Predicting herbicide movement across semi-permeable membranes using three phase partitioning.

Herbicide efficacy depends on herbicides crossing cell and organelle membranes. We evaluated an artificial membrane system to understand how herbicides cross biological membranes. This understanding aids in predicting herbicide behavior in planta and, consequently, efficacy, mode of action, and whether active transporter-based herbicide resistance mechanisms may be possible. Five herbicides with different log Kow and pKa values were assessed: glyphosate, 2,4-D, clopyralid, sulfentrazone and glufosinate. The artificial membrane apparatus included four semipermeable membranes containing buffers with pH 2.7, 5 and/or 7.4, floating in a bath of diethyl ether. These conditions were based on the pH from different cellular compartments and the pKa for these herbicides. Changes in herbicide concentration due to movement were measured using radioactivity or liquid chromatography mass spectrometry. In general, herbicide behavior followed the pattern predicted by their calculated pKa and log Kow. Herbicides added to an acidic phase (pH 2.7) were more mobile than when they were added to the more basic phase (pH 7.4), except when herbicide's pKa was lower than the pH of the starting phase. Clopyralid, 2,4-D, and sulfentrazone showed significant acid trapping behavior due to their weak acid functional groups. Sulfentrazone and 2,4-D had a high affinity for the nonpolar, diethyl ether bath, especially when they were protonated at low pH. Our findings illustrate the robustness of the system to provide predictions about herbicide behavior at the subcellular level.

Enhanced metabolic detoxification is associated with fluroxypyr resistance in Bassia scoparia.

Auxin-mimic herbicides chemically mimic the phytohormone indole-3-acetic-acid (IAA). Within the auxin-mimic herbicide class, the herbicide fluroxypyr has been extensively used to control kochia (Bassia scoparia). A 2014 field survey for herbicide resistance in kochia populations across Colorado identified a putative fluroxypyr-resistant (Flur-R) population that was assessed for response to fluroxypyr and dicamba (auxin-mimics), atrazine (photosystem II inhibitor), glyphosate (EPSPS inhibitor), and chlorsulfuron (acetolactate synthase inhibitor). This population was resistant to fluroxypyr and chlorsulfuron but sensitive to glyphosate, atrazine, and dicamba. Subsequent dose-response studies determined that Flur-R was 40 times more resistant to fluroxypyr than a susceptible population (J01-S) collected from the same field survey (LD50 720 and 20 g ae ha-1, respectively). Auxin-responsive gene expression increased following fluroxypyr treatment in Flur-R, J01-S, and in a dicamba-resistant, fluroxypyr-susceptible line 9,425 in an RNA-sequencing experiment. In Flur-R, several transcripts with molecular functions for conjugation and transport were constitutively higher expressed, such as glutathione S-transferases (GSTs), UDP-glucosyl transferase (GT), and ATP binding cassette transporters (ABC transporters). After analyzing metabolic profiles over time, both Flur-R and J01-S rapidly converted [14C]-fluroxypyr ester, the herbicide formulation applied to plants, to [14C]-fluroxypyr acid, the biologically active form of the herbicide, and three unknown metabolites. The formation and flux of these metabolites were faster in Flur-R than J01-S, reducing the concentration of phytotoxic fluroxypyr acid. One unique metabolite was present in Flur-R that was not present in the J01-S metabolic profile. Gene sequence variant analysis specifically for auxin receptor and signaling proteins revealed the absence of non-synonymous mutations affecting auxin signaling and binding in candidate auxin target site genes, further supporting our hypothesis that non-target site metabolic degradation is contributing to fluroxypyr resistance in Flur-R.

Gene amplification confers glyphosate resistance in Amaranthus palmeri.

The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F(2) populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology.

2,4-D and 2,4-D butoxyethyl ester behavior in Eurasian and hybrid watermilfoil (Myriophyllum spp.).

BACKGROUND: Hybrid watermilfoil is becoming more prevalent in many lakes where the invasive Eurasian (Myriophyllum spicatum, EWM) and native northern watermilfoil (M. sibiricum) co-occur. These Eurasian and northern watermilfoil hybrids (HWM) grow 30% faster and in many cases are less sensitive to 2,4-dichlorophenoxy acetic acid (2,4-D) than either parent. The mechanism(s) impacting 2,4-D tolerance in these hybrids was investigated by comparing the absorption, translocation, metabolism, and desorption of two 2,4-D formulations in EWM and HWM. RESULTS: 2,4-D absorption in EWM and HWM was 5.7 and 7.9 times the external herbicide concentration determined by the plant concentration factor, a metric used to determine herbicide bioaccumulation, and 2,4-D butoxyethyl ester absorption was 35.6 and 52.1 times the external concentration in EWM and HWM, respectively. Herbicide bioaccumulation was greater in HWM than in EWM. Herbicide translocation to HWM roots was limited at 192 HAT and herbicide desorption in HWM was slightly lower than EWM. No differences were found in herbicide metabolism between the two plant species. CONCLUSION: 2,4-D resistance in HWM is not due to non-target-site resistance as no differences in herbicide absorption, translocation, desorption and/or metabolism were identified; therefore, target-site resistance is the most likely resistance mechanism. More research is needed to identify the molecular basis for the 2,4-D-resistant trait in HWM. © 2021 Society of Chemical Industry.

