Protoporphyrinogen oxidase (PPO)-inhibiting herbicides are used to control weeds in a variety of crops. These herbicides inhibit heme and photosynthesis in plants. PPO-inhibiting herbicides are used to control Amaranthus palmeri (Palmer amaranth) especially those with resistance to glyphosate and acetolactate synthase (ALS) inhibiting herbicides. While investigating the basis of high fomesafen-resistance in A. palmeri , we identified a new amino acid substitution of glycine to alanine in the catalytic domain of PPO2 at position 399 (G399A) (numbered according to the protein sequence of A. palmeri ). G399 is highly conserved in the PPO protein family across eukaryotic species. Through combined molecular, computational, and biochemical approaches, we established that PPO2 with G399A mutation has reduced affinity for several PPO-inhibiting herbicides, possibly due to steric hindrance induced by the mutation. This is the first report of a PPO2 amino acid substitution at G399 position in a field-selected weed population of A. palmeri . The mutant A. palmeri PPO2 showed high-level in vitro resistance to different PPO inhibitors relative to the wild type. The G399A mutation is very likely to confer resistance to other weed species under selection imposed by the extensive agricultural use of PPO-inhibiting herbicides.

The widespread occurrence of Palmer amaranth resistant to acetolactate synthase inhibitors and/or glyphosate led to the increased use of protoporphyrinogen oxidase (PPO)-inhibiting herbicides. This research aimed to: (1) evaluate the efficacy of foliar-applied fomesafen to Palmer amaranth, (2) evaluate cross-resistance to foliar PPO inhibitors and efficacy of foliar herbicides with different mechanisms of action, (3) survey the occurrence of the PPO Gly-210 deletion mutation among PPO inhibitor–resistant Palmer amaranth, (4) identify other PPO target-site mutations in resistant individuals, and (5) determine the resistance level in resistant accessions with or without the PPO Gly-210 deletion. Seedlings were sprayed with fomesafen (263 gaiha−1), dicamba (280 gaiha−1), glyphosate (870 gaiha−1), glufosinate (549 g ai ha−1), and trifloxysulfuron (7.84 gaiha−1). Selected fomesafen-resistant accessions were sprayed with other foliar-applied PPO herbicides. Mortality and injury were evaluated 21 d after treatment (DAT). The PPX2L gene of resistant and susceptible plants from a selected accession was sequenced. The majority (70%) of samples from putative PPO-resistant populations in 2015 were confirmed resistant to foliar-applied fomesafen. The efficacy of other foliar PPO herbicides on fomesafen-resistant accessions was saflufenacil>acifluorfen=flumioxazin>carfentrazone=lactofen>pyraflufen-ethyl>fomesafen>fluthiacet-methyl. With small seedlings, cross-resistance occurred with all foliar-applied PPO herbicides except saflufenacil (i.e., 25% with acifluorfen, 42% with flumioxazin). Thirty-two percent of PPO-resistant accessions were multiple resistant to glyphosate and trifloxysulfuron. Resistance to PPO herbicides in Palmer amaranth occurred in at least 13 counties in Arkansas. Of 316 fomesafen survivors tested, 55% carried the PPO Gly-210 deletion reported previously in common waterhemp. The PPO gene (PPX2L) in one accession (15CRI-B), which did not encode the Gly-210 deletion, encoded an Arg-128-Gly substitution. The 50% growth reduction values for fomesafen in accessions with Gly-210 deletion were 8- to 15-fold higher than that of a susceptible population, and 3- to 10-fold higher in accessions without the Gly-210 deletion.

