Protoporphyrinogen oxidase (PPO)-inhibiting herbicides remain an important and useful chemistry 60 yr after their first introduction. In this review, based on topics introduced at the Weed Science Society of America 2021 symposium titled “A History, Overview, and Plan of Action on PPO Inhibiting Herbicides,” we discuss the current state of PPO-inhibiting herbicides. Renewed interest in the PPO-inhibiting herbicides in recent years, due to increased use and increased cases of resistance, has led to refinements in knowledge regarding the mechanism of action of PPO inhibitors. Herein we discuss the importance of the two isoforms of PPO in plants, compile a current knowledge of target-site resistance mechanisms, examine non–target site resistance cases, and review crop selectivity mechanisms. Consistent and reproducible greenhouse screening and target-site mutation assays are necessary to effectively study and compare PPO-inhibitor resistance cases. To this end, we cover best practices in screening to accurately identify resistance ratios and properly interpret common screens for point mutations. The future of effective and sustainable PPO-inhibitor use relies on development of new chemistries that maintain activity on resistant biotypes and the promotion of responsible stewardship of PPO inhibitors both new and old. We present the biorational design of the new PPO inhibitor trifludimoxazin to highlight the future of PPO-inhibitor development and discuss the elements of sustainable weed control programs using PPO inhibitors, as well as how responsible stewardship can be incentivized. The sustained use of PPO inhibitors in future agriculture relies on the effective and timely communication from mode of action and resistance research to agronomists, Extension workers, and farmers.

This study was developed based on a goosegrass [Eleusine indica (L.) Gaertn.] population from Primavera do Leste, MT, Brazil, with resistance to multiple herbicide modes of action (5-enol-pyruvylshikimate-3-phosphate synthase [EPSPS] inhibition: glyphosate; acetyl-coenzyme A carboxylase [ACCase] inhibition: aryloxyphenoxypropionate chemical group). The objective was to identify possible mechanisms of resistance associated or not with herbicide sites of action. Several experiments and analyses were carried out with the contribution of different laboratories and institutions. The results obtained allowed us to conclude that: (1) the Asp-2078-Gly mutation conferred resistance to ACCase inhibitors, without overexpression of ACCase or changes in herbicide absorption and translocation; (2) overexpression of EPSPS, Thr-102 and Pro-106 mutations, and changes in absorption and translocation are not involved in E. indica resistance to glyphosate; (3) the metabolism of glyphosate in resistant E. indica plants requires further studies to elucidate the final destination of this herbicide in these plants. The mechanism of resistance of E. indica biotypes to ACCase-inhibiting herbicides was elucidated: it involves a change in the action site. However, the mechanism of resistance to EPSPS inhibitors was not conclusive, indicating that some hypotheses, mainly those regarding the metabolism of glyphosate in resistant plants, require further testing.

The current assays to confirm herbicide resistance can be time- and labor-intensive (dose–response) or require a skill set/technical equipment (genetic sequencing). Stakeholders could benefit from a rapid assay to confirm herbicide-resistant weeds to ensure sustainable crop production. Because protoporphyrinogen oxidase (PPO)-inhibiting herbicides rapidly interfere with chlorophyll production/integrity; we propose a new, rapid assay utilizing spectral reflectance to confirm resistance. Leaf disks were excised from two PPO-inhibiting herbicide-resistant (target-site [TSR] and non–target site [NTSR]) and herbicide-susceptible redroot pigweed (Amaranthus retroflexus L.) populations and placed into a 24-well plate containing different concentrations (0 to 10 mM) of fomesafen for 48 h. A multispectral sensor captured images from the red (668 nm), green (560 nm), blue (475 nm), and red edge (717 nm) wavebands after a 48-h incubation period. The green leaf index (GLI) was utilized to determine spectral reflectance ratios of the treated leaf disks. Clear differences of spectral reflectance were observed in the red edge waveband for all populations treated with the 10 mM concentration in the dose–response assays. Differences of spectral reflectance were observed for the NTSR population compared with the TSR and susceptible populations treated with the 10 mM concentration in the green waveband and the GLI in the dose–response assay. Leaf disks from the aforementioned A. retroflexus populations and two additional susceptible populations were subjected to a similar assay with the discriminating concentration (10 mM). Spectral reflectance was different between the PPO-inhibiting herbicide-resistant and herbicide-susceptible populations in the red, blue, and green wavebands. Spectral reflectance was not distinctive between the populations in the red edge waveband and the GLI. The results provide a basis for rapidly (∼48 h) detecting PPO-inhibiting herbicide-resistant A. retroflexus via spectral reflectance. Discrimination between TSR and NTSR populations was possible only in the dose–response assay, but the assay still has utility in distinguishing herbicide-resistant plants from herbicide-susceptible plants.

