resistance in Lolium perenne L. is due to both target-site and non-target-site mechanisms

Application of herbicides inhibiting acetyl CoA carboxylase (ACCase) has been one of the main strategies for selectively controlling grass weed species such as perennial ryegrass (Lolium perenne L.) in wheat and barley crops in New Zealand. In this study, we have confirmed and characterized resistance to pinoxaden, an ACCase-inhibiting herbicide, in a population of L. perenne. Dose-response experiments were conducted to assess the level of pinoxaden resistance, and based on the LD50 values, the studied population was 41.4-times more resistant to pinoxaden than a susceptible population. Application of malathion, an inhibitor of the cytochrome P450s, preceding pinoxaden treatment reduced the level of resistance to 9.7-fold. However, pre-treatment with the glutathione S-transferase (GST) inhibitor 4-chloro7- nitrobenzoxadiazole prior to pinoxaden treatment did not affect pinoxaden resistance. Partial sequencing of the ACCase gene revealed that the resistant population had an isoleucine to valine replacement at position 2041. These results suggest that both cytochrome P450-based and target-site mechanisms are jointly associated with this instance of pinoxaden resistance in L. perenne. The pinoxaden-resistant L. perenne individuals were also resistant to quizalofop-p-ethyl (108.6-fold), but they were susceptible to clethodim, which can, therefore, be used to manage this pinoxaden-resistant L. perenne. This is the first report of a L. perenne population in which a rare target-site mutation works in concert with enhanced cytochrome P-450 activity to confer pinoxaden resistance. Evolution of resistance to ACCase-inhibiting herbicides in this L. perenne population indicates that integrated weed management practices are required to prevent widespread resistance developing in New Zealand cereal crop systems.

Structural basis for DNA targeting by the Tn7 transposon.

Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ adaptor (TnsC) to coordinate target-site selection with transpososome assembly and to prevent insertions at sites already containing a Tn7 element. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. TnsC forms an asymmetric ring on target DNA that segregates target-site selection and interaction with the paired-end complex to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.

Analysis of the Knockdown Resistance Locus (kdr) in Anopheles stephensi, An. arabiensis, and Culex pipiens s.l. for Insight Into the Evolution of Target-site Pyrethroid Resistance in Eastern Ethiopia.

ABSTRACT.The malaria vector, Anopheles stephensi, which is typically restricted to South Asia and the Middle East, was recently detected in the Horn of Africa. Addressing the spread of this vector could involve integrated vector control that considers the status of insecticide resistance of multiple vector species in the region. Previous reports indicate that the knockdown resistance mutations (kdr) in the voltage-gated sodium channel (vgsc) are absent in both pyrethroid-resistant and pyrethroid-sensitive An. stephensi in eastern Ethiopia; however, similar information about other vector species in the same areas is limited. In this study, kdr and the neighboring intron were analyzed in An. stephensi, An. arabiensis, and Culex pipiens s.l. collected between 2016 and 2017 to determine the evolutionary history of kdr in eastern Ethiopia. A sequence analysis revealed that all of Cx. pipiens s.l. (N = 42) and 71.6% of the An. arabiensis (N = 67) carried kdr L1014F, which is known to confer target-site pyrethroid resistance. Intronic variation was only observed in An. stephensi (six segregating sites, three haplotypes), which was previously shown to have no kdr mutations. In addition, no evidence of non-neutral evolutionary processes was detected at the An. stephensi kdr intron, thereby further supporting the target-site mechanism not being a major resistance mechanism in this An. stephensi population. Overall, these results show key differences in the evolution of target-site pyrethroid/dichlorodiphenyltrichloroethane resistance mutations in populations of vector species from the same region. Variations in insecticide resistance mechanism profiles between eastern Ethiopian mosquito vectors may lead to different responses to insecticides used in integrated vector control.

D376E, A205V and A122S substitutions recently found in A. palmeri confer cross-resistance to ALS-inhibiting herbicides

Background Amaranthus palmeri S. Watson is a serious problem in soybean crops from Argentina. This weed has evolved high level of resistance to (ALS)-inhibiting herbicides mainly due to a target-site mechanism by an altered ALS enzyme. In an A. palmeri population from Totoras with cross-resistance to (ALS)-inhibiting herbicides, six allelic versions of the ALS enzyme were identified. Objective The aim of this study was to evaluate plants from that resistant population carrying the ALS substitutions A122S, D376E or A205V, [...]