Identification of a Novel 2,4-D Metabolic Detoxification Pathway in 2,4-D-Resistant Waterhemp (Amaranthus tuberculatus).

A 2,4-dichlorophenoxyactic acid (2,4-D)-resistant population of Amaranthus tuberculatus (common waterhemp) from Nebraska, USA, was previously found to have rapid metabolic detoxification of the synthetic auxin herbicide 2,4-D. We purified the main 2,4-D metabolites from resistant and susceptible plants, solved their structures by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS), and synthesized the metabolites to determine their in planta toxicity. Susceptible plants conjugated 2,4-D to aspartate to form 2,4-D-aspartic acid (2,4-D-Asp), while resistant plants had a unique metabolic profile where 2,4-D was hydroxylated into 5-OH-2,4-D, followed by conjugation into a sugar metabolite (2,4-D-5-O-d-glucopyranoside) and subsequent malonylation into 2,4-D-(6'-O-malonyl)-5-O-d-glucopyranoside. Toxicological studies on waterhemp and Arabidopsis thaliana confirmed that the hydroxylated metabolite lost its auxinic action and toxicity. In contrast, the 2,4-D-Asp metabolite found in susceptible plants retained some auxinic action and toxicity. These results demonstrate that 2,4-D-resistant A. tuberculatus evolved novel detoxification reactions not present in susceptible plants to rapidly metabolize 2,4-D, potentially mediated by cytochrome P450 enzymes that perform the initial 5-hydroxylation reaction. This novel mechanism is more efficient to detoxify 2,4-D and produces metabolites with lower toxicity compared to the aspartic acid conjugation found in susceptible waterhemp.

Endothall behavior in Myriophyllum spicatum and Hydrilla verticillata.

BACKGROUND: Endothall has been used to control submersed aquatic plants since 1960, providing broad-spectrum control of aquatic weeds. Although endothall is considered a contact herbicide, many field observations suggest that it might have systemic activity. The goals of this research were to determine endothall's (i) absorption characteristics, (ii) translocation from shoots to roots, and (iii) potential for desorption in Eurasian watermilfoil (EWM), monoecious and dioecious hydrilla. RESULTS: Endothall absorption was linear in dioecious hydrilla up to 192 HAT, while in EWM and monoecious hydrilla absorption data best fit an asymptotic rise function. Endothall absorption in EWM, monoecious and dioecious hydrilla was 3.3, 6.6, and 11.0 times the external herbicide concentration determined by the plant concentration factor. Translocation to EWM roots reached 7.9% of total absorbed radioactivity by 192 HAT, while translocation to monoecious and dioecious hydrilla roots reached 17.8% and 16.4% by 192 HAT, respectively. For all three species, no more than 30% of absorbed endothall moved from the plant to clean water 96 HAT. CONCLUSION: Endothall is a very water soluble compound and yet it accumulated in these three important aquatic weeds at concentrations significantly higher than the external herbicide concentration. These data provide evidence that endothall could have systemic activity in these aquatic species. Following 14 C-endothall applications, more 14 C translocated from shoots to roots compared to the translocation of 14 C for other systemic aquatic herbicides. The final confirmation of endothall's systemic behavior requires that the radioactivity found in the root system of these aquatic plants is 14 C endothall. © 2019 Society of Chemical Industry.

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.

Indaziflam: a new cellulose-biosynthesis-inhibiting herbicide provides long-term control of invasive winter annual grasses.

