Introduction
Waterhemp [Amaranthus tuberculatus (Moq.) Sauer] is a North American native summer-annual weed (Sauer Reference Sauer1957; Waselkov and Olsen Reference Waselkov and Olsen2014) with many life-history traits favorable for adaptation, persistence, and spread in agricultural environments. These include a rapid growth rate, prolific seed production, small seed size, adaptability to diverse environments, and an ability to rapidly evolve herbicide resistance. Such characteristics have made A. tuberculatus one of the most common and most troublesome weed species in the U.S. Midwest, particularly in corn (Zea mays L.) and soybean [Glycine max (L.) Merr.] (Van Wychen Reference Van Wychen2022, Reference Van Wychen2023). In the United States, A. tuberculatus has evolved resistance to herbicides spanning seven sites of action (SOAs): acetolactate synthase (ALS), auxin mimics, photosynthesis at photosystem II–serine 264 binders (PSII), enolpyruvyl shikimate phosphate synthase (EPSPS), protoporphyrinogen oxidase (PPO), very-long-chain fatty-acid synthesis (VLCFA), and hydroxyphenyl pyruvate dioxygenase (HPPD) (Heap Reference Heap2025; Tranel Reference Tranel2021). In Wisconsin, A. tuberculatus resistance to ALS and EPSPS inhibitors is widespread, whereas resistance to auxin mimics, PSII inhibitors, and PPO inhibitors is present to a lesser extent (Faleco et al. Reference Faleco, Oliveira, Arneson, Renz, Stoltenberg and Werle2022a; Zimbric et al. Reference Zimbric, Stoltenberg, Renz and Werle2018).
The rapid adoption of glyphosate-resistant (GR) soybean in the late 1990s led to a significant decline in the use of PPO-inhibitor herbicides historically considered to be a key component for weed management in soybean (Dayan et al. Reference Dayan, Barker and Tranel2018). However, intense selection pressure associated with the extensive use of glyphosate has recently led to the evolution of several GR weeds, including A. tuberculatus and Palmer amaranth (Amaranthus palmeri S. Watson). This trend has since sparked a renewed interest in using PPO-inhibitor herbicides, particularly in soybean (Sarangi and Jhala Reference Sarangi and Jhala2019; Sarangi et al. Reference Sarangi, Sandell, Kruger, Knezevic, Irmak and Jhala2017; Tranel Reference Tranel2021; USGS 2024). PPO inhibitors are widely used both preemergence and postemergence in soybean. Additionally, in non-GMO soybean, PPO inhibitors are the main option for postemergence broadleaf weed control (Bradley and Bish Reference Bradley and Bish2020; Gerlach Reference Gerlach2021; Ifft Reference Ifft2019). Herbicides in this group disrupt the oxidation of protoporphyrinogen IX to protoporphyrin IX in both chloroplast and mitochondria causing protoporphyrinogen IX to accumulate in the cytosol. This ultimately leads to the generation of reactive oxygen species, resulting in membrane disruption and plant death (Borgato et al. Reference Borgato, Thiagarayaselvam, Peterson, Hay, Dille and Jugulam2024; Duke and Kenyon Reference Duke and Kenyon1987; Lehnen et al. Reference Lehnen, Sherman, Becerril and Duke1990).