The mitotic-inhibiting herbicide pronamide controls susceptible annual bluegrass (Poa annua L.) pre- and postemergence, but in some resistant populations, postemergence activity is compromised, hypothetically due to a target-site mutation, lack of root uptake, or an unknown resistance mechanism. Three suspected pronamide-resistant (LH-R, SC-R, and SL-R) and two pronamide-susceptible (BS-S and HH-S) populations were collected from Mississippi golf courses. Dose–response experiments were conducted to confirm and quantify pronamide resistance, as well as resistance to flazasulfuron and simazine. Target sites known to confer resistance to mitotic-inhibiting herbicides were sequenced, as were target sites for herbicides inhibiting acetolactate synthase (ALS) and photosystem II (PSII). Pronamide absorption and translocation were investigated following foliar and soil applications. Dose–response experiments confirmed pronamide resistance of LH-R, SC-R, and SL-R populations, as well as instances of multiple resistance to ALS- and PSII-inhibiting herbicides. Sequencing of the α-tubulin gene confirmed the presence of a mutation that substituted isoleucine for threonine at position 239 (Thr-239-Ile) in LH-R, SC-R, SL-R, and BS-S populations. Foliar application experiments failed to identify differences in pronamide absorption and translocation between the five populations, regardless of harvest time. All populations had limited basipetal translocation—only 3% to 13% of the absorbed pronamide—across harvest times. Soil application experiments revealed that pronamide translocation was similar between SC-R, SL-R, and both susceptible populations across harvest times. The LH-R population translocated less soil-applied pronamide than susceptible populations at 24, 72, and 168 h after treatment, suggesting that reduced acropetal translocation may contribute to pronamide resistance. This study reports three new pronamide-resistant populations, two of which are resistant to two modes of action (MOAs), and one of which is resistant to three MOAs. Results suggest that both target site– and translocation-based mechanisms may be associated with pronamide resistance. Further research is needed to confirm the link between pronamide resistance and the Thr-239-Ile mutation of the α-tubulin gene.

Palmer amaranth (Amaranthus palmeri S. Watson) is a troublesome weed in several cropping systems in the United States. The evolution of resistance to multiple herbicides is a challenge for the management of this weed. Recently, we reported metabolic resistance to 2,4-D possibly mediated by cytochrome P450 (P450) activity in a six-way-resistant A. palmeri population (KCTR). Plant growth temperature can influence the herbicide efficacy and level of resistance. The effect of temperature on 2,4-D resistance in A. palmeri is unknown. In the present research, we investigated the response of KCTR and a known susceptible (MSS) A. palmeri response to 2,4-D grown under low-temperature (LT, 24/14 C, day/night [d/n]) or high-temperature (HT, 34/24 C, d/n) regimes. When MSS and KCTR plants were 8- to 10-cm tall, they were treated with 0, 140, 280, 560 (field recommended dose), 1,120, and 2,240 g ai ha^–1 of 2,4-D. Further, 8- to 10-cm-tall MSS and KCTR plants grown at LT and HT were also treated with [¹⁴C]2,4-D to assess the metabolism of 2,4-D at LT and HT. The results of dose–response experiments suggest that KCTR A. palmeri exhibits 23 times more resistance to 2,4-D at HT than MSS. Nonetheless, at LT, the resistance to 2,4-D in KCTR was only 2-fold higher than in MSS. Importantly, there was enhanced metabolism of 2,4-D in both KCTR and MSS A. palmeri at HT compared with LT. Further, treatment with the P450 inhibitor malathion, followed by 2,4-D increased the susceptibility of KCTR at HT. Overall, rapid metabolism of 2,4-D increased KCTR resistance to 2,4-D at HT compared with LT. Therefore, the application of 2,4-D when temperatures are cooler can improve control of 2,4-D–resistant A. palmeri.