BACKGROUND: Indaziflam is a cellulose-biosynthesis-inhibiting (CBI) herbicide that is a unique mode of action for resistance management and has broad spectrum activity at low application rates. This research further explores indaziflam's activity on monocotyledons and dicotyledons and evaluates indaziflam's potential for restoring non-crop sites infested with invasive winter annual grasses. RESULTS: Treated Arabidopsis, downy brome, feral rye and kochia were all susceptible to indaziflam in a dose-dependent manner. We confirmed that indaziflam has increased activity on monocots (average GR50  = 231 pm and 0.38 g AI ha-1 ) at reduced concentrations compared with dicots (average GR50  = 512 pm and 0.87 g AI ha-1 ). Fluorescence microscopy confirmed common CBI symptomologies following indaziflam treatments, as well as aberrant root and cell morphology. Across five application timings, indaziflam treatments resulted in superior invasive winter annual grass control 2 years after treatment (from 84 ± 5.1% to 99 ± 0.5%) compared with imazapic (36% ± 1.2%). Indaziflam treatments significantly increased biomass and species richness of co-occurring species 2 years after treatment. CONCLUSION: Indaziflam's increased activity on monocots could provide a new alternative management strategy for long-term control of multiple invasive winter annual grasses that invade >23 million ha of US rangeland. Indaziflam could potentially be used to eliminate the soil seed bank of these invasive grasses, reduce fine fuel accumulation and ultimately increase the competitiveness of perennial co-occuring species. © 2017 Society of Chemical Industry.

A KASP Genotyping Method to Identify Northern Watermilfoil, Eurasian Watermilfoil, and Their Interspecific Hybrids.

The invasive aquatic plant Eurasian watermilfoil (Myriophyllum spicatum L.) can hybridize with the related North American native species northern watermilfoil (M. sibiricum Kom.). Hybrid watermilfoil (M. spicatum × M. sibiricum) populations have higher fitness and reduced sensitivity to some commonly used aquatic herbicides, making management more difficult. There is growing concern that management practices using herbicides in lakes with mixed populations of watermilfoil species may further select for hybrid individuals due to the difference in herbicide sensitivity. Accurate and cost-effective identification of rare hybrid individuals within populations is therefore critical for herbicide management decisions. Here we describe KASP assays for three SNPs in the ITS region to genotype individuals from both parental watermilfoil species and their hybrid, using synthesized plasmids containing the respective sequences as positive controls. Using KASP we genotyped 16 individuals from one lake and 23 individuals from a second lake, giving a highly accurate picture of Myriophyllum species distribution dynamics. We identified one hybrid individual among 16 samples from one lake, a discovery rate of <10%. Discriminant analysis showed that while a single SNP was generally sufficient for genotyping an individual, using multiple SNPs increased the reliability of genotyping. In the future, the ability to genotype many samples will provide the ability to identify the presence of rare individuals, such as a less common parental species or the inter-specific hybrid. Lakes with complex species distribution dynamics, such as a low proportion of hybrids, are where herbicide application must be carefully chosen so as not to select for the more vigorous and less herbicide-sensitive hybrid individuals.

Influence of soil properties and soil moisture on the efficacy of indaziflam and flumioxazin on Kochia scoparia L.

BACKGROUND: Kochia (Kochia scoparia L.) is a highly competitive, non-native weed found throughout the western United States. Flumioxazin and indaziflam are two broad-spectrum pre-emergence herbicides that can control kochia in a variety of crop and non-crop situations; however, under dry conditions, these herbicides sometimes fail to control this important weed. There is very little information describing the effect of soil properties and soil moisture on the efficacy of these herbicides. RESULTS: Soil organic matter (SOM) explained the highest proportion of variability in predicting the herbicide dose required for 80% kochia growth reduction (GR80 ) for flumioxazin and indaziflam (R2 = 0.72 and 0.79 respectively). SOM had a greater impact on flumioxazin phytotoxicity compared to indaziflam. Flumioxazin and indaziflam kochia phytotoxicity was greatly reduced at soil water potentials below -200 kPa. CONCLUSION: Kochia can germinate at soil moisture potentials below the moisture required for flumioxazin and indaziflam activation, which means that kochia control is greatly influenced by the complex interaction between soil physical properties and soil moisture. This research can be used to gain a better understanding of how and why some weeds, like kochia, are so difficult to manage even with herbicides that normally provide excellent control. © 2016 Society of Chemical Industry.

Efficacy and environmental fate of imazapyr from directed helicopter applications targeting Tamarix species infestations in Colorado.

BACKGROUND: Aerial imazapyr applications are the most common and cost-effective method for controlling invasive tamarisk, but few studies have investigated whether or how infestation and site characteristics influence control and non-target impacts. This study used vertical stands with filter papers, plus soil and tree canopy sampling, to investigate how tamarisk canopies affected retention of applied imazapyr, soil herbicide residues and tree mortality. RESULTS: Tamarisk canopies captured 71% of aerially applied imazapyr, resulting in significantly lower soil residues beneath the tree canopy. Although initial imazapyr soil residue levels outside the tree canopy were 4 times greater than those inside, soil degradation occurred 2.4 times faster outside the tamarisk canopy and resulted in lower herbicide residues. Tamarisk mortality within 3 years was 70%, but variability in control appeared to be affected by non-linear stand boundaries and tall site obstructions. These same factors also increased variability in the actual quantity of herbicide applied, exacerbating collateral impacts on desirable understory species. CONCLUSION: While aerial imazapyr applications are highly effective in controlling tamarisk, our study provides evidence for the importance of evaluating overall site suitability for this management strategy so the probability of unintended ecological effects can be minimized.