Amaranthus tuberculatus was the first confirmed weed species to evolve resistance to PPO inhibitors (Shoup et al. Reference Shoup, Al-Khatib and Peterson2003; Tranel Reference Tranel2021), with two target site–resistance (TSR) mechanisms reported: a ΔG210 deletion (Evans et al. Reference Evans, Strom, Riechers, Davis, Tranel and Hager2019; Faleco et al. Reference Faleco, Oliveira, Arneson, Renz, Stoltenberg and Werle2022a; Patzoldt et al. Reference Patzoldt, Hager, McCormick and Tranel2006) and an R128G/I substitution (Nie et al. Reference Nie, Mansfield, Harre, Young, Steppig and Young2019), both in the protoporphyrinogen oxidase that is encoded by PPX2 (PPO2). Mutations conferring PPO-inhibitor resistance have also been reported in other weed species. These include the A212T substitution in PPO1 (encoded by PPX1) in goosegrass [Eleusine indica (L.) Gaertn.] (Bi et al. Reference Bi, Wang, Coleman, Porri, Peppers, Patel, Betz, Lerchl and McElroy2020); and other substitutions in PPO2, including R98L in common ragweed (Ambrosia artemisiifolia L.) (Rousonelos et al. Reference Rousonelos, Lee, Moreira, VanGessel and Tranel2012), giant ragweed (Ambrosia trifida L.) (Faleco et al. Reference Faleco, Machado, Bobadilla, Tranel, Stoltenberg and Werle2024), and wild poinsettia (Euphorbia heterophylla L.) (Mendes et al. Reference Mendes, Takano, Adegas, Oliveira, Gaines and Dayan2020); R128G/M (Copeland et al. Reference Copeland, Giacomini, Tranel, Montgomery and Steckel2018; Giacomini et al. Reference Giacomini, Umphres, Nie, Mueller, Steckel, Young, Scott and Tranel2017; Varanasi et al. Reference Varanasi, Brabham, Norsworthy, Nie, Young, Houston, Barber and Scott2018b), G399A (Rangani et al. Reference Rangani, Salas-Perez, Aponte, Knapp, Craig, Mietzner, Langaro, Noguera, Porri and Roma-Burgos2019), and V361A (Nie et al. Reference Nie, Harre and Young2023) in A. palmeri; and R128G in redroot pigweed (Amaranthus retroflexus L.) (Cao et al. Reference Cao, Huang, Wei, Lan, Li, Sun, Wang and Huang2022; Du et al. Reference Du, Li, Jiang, Ju, Guo, Li, Qu and Qu2021; Huang et al. Reference Huang, Cui, Wang, Wu, Zhang, Huang and Wei2020). Additionally, non–target site resistance (NTSR) mechanisms associated with enhanced herbicide detoxification mediated by cytochrome P450 monooxygenases (P450s) and glutathione S-transferases (GSTs) were identified in A. tuberculatus and A. palmeri, respectively (Borgato et al. Reference Borgato, Thiagarayaselvam, Peterson, Hay, Dille and Jugulam2024; Obenland et al. Reference Obenland, Ma, O’Brien, Lygin and Riechers2019; Varanasi et al. Reference Varanasi, Brabham and Norsworthy2018a, Reference Varanasi, Brabham, Korres and Norsworthy2019).
In the summer of 2021, a suspected PPO inhibitor–resistant A. tuberculatus population (A92 accession) from Dodge County, WI, USA, was reported to the Wisconsin Cropping Systems Weed Science Program. This field had been under a corn–soybean rotation for more than 6 yr, with sulfentrazone (Spartan® 4F, FMC, Philadelphia, PA, USA), a PPO-inhibitor herbicide, being applied preemergence at 280 g ai ha−1 in each soybean-growing year until a lack of A. tuberculatus control was noticed. Therefore, our objective was to characterize resistance to PPO inhibitors applied preemergence or postemergence to the A92 accession, along with two PPO inhibitor–susceptible control accessions (A66 and A82). Given that PPO-inhibitor resistance in A. tuberculatus is predominantly conferred through TSR mechanisms, we hypothesized that PPO inhibitors resistance in A92 was driven by TSR mechanisms.
Materials and Methods
Amaranthus tuberculatus Seed Collection and Research Sites
Seed samples from A. tuberculatus plants were harvested in the fall of 2021 and pooled among plants within the same field to compose the A92 accession (Dodge County, WI, USA). A PPO inhibitor–susceptible control accession (A66), harvested in 2018 from Grant County, WI, USA (Faleco et al. Reference Faleco, Oliveira, Arneson, Renz, Stoltenberg and Werle2022a) was included for treatment comparisons in the preemergence experiment. Seed availability of the A66 accession was limited following the preemergence experiment. Therefore, an alternative PPO inhibitor–susceptible control accession (A82), harvested in 2018 from Chippewa County, WI, USA (Faleco et al. Reference Faleco, Oliveira, Arneson, Renz, Stoltenberg and Werle2022a) was included for treatment comparisons in the postemergence experiment.
