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.

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.

Palmer amaranth (Amaranthus palmeri S. Wats.) is a highly competitive weed that can be difficult to manage in many cropping systems. Research to date has not quantified the growth and development of A. palmeri in a manner that allows direct comparisons across cropping systems. Research was conducted to compare the growth, development, and seed production of A. palmeri when competing with corn (Zea mays L.), cotton (Gossypium hirsutum L.), peanut (Arachis hypogaea L.), and soybean [Glycine max (L.) Merr.] when emerging with crops or emerging three weeks after crops emerge. Regardless of when A. palmeri emerged, seed production was greatest and similar in cotton and peanut and exceeded that of corn and soybean; seed production in soybean exceeded that of corn. However, seed production was approximately 10-fold greater when A. palmeri emerged with crops compared with emergence three weeks later. These results illustrate the importance of controlling weeds during the first three weeks of the season relative to contributions of A. palmeri to the weed seed bank and is the first report comparing seed production in presence of these crops in a manner allowing a statistical comparison of seed production and highlighting the importance of crop sequence for seed bank management.

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.

Herbicide-resistant Echinochloa spp. pose a significant threat to U.S. rice production. Two surveys were conducted to characterize Echinochloa resistance to common rice herbicides and provide important demographic information on the populations in Arkansas: one was the Echinochloa Herbicide Resistance Confirmation Survey conducted annually since 2006; the other was the Echinochloa Herbicide Resistance Demographics Survey conducted since 2010. The Resistance Confirmation Survey showed that resistance to propanil (50%) was most prevalent, followed by quinclorac (23%), imazethapyr (13%), and cyhalofop (3%). Multiple resistance increased with time, with 27% of accessions being multiple-resistant, mostly to propanil+quinclorac (12%). The parallel Resistance Demographics Survey tested resistance by species. Of the 264 accessions collected, 73% were junglerice, 14% were rough barnyardgrass, and 11% were barnyardgrass. Overall, this survey also showed resistance to propanil (53%) and quinclorac (28%) being most prevalent, with low frequencies of resistance to cyhalofop (12%) and imazethapyr (6%). Resistance to herbicides was less frequent with barnyardgrass (54%) and rough barnyardgrass (28%) than with junglerice (73%). Multiple resistance was most frequent with junglerice (33%) and least frequent with rough barnyardgrass (8%). Across both surveys, the resistance cases were clustered in the northeast and Grand Prairie regions of the state. Herbicide resistance among Echinochloa populations in rice fields is continuing to increase in frequency and complexity. This is a consequence of sequential selection with different major herbicide sites of action, starting with propanil followed by quinclorac and others.