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.

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.

BACKGROUND Protoporphyrinogen IX oxidase 2 (PPO2) inhibitors are important for the management of glyphosate‐ and acetolactate synthase‐resistant Palmer amaranth [Amaranthus palmeri (S.) Wats.]. The evolving resistance to PPO inhibitors is of great concern. We surveyed the evolution of resistance to fomesafen in the US Mid‐south and determined its correlation with the known functional PPO2 target‐site mutations (TSM). RESULTS The 167 accessions analyzed were grouped into five categories, four resistant (147) and one susceptible (20). Arkansas accessions constituted 100% of the susceptible group while the Missouri accessions comprised 60% of the most resistant category. The majority of Mississippi accessions (88%) clustered in the high‐survival‐high‐injury category, manifesting an early‐stage resistance evolution. One hundred and fifteen accessions were genotyped for four known TSMs; 74% of accessions carried at least one TSM. The most common single TSM was ΔG210 (18% of accessions) and the predominant double mutation was ΔG210 + G399A (17%). Other mutations are likely less favorable, hence are rare. All TSMs were detected in three accessions. Further examination revealed that 9 and two individuals carried G399A + G210 and G399A + R128G TSM in the same allele, respectively. The existence of these combinations is supported by molecular modeling. CONCLUSIONS Resistance to PPO inhibitors is widespread across the Mid‐southern USA. Highly resistant field populations have plants with multiple mutations. G399A is the most prone to co‐occur with other ppo2 mutations in the same allele. Mutation at R128 in the configuration of the PPO2 catalytic domain restrains the co‐occurrence of R128G with ΔG210, making ΔG210 + G399A the most plausible, tolerable functional mutation combination to co‐occur in the same ppo2 allele.

Echinochloa colona and other species in this genus are a threat to global rice production and food security. Quinclorac, an auxin mimic, is a common herbicide for grass weed control in rice, and Echinochloa spp. have evolved resistance to it. The complete mode of quinclorac action and subsequent evolution of resistance is not fully understood. We analyzed the de novo transcriptome of multiple-herbicide-resistant (ECO-R) and herbicide-susceptible genotypes in response to quinclorac. Several biological processes were constitutively upregulated in ECO-R, including carbon metabolism, photosynthesis, and ureide metabolism, indicating improved metabolic efficiency. The transcriptional change in ECO-R following quinclorac treatment indicates an efficient response, with upregulation of trehalose biosynthesis, which is also known for abiotic stress mitigation. Detoxification-related genes were induced in ECO-R, mainly the UDP-glycosyltransferase (UGT) family, most likely enhancing quinclorac metabolism. The transcriptome data also revealed that many antioxidant defense elements were uniquely elevated in ECO-R to protect against the auxin-mediated oxidative stress. We propose that upon quinclorac treatment, ECO-R detoxifies quinclorac utilizing UGT genes, which modify quinclorac using the sufficient supply of UDP-glucose from the elevated trehalose pathway. Thus, we present the first report of upregulation of trehalose synthesis and its association with the herbicide detoxification pathway as an adaptive mechanism to herbicide stress in Echinochloa, resulting in high resistance.