Seeds from each A. tuberculatus accession were threshed apart from other plant material and cleaned using a seed blower separator (Oregon Seed Blower, Hoffman Manufacturing Inc, Corvallis, OR, USA). To increase the germination rate, each accession was stratified by placing all seeds in a glass container, floated on a thin layer of water, and stored in a dark environment at 5 C for 2 wk. After this period, seeds were washed with water using a soil sieve mesh to retain the seeds and dried on paper towels at room temperature for 24 h (Faleco et al. Reference Faleco, Oliveira, Arneson, Renz, Stoltenberg and Werle2022a). Seeds were placed in plastic bags and stored at 5 C until the onset of dose–response experiments, which were conducted at the University of Wisconsin–Madison Walnut Street Greenhouses, Madison, WI, USA, in 2022 and 2023.
The A92 Response to PPO-Inhibitor Herbicides Applied Preemergence
A dose–response greenhouse experiment was conducted to quantify the sensitivity of A92 (suspected PPO inhibitor–resistant) and A66 (PPO inhibitor–susceptible control) accessions to sulfentrazone, fomesafen, and flumioxazin applied preemergence. Treatments were evaluated in a completely randomized design (CRD), with four replications per treatment, and repeated over time (two experimental runs). For the A92 accession, herbicide rates were 0× (non-treated control [NTC]), 0.015×, 0.031×, 0.062×, 0.125×, 0.25×, 0.5×, 1×, 2×, 4×, and 8× the label rate of sulfentrazone (Spartan® 4F, FMC; 1×: 280 g ai ha−1), fomesafen (Flexstar® 1.88 SL, Syngenta Crop Protection, Greensboro, NC, USA; 1×: 263 g ai ha−1), and flumioxazin (Valor EZ®, Valent USA, Walnut Creek, CA, USA; 1×: 105 g ai ha−1). For A66, herbicide rates were 0×, 0.015×, 0.031×, 0.062×, 0.125×, 0.25×, 0.5×, and 1× the label rate of the herbicides described.
Experimental units consisted of approximately 190 seeds (measured by volume using a scoop) planted 1.5 cm deep in 360-ml pots (8.9 cm Kord Traditional Square Pot, HC Companies, Twinsburg, OH, USA) filled with non-sterilized field soil (silty clay loam; 6.4 pH; 3.0% organic matter; 18% sand, 53% silt, and 30% clay by weight). The soil was watered immediately before herbicide application to promote seed germination and preemergence herbicide activation. Preemergence herbicide treatments were applied using a single-nozzle research track spray chamber (DeVries, Hollandale, MN, USA) equipped with a DG9502EVS nozzle (TeeJet® Technologies, Spraying Systems, Wheaton, IL, USA). A carrier volume of 140 L ha−1 was used for all applications. Plants were maintained in a greenhouse with temperatures ranging from 20 to 35 C (minimum/maximum), and a natural ventilation system. Relative humidity ranged from 20% to 100% and was not controlled, allowing for its natural variation within the greenhouse. Natural lighting was supplemented with artificial lighting from 400-W high-pressure sodium light bulbs (50,000 lumens; LU400/ECO, Ledvance, Wilmington, MA, USA), providing a 16-h photoperiod. Plants were top-watered daily and fertigated weekly with 20-10-20 water-soluble fertilizer (Peters Professional, ICL Fertilizers, Dublin, OH, USA) delivering 500 ppm of nitrogen (N) and potassium (K) each and 250 ppm of phosphorus (P).
At 28 d after treatment (DAT), emerged plants per experimental unit were counted. Data were converted into percent plant count compared with the respective NTC using Equation 1 (adapted from Faleco et al. Reference Faleco, Oliveira, Arneson, Renz, Stoltenberg and Werle2022b):
$${\rm{Plant\;Count\;}}\left( {\rm{\% }} \right) = {{\rm{\;}}{{{\rm{PCEU}}}}\over{{\overline {{\rm{PCNTC}}} }}}$$
where
$\;{\rm{PCEU}}$
represents the plant count of the experimental unit and
$\overline {{\rm{PCNTC}}} \;$
represents the plant count mean of the respective NTC.