Confirmation and mechanism of glyphosate resistance in tall windmill grass (Chloris elata) from Brazil.

BACKGROUND: Overreliance on glyphosate as a single tool for weed management in agricultural systems in Brazil has selected glyphosate-resistant populations of tall windmill grass (Chloris elata Desv.). RESULTS: Two C. elata populations, one glyphosate resistant (GR) and one glyphosate susceptible (GS), were studied in detail for a dose-response experiment and for resistance mechanism. The dose causing 50% reduction in dry weight was 620 g a.e. ha(-1) for GR and 114 g ha(-1) for GS, resulting in an R/S ratio of 5.4. GS had significantly higher maximum (14) C-glyphosate absorption into the treated leaf (51.3%) than GR (39.5%), a difference of 11.8% in maximum absorption. GR also retained more (14) C-glyphosate in the treated leaf (74%) than GS (51%), and GR translocated less glyphosate (27%) to other plant parts (stems, roots and root exudation) than GS (36%). There were no mutations at the Pro106 codon in the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). There was no difference in EPSPS genomic copy number or EPSPS transcription between GS and GR populations. CONCLUSION: Based on these data, reduced glyphosate absorption and increased glyphosate retention in the treated leaf contribute to glyphosate resistance in this C. elata population from Brazil. © 2015 Society of Chemical Industry.

Effect of Commercial Adjuvants on Vegetable Crop Fungicide Coverage, Absorption, and Efficacy.

The addition of an appropriate adjuvant with foliar fungicide can significantly improve coverage, absorption, and efficacy. Laboratory and field studies evaluated coverage, absorption, and efficacy of commercial adjuvants with diverse chemistries on multiple host-pathogen systems. Organosilicone-based adjuvants improved coverage by 26 to 38% compared with a latex spreader-sticker and water. Significant crop by coverage interaction effects were also detected. The organosilicone/methylated seed oil-based adjuvant, Aero Dyne-Amic, significantly improved total [14C]azoxystrobin absorption on onion and potato by 30 and 21%, respectively, compared with water. The spreader-sticker, Bond, improved [14C]azoxystrobin absorption on onion and dry bean by 41 and 39%, respectively, compared with water. In experimental field plots, dry bean rust incidence was reduced by 52% when Kinetic or Latron AG-98 was added to maneb compared with maneb alone. The area under the potato early blight disease progress curve was reduced 29, 24, or 21% when Kinetic, Bond, or Latron AG-98 was added to maneb, respectively, compared with maneb applications alone.

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.

Herbicide-resistant weeds: from research and knowledge to future needs.

Synthetic herbicides have been used globally to control weeds in major field crops. This has imposed a strong selection for any trait that enables plant populations to survive and reproduce in the presence of the herbicide. Herbicide resistance in weeds must be minimized because it is a major limiting factor to food security in global agriculture. This represents a huge challenge that will require great research efforts to develop control strategies as alternatives to the dominant and almost exclusive practice of weed control by herbicides. Weed scientists, plant ecologists and evolutionary biologists should join forces and work towards an improved and more integrated understanding of resistance across all scales. This approach will likely facilitate the design of innovative solutions to the global herbicide resistance challenge.

A lack of evidence for an ecological role of the putative allelochemical (+/-)-catechin in spotted knapweed invasion success.

Allelopathy is a notoriously difficult mechanism to demonstrate. There has been a recent resurgence of interest in allelopathy because of the work done on the invasive weed spotted knapweed and its putative allelochemical, (+/-)-catechin. In this study we collected and analyzed soil samples taken from three, long-term knapweed infested sites in Montana, USA during the summer and fall of 2005. We only detected catechin in all the soil cores at one time point (August, 2005) at two of the sites. Field levels from these two sites were nearly three orders of magnitude lower than what has previously been reported to cause reduced growth in a sensitive native species. Fourteen percent of the remaining soil cores contained low but detectable levels (<0.11 ppm) of (+/-)-catechin. Additional experiments indicated that soil moisture appears to play a significant role in whether or not catechin degrades rapidly or remains in the soil. Adding to previous work, this paper sheds doubt on the importance of this chemical in spotted knapweed invasion success.