Shortawn foxtail (Alopecurus aequalis Sobol.) is an obligate wetland plant that is widely distributed throughout Europe, temperate Asia, and North America. In China, it is widespread in the middle and lower reaches of the Yangtze River as a noxious weed in winter cropping fields with a rice (Oryza sativa L.) rotation. The acetolactate synthase (ALS)-inhibiting herbicide mesosulfuron-methyl has been widely used to control annual grass and broadleaf weeds, including A. aequalis, in wheat (Triticum aestivum L.) fields, leading to the selection of herbicide-resistant weeds. In this study, an A. aequalis population, AHFT-4, that survived mesosulfuron-methyl at the field-recommended rate (9 g ai ha^–1) was collected in Anhui Province. Single-dose testing confirmed that the suspected resistant AHFT-4 had evolved resistance to mesosulfuron-methyl. Target gene sequencing revealed a resistance mutation of Pro-197-Ala in ALS1 of the resistant plants, and a derived cleaved amplified polymorphic sequence marker was developed to specifically detect the mutation. A relative expression assay showed no significant difference in ALS expression between AHFT-4 and a susceptible population without or with mesosulfuron-methyl treatment. Whole-plant dose–response bioassays indicated that AHFT-4 had evolved broad-spectrum cross-resistance to ALS-inhibiting herbicides of all five chemical families tested, with GR₅₀ resistance index (RI) values ranging from 21 to 206. However, it remained susceptible to the photosystem II inhibitor isoproturon. Pretreatment with the cytochrome P450 inhibitor malathion or the glutathione S-transferase inhibitor 4-chloro-7-nitrobenzoxa-diazole had no significant effects on the resistance of AHFT-4 to mesosulfuron-methyl. To our knowledge, this study reports for the first time the ALS gene Pro-197-Ala substitution conferring broad-spectrum cross-resistance to ALS-inhibiting herbicides in A. aequalis.

In the transition zone, turfgrass managers generally utilize the dormancy period of warm-season turfgrass to apply herbicides for managing winter annual weeds. Although this weed control strategy is common in bermudagrass [Cynodon dactylon (L.) Pers.], it has been less adopted in zoysiagrass (Zoysia spp.) due to variable turfgrass injury during post-dormancy transition. Previous research reported that air temperature could affect weed control and crop safety from herbicides. Growth-chamber studies were conducted to evaluate zoysiagrass response to glyphosate and glufosinate as influenced by three different temperature regimes during and after treatment. A field research study was conducted at four site-years to assess the influence of variable heat-unit accumulation on zoysiagrass response to seven herbicides. In the growth-chamber study, glufosinate injured zoysiagrass more than glyphosate and reduced time to reach 50% green cover reduction, regardless of the rate, when incubated for 7 d under different temperature levels. When green zoysiagrass sprigs were incubated for 7 d at 10 C, the rate of green cover reduction was slowed for both herbicides; however, green cover was rapidly reduced under 27 C. After treated zoysiagrass plugs having 5% green cover were incubated at 10 C for 14 d, glyphosate-treated plugs reached 50% green cover in 22 d, similar to nontreated plugs but less than the 70 d required for glufosinate-treated plugs. Zoysiagrass response to glyphosate was temperature dependent, but glufosinate injured zoysiagrass unacceptably regardless of temperature regime. Diquat, flumioxazin, glufosinate, and metsulfuron + rimsulfuron injured zoysiagrass at 200 or 300 growing-degree days at base 5 C (GDD_5C) application timings, but foramsulfuron and oxadiazon did not injure zoysiagrass regardless of GDD_5C. The relationship of leaf density to green turf cover is dependent on zoysiagrass mowing height, and both metrics are reduced by injurious herbicides. Research indicates that glufosinate injures zoysiagrass more than glyphosate, and the speed and magnitude of herbicide injury generally increase with temperature.