The A92 Response to PPO-Inhibitor Herbicides Applied Postemergence
A dose–response greenhouse experiment was conducted to quantify the sensitivity of the A92 (suspected PPO inhibitor–resistant) and A82 (PPO inhibitor–susceptible control) accessions to lactofen and fomesafen applied postemergence. Treatments were evaluated in a CRD experiment, with four replications per treatment, and repeated once over time (two experimental runs). Herbicide rates were 0×, 0.015×, 0.031×, 0.062×, 0.125×, 0.25×, 0.5×, 1×, 2×, 4×, 8×, and 16× the label rate of lactofen (Cobra® 2EC, Valent USA; 1×: 219 g ai ha−1 + 0.4 v/v % high surfactant oil concentrate [HSOC] + 2,804 g ha−1 ammonium sulfate [AMS]) and fomesafen (Flexstar® 1.88 SL, Syngenta Crop Protection; 1×: 263 g ai ha−1 + 0.5 v/v % HSOC + 1,430 g ha−1 AMS).
Amaranthus tuberculatus seeds were planted 1.5 cm deep in potting mix (Promix® HP Mycorrhizae, Premier Tech Horticulture, Rivière-du-Loup, QC, Canada) contained in 8,600-ml plastic flats (1020 Standard Full Depth Vacuum Flat, HC Companies). Seedlings at the 4 true-leaf stage were transplanted into 656-ml pots (D40H Deepots™, Stuewe & Sons, Tangent, OR, USA) filled with potting mix. The experimental unit was 1 seedling pot−1. Herbicides were applied when plants reached 5 to 10 cm in height using the spray chamber, nozzle, and carrier volume described earlier. Plants were maintained in the greenhouse under the same conditions described earlier.
At 21 DAT, aboveground biomass was harvested and forced-air dried at 50 C to constant mass. The biomass data were converted into percent biomass compared with respective NTC using Equation 2 (adapted from Faleco et al. Reference Faleco, Oliveira, Arneson, Renz, Stoltenberg and Werle2022b):
$${\rm{Biomass\;}}\left( {\rm{\% }} \right) = {{\rm{\;}}{{{\rm{BEU}}}}\over{{\overline {{\rm{BNTC}}} }}}$$
where
$\;{\rm{BEU}}$
represents the biomass of the experimental unit and
$\overline {{\rm{BNTC}}} \;$
represents the biomass mean of the respective NTC.
Assessment of TSR Mechanism for PPO-Inhibitor Herbicides
The presence of target-site mutations conferring resistance to PPO-inhibitor herbicides was evaluated in the A92 accession using an RNA sequencing approach. Leaf tissues were collected from 15 untreated A92 seedlings, immediately frozen in liquid nitrogen, and stored at −80 C until RNA extraction. Total RNA was extracted using a Trizol-based method (Simms et al. Reference Simms, Cizdziel and Chomczynski1993), followed by DNase I (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) treatment to remove contaminating DNA. After extraction, three RNA pools were prepared, each consisting of equal amounts of RNA from five plants randomly selected from the 15 untreated A92 seedlings. The RNA quality and quantity of the pooled samples were assessed using gel electrophoresis (1% agarose) and a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA), respectively. The RNA samples were then sent to the Roy J. Carver Biotechnology Center at the University of Illinois for Illumina library construction and sequencing. Libraries were prepared with the WatchMaker mRNAseq Prep Poly A+ selection kit (Watchmaker Genomics, Boulder, CO, USA) and sequenced on an Illumina NovaSeq X 25B lane generating 150-bp paired-end reads. Sequenced read quality was assessed using FastQC v. 0.12.0 (Andrews Reference Andrews2010) and summarized with MultiQC v. 1.12 (Ewels et al. Reference Ewels, Magnusson, Lundin and Käller2016).