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Amaranthus palmeri (Amaranthaceae) is a noxious weed in several agroecosystems and in some cases seriously threatens the sustainability of crop production in North America. Glyphosate-resistant Amaranthus species are widespread, prompting the use of alternatives to glyphosate such as glufosinate, in conjunction with glufosinate-resistant crop cultivars, to help control glyphosate-resistant weeds. An experiment was conducted to analyze the transcriptome of A. palmeri plants that survived exposure to 0.55 kg ha-1 glufosinate. Since there was no record of glufosinate use at the collection site, survival of plants within the population are likely due to genetic expression that pre-dates selection; in the formal parlance of weed science this is described as natural tolerance. Leaf tissues from glufosinate-treated and non-treated seedlings were harvested 24 h after treatment (HAT) for RNA-Seq analysis. Global gene expression was measured using Illumina DNA sequence reads from non-treated and treated surviving (presumably tolerant, T) and susceptible (S) plants. The same plants were used to determine the mechanisms conferring differential tolerance to glufosinate. The S plants accumulated twice as much ammonia as did the T plants, 24 HAT. The relative copy number of the glufosinate target gene GS2 did not differ between T and S plants, with 1 to 3 GS2 copies in both biotypes. A reference cDNA transcriptome consisting of 72,780 contigs was assembled, with 65,282 sequences putatively annotated. Sequences of GS2 from the transcriptome assembly did not have polymorphisms unique to the tolerant plants. Five hundred sixty-seven genes were differentially expressed between treated T and S plants. Of the upregulated genes in treated T plants, 210 were more highly induced than were the upregulated genes in the treated S plants. Glufosinate-tolerant plants had greater induction of ABC transporter, glutathione S-transferase (GST), NAC transcription factor, nitronate monooxygenase (NMO), chitin elicitor receptor kinase (CERK1), heat shock protein 83, ethylene transcription factor, heat stress transcription factor, NADH-ubiquinone oxidoreductase, ABA 8’-hydroxylase, and cytochrome P450 genes (CYP72A, CYP94A1). Seven candidate genes were selected for validation using quantitative real time-PCR. While GST was upregulated in treated tolerant plants in at least one population, CYP72A219 was consistently highly expressed in all treated tolerant biotypes. These genes are candidates for contributing tolerance to glufosinate. Taken together, these results show that differential induction of stress-protection genes in a population can enable some individuals to survive herbicide application. Elevated expression of detoxification-related genes can get fixed in a population with sustained selection pressure, leading to evolution of resistance. Alternatively, sustained selection pressure could select for mutation(s) in the GS2 gene with the same consequence.

Herbicides are major tools for effective weed management. The evolution of resistance to herbicides in weedy species, especially contributed by non-target-site-based resistance (NTSR) is a worrisome issue in crop production globally. Glyphosate-resistant Palmer amaranth (Amaranthus palmeri) is one of the extremely difficult weeds in southern US crop production. In this study, we present the level and molecular basis of resistance to the chloroacetamide herbicide, S-metolachlor, in six field-evolved A. palmeri populations that had survivors at the recommended field-dose (1.1 kg ai ha−1). These samples were collected in 2014 and 2015. The level of resistance was determined in dose-response assays. The effective dose for 50% control (ED50) of the susceptible population was 27 g ai ha−1, whereas the ED50 of the resistant populations ranged from 88 to 785 g ai ha−1. Therefore, A. palmeri resistance to S-metolachlor evolved in Arkansas as early as 2014. Metabolic-inhibitor and molecular assays indicated NTSR in these populations, mainly driven by GSTs. To understand the mechanism of resistance, selected candidate genes were analyzed in leaves and roots of survivors (with 1 × S-metolachlor). Expression analysis of the candidate genes showed that the primary site of S-metolachlor detoxification in A. palmeri is in the roots. Two GST genes, ApGSTU19 and ApGSTF8 were constitutively highly expressed in roots of all plants across all resistant populations tested. The expression of both GSTs increased further in survivors after treatment with S-metolachlor. The induction level of ApGSTF2 and ApGSTF2like by S-metolachlor differed among resistant populations. Overall, higher expression of ApGSTU19, ApGSTF8, ApGSTF2, and ApGSTF2like, which would lead to higher GST activity in roots, was strongly associated with the resistant phenotype. Phylogenetic relationship and analysis of substrate binding site of candidate genes suggested functional similarities with known metolachlor-detoxifying GSTs, effecting metabolic resistance to S-metolachlor in A. palmeri. Resistance is achieved by elevated baseline expression of these genes and further induction by S-metolachlor in resistant plants.

