During the 2017 to 2019 growing seasons, samples of waterhemp and Palmer amaranth that had reportedly survived field-rate applications of protoporphyrinogen oxidase (PPO)–inhibiting herbicides were collected from the American Midwest and tested for target-site mutations known at the time to confer resistance. Target-site resistance was identified in nearly all (135 of 145) tested common waterhemp populations but in only 8 of 13 Palmer amaranth populations. Follow-up research on one population of Palmer amaranth (W-8), which tested negative for all such mutations, confirmed it was resistant to lactofen, with a magnitude of resistance comparable to that conferred by the ΔG210 PPO2 mutation. Gene sequences from both isoforms of PPO (PPO1 and PPO2) were compared between W-8 and known PPO inhibitor–sensitive sequence. A glycine-to-alanine substitution at the 399th amino acid position (G399A) of PPO2, recently identified to reduce target-site herbicide sensitivity, was observed in a subset of resistant W-8 plants. Because no missense mutation completely delimited resistant and sensitive sequences, we initially suspected the presence of a secondary, non-target-site resistance mechanism in this population. To isolate G399A, a segregating F2 population was produced and screened with a delimiting rate of lactofen. χ2 goodness-of-fit analysis of dead/alive ratings indicated single-locus inheritance of resistance in the F2 population, and molecular markers for the W-8 parental PPO2 coding region co-segregated tightly, but not perfectly, with resistance. More research is needed to fully characterize Palmer amaranth PPO inhibitor–resistance mechanisms, which appear to be more diverse than those found in common waterhemp.

A survey questionnaire was sent to cotton consultants of Arkansas and Mississippi through direct mail and Louisiana and Tennessee consultants through on-farm visits in fall of 2011. The survey was returned by a total of 22 Arkansas, 17 Louisiana, 10 Mississippi, and 11 Tennessee cotton consultants, representing 26, 53, 13, and 38% of total cotton planted in these states in 2011, respectively. Collectively, the area planted to glyphosate-resistant (Roundup Ready®, RR) cotton was 97%, glyphosate plus glufosinate-resistant (Widestrike® Flex, WRF) cotton was 30%, and glufosinate-resistant (Liberty Link, LL) cotton was 2.6% of the total cotton surveyed in 2011. Seventy percent of area in all states is still under continuous RR/WRF cotton. Average cost of herbicides in RR systems was $114 ha−1 and in LL systems was $137 ha−1. Across the states, cotton planted under no-tillage, conservation tillage, and conventional tillage was 31, 36, and 33%, respectively, of total scouted cotton. Area under conventional tillage increased and conservation tillage decreased in Arkansas compared with a previous survey conducted in 2006. Palmer amaranth, morningglories, and horseweed in the order of listing were the most problematic weeds of cotton across Arkansas, Mississippi, and Tennessee. In Louisiana, however, morningglories were the most problematic weed followed by Palmer amaranth and common waterhemp. Glyphosate-resistant (GR) Palmer amaranth infested only 13% of scouted cotton area in Louisiana compared with 75% in the remaining three states, and consequently, hand-weeding to control GR Palmer amaranth is practiced on only 2.5% of total scouted area of Louisiana and 49% of the scouted area of the remaining three states. Hand-weeding added an additional $12 to 371 ha−1 to weed-management costs. One-half (50%) of the cotton consultants emphasized the need for more research on residual herbicides that can control GR Palmer amaranth effectively.

A waterhemp population (MCR) previously characterized as resistant to 4-hydroxyphenylpyruvate dioxygenase and photosystem II inhibitors demonstrated both moderate and high levels of resistance to acetolactate synthase (ALS) inhibitors. Plants from the MCR population exhibiting high resistance to ALS inhibitors contained the commonly found Trp574Leu ALS amino acid substitution, whereas plants with only moderate resistance did not have this substitution. A subpopulation (JG11) was derived from the MCR population in which the moderate-resistance trait was isolated from the Trp574Leu mutation. Results from DNA sequencing and ALS enzyme assays demonstrated that resistance to ALS inhibitors in the JG11 population was not due to an altered site of action. This nontarget-site ALS-inhibitor resistance was characterized with whole-plant dose–response experiments using herbicides from each of the five commercialized families of ALS-inhibiting herbicides. Resistance ratios ranging from 3 to 90 were obtained from the seven herbicides evaluated. Nontarget-site resistance to ALS has been rarely documented in eudicot weeds, and adds to the growing list of resistance traits evolved in waterhemp.

A survey of soybean fields containing waterhemp infestations was conducted just prior to harvest in 2008 and 2009 to determine the frequency and distribution of glyphosate-resistant waterhemp in Missouri, and to determine if there are any in-field parameters that may serve as indicators of glyphosate resistance in this species in future crop production systems. Glyphosate resistance was confirmed in 99 out of 144, or 69%, of the total waterhemp populations sampled, which occurred in 41 counties of Missouri. Populations of glyphosate-resistant waterhemp were more likely to occur in fields with no other weed species present at the end of the season, continuous cropping of soybean, exclusive use of glyphosate for several consecutive seasons, and waterhemp plants showing obvious signs of surviving herbicide treatment compared to fields characterized with glyphosate-susceptible waterhemp. Therefore, it is suggested that these four site parameters, and certain combinations of these parameters, serve as predictors of glyphosate resistance in future waterhemp populations.