The reads were then aligned to an available A. tuberculatus reference genome (Raiyemo et al. Reference Raiyemo, Cutti, Patterson, Llaca, Fengler, Montgomery, Morran, Gaines and Tranel2024) using STAR v. 2.710b (Dobin et al. Reference Dobin, Davis, Schlesinger, Drenkow, Zaleski, Jha, Batut, Chaisson and Gingeras2013). The sequence data were examined at specific codons in PPX1 (A212, corresponding to A235 in A. tuberculatus) and PPX2 (G210, R128, V361, and G399), which have been previously associated with resistance to PPO-inhibitor herbicides in several weed species (Bi et al. Reference Bi, Wang, Coleman, Porri, Peppers, Patel, Betz, Lerchl and McElroy2020; Copeland et al. Reference Copeland, Giacomini, Tranel, Montgomery and Steckel2018; Du et al. Reference Du, Li, Jiang, Ju, Guo, Li, Qu and Qu2021; Faleco et al. Reference Faleco, Machado, Bobadilla, Tranel, Stoltenberg and Werle2024; Giacomini et al. Reference Giacomini, Umphres, Nie, Mueller, Steckel, Young, Scott and Tranel2017; Huang et al. Reference Huang, Cui, Wang, Wu, Zhang, Huang and Wei2020; Mendes et al. Reference Mendes, Takano, Adegas, Oliveira, Gaines and Dayan2020; Nie et al. Reference Nie, Mansfield, Harre, Young, Steppig and Young2019, Reference Nie, Harre and Young2023; Patzoldt et al. Reference Patzoldt, Hager, McCormick and Tranel2006; Rangani et al. Reference Rangani, Salas-Perez, Aponte, Knapp, Craig, Mietzner, Langaro, Noguera, Porri and Roma-Burgos2019; Rousonelos et al. Reference Rousonelos, Lee, Moreira, VanGessel and Tranel2012; Varanasi et al. Reference Varanasi, Brabham, Norsworthy, Nie, Young, Houston, Barber and Scott2018b). Specific codons within PPX1 and PPX2 were identified in the reference genome through a local BLASTn search using available resistance alleles as the query. Read alignments at these codons were examined, and allelic frequencies were calculated using the Integrative Genomics Viewer v. 2.11.9 genome browser (Robinson et al. Reference Robinson, Thorvaldsdóttir, Winckler, Guttman, Lander, Getz and Mesirov2011).
To identify potential new mutations that confer resistance, consensus sequences of the PPX1 and PPX2 genes were generated from each pool using a custom script and further evaluated. A multi-sequence alignment was performed with the translated protein sequences from each pool, along with homologous proteins from nine different plant species, using CLC Sequence Viewer v. 8.0 (CLC Bio, Aarhus, Denmark). Amino acid substitutions unique to the pool sequences and not present in other species were further investigated to determine their localization relative to the catalytic domain by comparing the respective residues in the crystallized PPO2 (1SEZ) from tobacco (Nicotiana tabacum L.) (Koch et al. Reference Koch, Breithaupt, Kiefersauer, Freigang, Huber and Messerschmidt2004). The ConSurf server v. 3.0 was used to determine the conservation of amino acid residues of the PPO proteins based on phylogenetic relationships among each protein and 149 homologous sequences collected from the UniRef database (Landau et al. Reference Landau, Mayrose, Rosenberg, Glaser, Martz, Pupko and Ben-Tal2005).
Statistical Analyses
Dose–response models were fit to the percent plant count data (preemergence experiment) and to the percent biomass data (postemergence experiment) using the drm function from drc package v. 3.0-1 (Ritz et al. Reference Ritz, Baty, Streibig and Gerhard2015) in R v. 4.3.2 (R Core Team 2023) and RStudio v. 2023.9.1.494 (Posit Team 2023). Based on the lowest Akaike information criterion (AIC) from the mselect function (drc package), the two-parameter Weibull type 1 model (W1.2; Equation 3) was selected and used in all analyses:
$$f\left( x \right) = {\rm{exp}}\left\{ - \exp \left[ {b\left( {\log \left( x \right) - {\rm{log}}(e} \right)} \right)]\right\}$$
where
$b$
is the relative slope around the inflection point
$e$
(Keshtkar et al. Reference Keshtkar, Kudsk and Mesgaran2021; Ritz et al. Reference Ritz, Baty, Streibig and Gerhard2015).