We conducted a greenhouse study to evaluate the differential response of Palmer amaranth to glyphosate and mesotrione and to quantify the level of tolerance to mesotrione in recalcitrant (difficult-to-control) accessions and their offspring. Seeds were collected from 174 crop fields (corn, cotton, and soybean) across Arkansas between 2008 and 2016. Palmer amaranth seedlings (7 to 10 cm tall) were treated with glyphosate at 840 g ae ha–1or mesotrione at 105 g ha–1. Overall, 47% of the accessions (172) were resistant to glyphosate with 68% survivors. Almost 35% of accessions were highly resistant, with 90% survivors. The majority of survivors from glyphosate application incurred between 31% and 60% injury. Mesotrione killed 66% of the accessions (174); the remaining accessions had survivors with injury ranging from 61% to 90%. Accessions with the least response to mesotrione were selected to determine tolerance level. Dose–response assays were conducted with four recalcitrant populations and their F1progeny. The average effective doses (ED50) for the parent accessions and F1progeny of survivors were 21.5 g ha–1and 27.5 g ha–1, respectively. The recalcitrant parent populations were three- to five-fold more tolerant to mesotrione than the known susceptible population, as were the F1progeny.

BACKGROUND Palmer amaranth (Amaranthus palmeri S. Wats) is one of the most common and troublesome weeds in the USA. Palmer amaranth resistance to acetolactate synthase (ALS) inhibitors is widespread in the USA, as in Arkansas. The cross‐resistance patterns and mechanism of resistance are not known. Experiments were conducted to determine cross‐resistance to ALS inhibitors and identify target‐site mutations in 20 Palmer amaranth localities from 13 counties in Arkansas. RESULTS All Palmer amaranth localities tested had plants cross‐resistant to imazethapyr, flumetsulam, primisulfuron, pyrithiobac and trifloxysulfuron. The dose of trifloxysulfuron that caused 50% control was 21–56‐fold greater for resistant accessions than for susceptible ones. All but three resistant plants analyzed had one or two relative copies of ALS; one plant had seven relative copies. All resistant plants tested (18 localities) carried the Trp574Leu mutation, which is known to confer broad resistance to ALS inhibitors, supporting the cross‐resistance pattern observed. Besides the Trp574Leu mutation, 30% of localities had individuals with one additional resistance‐conferring mutation including Ala122Thr, Pro197Ala or Ser653Asn. CONCLUSION The Trp574Leu mutation in ALS is the primary mechanism of resistance to ALS inhibitors in Palmer amaranth from Arkansas, USA. In some localities, multiple mutations have accumulated in one plant. All localities tested contained plants with resistance to five families of ALS inhibitors. Localities with extremely high resistance to ALS inhibitors, and those outside the subset we studied, may harbor non‐target site resistance mechanisms. ALS inhibitors are generally no longer effective on Palmer amaranth in these localities from the US mid‐south. © 2018 Society of Chemical Industry

Changes in the environment, specifically rising temperature and increasing atmospheric carbon dioxide concentration [CO 2 ], can alter the growth and physiology of weedy plants. These changes could alter herbicide efficacy, crop-weed interaction, and weed management. The objectives of this research were to quantify the effects of increased atmospheric [CO 2 ] and temperature on absorption, translocation and efficacy of cyhalofop-butyl on multiple-resistant (MR) and susceptible (S) Echinochloa colona genotypes. E. colona , or junglerice, is a troublesome weed in rice and in agronomic and horticultural crops worldwide. Cyhalofop-butyl is a grass herbicide that selectively controls Echinochloa spp. in rice. Maximum 14 C-cyhalofop-butyl absorption occurred at 120 h after herbicide treatment (HAT) with >97% of cyhalofop-butyl retained in the treated leaf regardless of [CO 2 ], temperature, or genotype. Neither temperature nor [CO 2 ] affected herbicide absorption into the leaf. The translocation of herbicide was slightly reduced in the MR plants vs. S plants either under elevated [CO 2 ] or high temperature. Although plants grown under high [CO 2 ] or high temperature were taller than those in ambient conditions, neither high [CO 2 ] nor high temperature reduced the herbicide efficacy on susceptible plants. However, herbicide efficacy was reduced on MR plants grown under high [CO 2 ] or high temperature about 50% compared to MR plants at ambient conditions. High [CO 2 ] and high temperature increased the resistance level of MR E. colona to cyhalofop-butyl. To mitigate rapid resistance evolution under a changing climate, weed management practitioners must implement measures to reduce the herbicide selection pressure. These measures include reduction of weed population size through reduction of the soil seedbank, ensuring complete control of current infestations with multiple herbicide modes of action in mixture and in sequence, augmenting herbicides with mechanical control where possible, rotation with weed-competitive crops, use of weed-competitive cultivars, use of weed-suppressive cover crops, and other practices recommended for integrated weed management.