Cover crops (CCs) have shown great potential for suppressing annual weeds within agronomic cropping systems across the United States. However, the weed suppressive potential of CCs may be moderated by environmental and management factors that are specific to certain geographic areas and their associated characteristics. This may be particularly true within the U.S. Southeast, where higher mean annual temperature and precipitation generate favorable conditions for both CC and weed growth. To understand the effects of this regional context on CCs and weeds, a meta-analysis examining paired comparisons of weed biomass and/or weed density under CC and bare ground conditions from studies conducted within the Southeast was conducted. Data were identified and extracted from 28 journal articles in which weed biomass and/or weed density were measured along with cash crop yield data, if they were provided. Fourteen studies provided 142 comparisons for weed biomass; 23 studies provided 139 comparisons for weed density; and 22 studies, pooled over both weed response variables, provided 144 comparisons for cash crop yield. CCs had a negative effect on weed density (P = 0.0016) but no effect on either weed biomass (P = 0.16) or cash crop yield (P = 0.88). The mean relative reduction in weed density under CCs was 44%. Subsequent analyses indicated that CC biomass was the key factor associated with this reduction. Weed density suppression was linearly related to CC biomass; a 50% decrease in weed density was associated with 6,600 kg ha^–1 of CC biomass. Edaphic, geographic, and other management factors had no bearing on this suppressive effect. This highlights the importance of generating adequate CC biomass if weed suppression is the primary objective of CC use and the potential for CCs to reduce weed density over diverse soil, climate, and farm management conditions.

Field studies were conducted in 2021 in Kibler and Augusta, AR, to determine the effect of winter cover crops and cultivar selection on weed suppression and sweetpotato [Ipomoea batatas (L.) Lam.] yield. The split-split-plot studies evaluated three cover crops [cereal rye (Secale cereale L.) + crimson clover (Trifolium incarnatum L.)], [winter wheat (Triticum aestivum L.) + crimson clover], and fallow; weeding (with or without); and four sweetpotato cultivars (‘Heartogold’, ‘Bayou-Belle-6’, ‘Beauregard-14’, and ‘Orleans’). Heartogold had the tallest canopy, while Beauregard-14 and Bayou Belle-6 had the longest vines at 5 and 8 wk after sweetpotato transplanting. Sweetpotato canopy was about 20% taller in weedy plots compared with the hand-weeded treatment, and vines were shorter under weed interference. Canopy height and vine length of sweetpotato cultivars were not related to weed biomass suppression. However, vine length was positively correlated to all yield grades (r > 0.5). Weed biomass decreased 1-fold in plots with cover crops compared with bare soil at Augusta. Cover crop biomass was positively correlated with jumbo (r = 0.29), no. 1 (r = 0.33), and total sweetpotato yield (r = 0.34). Jumbo yield was affected the most by weed pressure. On average, sweetpotato total yield was reduced by 80% and 60% with weed interference in Augusta and Kibler, respectively. Bayou Belle-6 was the high-yielding cultivar without weed interference in both locations. Bayou Belle-6 and Heartogold were less affected by weed interference than Beauregard-14 and Orleans.

Damage to non–dicamba resistant (non-DR) soybean [Glycine max (L.) Merr.] has been frequent in geographies where dicamba-resistant (DR) soybean and cotton (Gossypium hirsutum L.) have been grown and sprayed with the herbicide in recent years. Off-target movement field trials were conducted in northwest Arkansas to determine the relationship between dicamba concentration in the air and the extent of symptomology on non-DR soybean. Additionally, the frequency and concentration of dicamba in air samples at two locations in eastern Arkansas and environmental conditions that impacted the detection of the herbicide in air samples were evaluated. Treatment applications included dicamba at 560 g ae ha–1 (1X rate), glyphosate at 860 g ae ha^–1, and particle drift retardant at 1% v/v applied to 0.37-ha fields with varying degrees of vegetation. The relationship between dicamba concentration in air samples and non-DR soybean response to the herbicide was more predictive with visible injury (generalized R² = 0.82) than height reduction (generalized R² = 0.43). The predicted dicamba air concentration resulting in 10% injury to soybean was 1.60 ng m^–3 d–1 for a single exposure. The predicted concentration from a single exposure to dicamba resulting in a 10% height reduction was 3.78 ng m^–3 d^–1. Dicamba was frequently detected in eastern Arkansas, and daily detections above 1.60 ng m^–3 occurred 17 times in the period sampled. The maximum concentration of dicamba recorded was 7.96 ng m^–3 d^–1, while dicamba concentrations at Marianna and Keiser, AR, were ≥1 ng m^–3 d^–1 in six samples collected in 2020 and 22 samples in 2021. Dicamba was detected consistently in air samples collected, indicating high usage in the region and the potential for soybean damage over an extended period. More research is needed to quantify the plant absorption rate of volatile dicamba and to evaluate the impact of multiple exposures of gaseous dicamba on non-targeted plant species.