In asymmetrical models, such as W1.2, the parameter
$e$
(inflection point) does not correspond to the ED50 (here, defined as the estimated herbicide rate that decreased percent plant count [preemergence experiment] or percent biomass [postemergence experiment] by 50% compared with the respective NTC). Therefore, the absolute ED50 (rather than the commonly used relative ED50) (Keshtkar et al. Reference Keshtkar, Kudsk and Mesgaran2021) was estimated using the ED.drc function (drc package). The EDcomp function (drc package), which performs a Student’s t-test, was used to estimate the resistance index (RI) by comparing the absolute ED50 of the A92 accession (suspected PPO inhibitor–resistant) versus the absolute ED50 of the PPO inhibitor–susceptible control accession (A66 in the preemergence experiment and A82 in the postemergence experiment). Because our objective was to assess the response of the A. tuberculatus accessions evaluated herein without generating plants that were homozygous for resistance, a significance level of α = 0.1 was used in all comparisons.
Results and Discussion
The A92 Response to PPO-Inhibitor Herbicides Applied Preemergence
The A92 accession, compared with A66 (control), was resistant to sulfentrazone (RI = 3.1; P-value = 0.0278; Table 1; Figure 1A) and fomesafen (RI = 3.1; P-value = 0.0745; Table 1; Figure 1B) and susceptible to flumioxazin (P-value = 0.3853; Table 1; Figure 1C).
Dose–response modeling output comparing the protoporphyrinogen oxidase (PPO) inhibitor–resistant (A92) and the PPO inhibitor–susceptible control (A66) Amaranthus tuberculatus accessions from Wisconsin at 28 d after treatment with sulfentrazone, fomesafen, and flumioxazin applied preemergence.
a ED50 percent plant count: estimated herbicide rate that decreased percent plant count by 50% compared with the respective non-treated control. Standard errors are shown in parentheses. For each herbicide, estimates followed by the same letter within a column did not differ by Student’s t-test (α = 0.1).
b ED50 RI: resistance index calculated by dividing the ED50 of the PPO inhibitor–resistant accession by the ED50 of the control accession. A dash (—) indicates that the ED50 RI was not calculated because ED50 RI P-value was not significant.
Dose–response curves comparing percent plant count of the protoporphyrinogen oxidase (PPO) inhibitor–resistant (A92) and the PPO inhibitor–susceptible control (A66) Amaranthus tuberculatus accessions from Wisconsin at 28 d after treatment with sulfentrazone (A), fomesafen (B), and flumioxazin (C) applied preemergence. Vertical dash-dot lines indicate the respective 1× herbicide label rate. Asterisks indicate the ED50 percent plant count (estimated herbicide rate that decreased percent plant count by 50% compared with the respective non-treated control).
The A92 Response to PPO-Inhibitor Herbicides Applied Postemergence
The A92 accession, compared with A82 (control), was resistant to lactofen (RI = 18.6; P-value = 0.0003; Table 2; Figure 2A) and to fomesafen (RI = 5.9; P-value = < 0.0001; Table 2; Figure 2B).
Dose–response modeling output comparing the protoporphyrinogen oxidase (PPO) inhibitor–resistant (A92) and the PPO inhibitor–susceptible control (A82) Amaranthus tuberculatus accessions from Wisconsin at 21 d after treatment with lactofen and fomesafen applied postemergence.
a ED50 percent biomass: estimated herbicide rate that decreased percent biomass by 50% compared with the respective non-treated control. Standard errors are shown in parentheses. For each herbicide, estimates followed by the same letter within a column did not differ by Student’s t-test (α = 0.1).b ED50 RI: resistance index calculated by dividing the ED50 of the PPO inhibitor–resistant accession by the ED50 of the control accession.
Dose–response curves comparing percent biomass of the protoporphyrinogen oxidase (PPO) inhibitor–resistant (A92) and the PPO inhibitor–susceptible control (A82) Amaranthus tuberculatus accessions from Wisconsin at 21 d after treatment with lactofen (A) and fomesafen (B) applied postemergence. Vertical dash-dot lines indicate the respective 1× herbicide label rate. Asterisks indicate the ED50 percent biomass (estimated herbicide rate that decreased percent biomass by 50% compared with the respective non-treated control).
The efficacy of many herbicides, including PPO inhibitors, is influenced by weed size and growth stage at the time of application. For example, younger weeds typically have less-developed leaf cuticular wax, which facilitates herbicide absorption and improves control efficacy (Ivaschenko and Ivaschenko Reference Ivaschenko and Ivaschenko2019). Furthermore, PPO inhibitors are sometimes more effective when applied preemergence rather than postemergence (Falk et al. Reference Falk, Shoup, Al-Khatib and Peterson2006; Harder et al. Reference Harder, Nelson and Smeda2012). Consequently, PPO inhibitors applied preemergence typically provide partial control even of resistant populations. This has led to the misconception that PPO-inhibitor resistance in A. tuberculatus is limited only to postemergence applications (Dayan et al. Reference Dayan, Barker and Tranel2018; Tranel Reference Tranel2021). Moreover, as herbicide concentrations in the soil decrease over time due to factors such as microbial degradation, leaching, and volatilization, it can be difficult to directly attribute the reduced residual control of resistant biotypes to herbicide resistance (Tranel Reference Tranel2021). However, research has shown that while the RI may increase from preemergence to postemergence applications, fomesafen-resistant Amaranthus species are less sensitive to the herbicide regardless of the application timing (Lillie et al. Reference Lillie, Giacomini and Tranel2020; Wuerffel et al. Reference Wuerffel, Young, Matthews and Young2015). Similarly, our results demonstrated that the A92 accession, compared with control accessions, was less sensitive to fomesafen regardless of the application timing (Tables 1 and 2; Figures 1B and 2B).
Despite the significant decline in the use of PPO inhibitors after the introduction of GR soybean, particularly postemergence (Dayan et al. Reference Dayan, Barker and Tranel2018), their continued preemergence use may have contributed to a gradual increase in the frequency of resistance to PPO inhibitors in A. tuberculatus. Moreover, the evolution PPO-inhibitor resistance in A. tuberculatus is a major concern that should be monitored and remediated (Barker et al. Reference Barker, Pawlak, Duke, Beffa, Tranel, Wuerffel, Young, Porri, Liebl, Aponte, Findley, Betz, Lerchl, Culpepper, Bradley and Dayan2023), particularly in a scenario with novel PPO-inhibitor herbicides, including trifludimoxazim and epyrifenacil (Porri et al. Reference Porri, Betz, Seebruck, Knapp, Johnen, Witschel, Aponte, Liebl, Tranel and Lerchl2023; Sada et al. Reference Sada, Jin, Hikosaka and Ido2025), and PPO inhibitor–resistant crop traits (Larue et al. Reference Larue, Ream, Zhou, Moshiri, Howe, Goley, Sparks, Voss, Hall, Ellis, Weihe, Qi, Ribeiro, Wei and Guo2020) expected to be marketed in the near future.
Assessment of TSR Mechanism for PPO-Inhibitor Herbicides
Resistance to PPO-inhibiting herbicides in A. tuberculatus has most commonly been associated with a deletion at position G210, but more recently, substitutions at R128 have also been associated with resistance (Tranel Reference Tranel2021). In the A92 accession, we identified a synonymous substitution (AGA to AGG) at the PPX2 R128 codon, which was seen previously in other A. tuberculatus populations (Nie et al. Reference Nie, Mansfield, Harre, Young, Steppig and Young2019). According to our results, this substitution was identified at low frequencies in pools 2 (minor allele frequency [MAF] = 0.190) and 3 (MAF = 0.112) (Supplementary Figure S1). However, no previously reported mutations corresponding to resistance were identified at the G210 or R128 codons (Figure 3; Supplementary Figure S1).
Multiple sequence alignment of the protoporphyrinogen oxidase (PPO) inhibitor–resistant Amaranthus tuberculatus accession (A92) consensus sequences with related homologues across amino acid residues forming the catalytic domains of PPO2 (A) and PPO1 (B). Residue changes conferring resistance to PPO inhibitors in PPO2 (at R128, G210, V361, and G399) and PPO1 (at A235) are indicated with a red box, and residues reported to be involved in substrate binding are indicated with a blue box.
A single nonsynonymous mutation resulting in a substitution of P to L at position 340 was identified within PPX1 in two of the three A92 pools (Supplementary Table S1). Conversely, 10 nonsynonymous mutations were identified in PPX2 across all three pools. A comparison of the PPO consensus sequences with homologous proteins showed that of the predicted amino acid substitutions identified, only four in PPO2 were unique to the A92 accession. These substitutions included S42T, A172V, E253G, and S476C. Further investigation revealed that these substitutions were consistently observed in all three A92 pools at varying frequencies. Notably, however, none of the substitutions were near the catalytic domain when compared with the respective residues in the crystallized PPO2 protein from tobacco (Figure 3; Supplementary Figure S2). Additionally, none of these substitutions occurred at highly conserved residues, suggesting that they are unlikely to disrupt the structural or functional properties of the proteins in a way that could lead to herbicide resistance (Supplementary Figures S3 and S4).
In the A92 accession, nonsynonymous mutations in PPX1 and PPX2 were identified, but these mutations do not appear to affect the binding domains or conserved functional regions of the encoded proteins. This observation suggests that an NTSR mechanism may be associated with resistance to PPO-inhibiting herbicides in this accession, as has been suggested in previous studies in Amaranthus spp. For example, metabolic NTSR to PPO inhibitors was recently suggested in an A. tuberculatus population from Stanford, IL (SIR), which lacked any target-site mutations but was resistant to carfentrazone-ethyl but not to other PPO inhibitors (Obenland et al. Reference Obenland, Ma, O’Brien, Lygin and Riechers2019). This population was previously reported to exhibit metabolic resistance to HPPD inhibitors, likely mediated by enhanced P450 activity (Ma et al. Reference Ma, Kaundun, Tranel, Riggins, McGinness, Hager, Hawkes, McIndoe and Riechers2013). Further research exhibited a reversal of carfentrazone-ethyl resistance following treatment with malathion, a P450 inhibitor, supporting the hypothesis of a P450-mediated metabolic resistance mechanism contributing to cross-resistance.
In related Amaranthus species, NTSR to PPO inhibitors has also been observed. For instance, both P450s and GSTs were associated with fomesafen resistance in an A. palmeri population from Arkansas, following reversal of resistance with malathion (P450 inhibitor) and NBD-Cl (GST inhibitor) (Varanasi et al. Reference Varanasi, Brabham and Norsworthy2018a). In A. retroflexus, P450-mediated metabolic resistance was found to coexist with the R128G mutation after high-performance liquid chromatography-mass spectrometry analysis indicated that fomesafen was metabolized more rapidly in resistant plants compared with susceptible plants (Cao et al. Reference Cao, Huang, Wei, Lan, Li, Sun, Wang and Huang2022). Although there is no direct evidence of NTSR in the A92 accession at this time, further experiments are necessary to determine whether such mechanisms could be associated with resistance to PPO herbicides in this accession, including experiments using P450 and GST inhibitors as well as herbicide metabolite profiling.
In conclusion, according to our results, the A92 accession is resistant to sulfentrazone (3.1-fold; P-value = 0.0278) and fomesafen (3.1-fold; P-value = 0.0745) applied preemergence and to lactofen (18.6-fold; P-value = 0.0003) and fomesafen (5.9-fold; P-value = < 0.0001) applied postemergence. Resistance to PPO inhibitors in A92 was not explained by the presence of any known target-site mutations in PPX1 or PPX2 genes. Moreover, A92 represents the first confirmed case of an A. tuberculatus accession resistant to PPO inhibitors applied preemergence in Wisconsin, with resistance possibly being conferred through some NTSR mechanism. Our results corroborate previous research (Lillie et al. Reference Lillie, Giacomini and Tranel2020; Wuerffel et al. Reference Wuerffel, Young, Matthews and Young2015) demonstrating that the A92 accession, compared with control accessions, was less sensitive to fomesafen regardless of the application timing. Further research is necessary to identify other potential PPO-inhibitor resistance mechanisms in the A92 accession, including NTSR mechanisms associated with P450s or GSTs.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/wsc.2025.10044
Acknowledgments
The authors thank independent crop consultant Bill Stangel for collecting and submitting seed samples from the A92 Amaranthus tuberculatus accession, and the UW-Madison Walnut Street Greenhouse Staff and Cropping Systems Weed Science Program for their technical assistance.
Funding statement
The authors thank the Wisconsin Soybean Marketing Board for funding FAF’s graduate research assistantship.
Competing interests
The authors declare no conflicts of interest.
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