The effect of application technology on efficacy has been thoroughly investigated for sprays of chemical pesticide but not biological pesticides. This study investigates the effect of applying conidia of Microsphaeropsis amaranthi, a candidate bioherbicide for Amaranthus spp., with a range of different hydraulic nozzle tips. The nozzle tips were selected to deliver sprays with different spectra of droplet sizes deployed at different angles. We found that sprays of large droplets gave poor coverage of the target and resulted in low levels of disease severity on common waterhemp. The most effective nozzle tip tested was a hollow cone nozzle tip, such as is commonly used for the application of fungicides. This nozzle tip deployed large numbers of fine droplets that swirled within the plant canopy, provided good coverage of all plant parts, and resulted in the highest levels of disease severity, particularly on stems.

Common waterhemp is one of the most commonly encountered and troublesome weeds in the midwestern United States. It is well known that water stress adversely affects crop growth and yield; however, the effects of water stress on weed growth and seed production are poorly understood. The objective of this study was to determine the effects of degree and duration of water stress on growth, development, and fecundity of two common waterhemp biotypes in greenhouse experiments conducted at the University of Nebraska–Lincoln. No difference was observed in growth, development, and seed production between two biotypes in response to degree and duration of water stress; therefore, data were combined. The degree of water stress study included five treatments, where the amount of water applied to each pot at 2-d interval was equivalent to 100, 75, 50, 25, and 12.5% of pot (soil) water content. The highest plant height (163 cm), number of leaves (231 plant−1), and growth index (4.4 × 105 cm3) were recorded at 100% of pot water content (no water stress). Similarly, aboveground biomass, total leaf area, and seed production reached their maximum at 100% of pot water content treatment, whereas they were reduced as degree of water stress increased. The study of water stress duration included five treatments, where amount of water applied to each pot at 2-, 4-, 6-, 8-, and 10-d intervals was equivalent to 100% of pot water content. The highest plant height (150 cm), number of leaves (210 plant−1), and growth index (3.8 × 105 cm3) were observed at 2-d interval of water stress, whereas seed production was similar at 2-d (36,549 seeds plant−1) and 4-d (34,176 seeds plant−1) intervals. This study shows that common waterhemp has capacity to survive and reproduce even under a higher degree and duration of water stress.

Waterhemp resistance to foliar applications of protoporphyrinogen oxidase (PPO)–inhibiting herbicides has become increasingly disconcerting given the widespread distribution of glyphosate resistance. Fortunately, soil-residual PPO-inhibiting herbicides remain efficacious in waterhemp populations resistant to PPO-inhibiting herbicides; however, these herbicides should theoretically select for the resistant biotype as herbicide concentrations diminish in the soil. Accordingly, the objectives of this research were twofold: (1) evaluate the efficacy of three PPO-inhibiting herbicides, foliar- and soil-applied, on PPO-resistant (PPO-R) and PPO-susceptible (PPO-S) waterhemp, and (2) investigate the differential effects of PPO-inhibiting herbicides on an R biotype and an S biotype during several discrete developmental events relevant to soil–residual herbicide activity (i.e., radicle protrusion, radicle elongation, and waterhemp emergence). Greenhouse and growth chamber experiments indicated that the R biotype was least sensitive to the diphenylether herbicide fomesafen, followed by sulfentrazone and flumioxazin; however, fomesafen plus s-metolachlor improved soil-residual efficacy over fomesafen alone. Growth stage considerably influenced the R : S ratio, decreasing from 38× to 3.4×, when comparing ratios generated from foliar applications and soil-residual applications measuring radicle protrusion, respectively. Overall, this research supports the use of full soil-residual herbicide rates, reinforcing the importance of best management practices to manage the spread of herbicide resistance.

The extensive use of foliar-applied protoporphyrinogen oxidase (PPO)-inhibiting herbicides indisputably contributes to the continued selection of waterhemp resistant to PPO-inhibiting herbicides (PPO-R). However, the role of soil-residual applications of PPO-inhibiting herbicides in the selection of PPO-R has been clouded by the efficacy that these herbicides have on PPO-R waterhemp. The aim of the present study was to understand if soil-residual PPO-inhibiting herbicides have the potential to influence the proportion of resistant waterhemp in emerging plants as herbicide concentrations diminish in the soil. Greenhouse and field experiments were conducted in a PRE dose-response experiment testing fomesafen or fomesafen plus s-metolachlor in the presence of mixed seed populations of PPO-R and -susceptible (S) waterhemp. The first 20 (greenhouse) or 10 (field) waterhemp plants that survived the residual herbicide treatment were sampled for genotypic analysis to detect the presence of the allele responsible for PPO-R in waterhemp (ΔG210). Relative to the nontreated control, the highest rate of fomesafen increased the frequency of resistance (FOR) by 70% in the greenhouse experiments and 20% in the field experiments. The addition of s-metolachlor did not reduce the fomesafen-induced increase in the FOR in the surviving plants from field or greenhouse experiments. However, the additional herbicide from an alternate site of action (s-metolachlor) substantially improved soil-residual herbicide efficacy over fomesafen alone, which limited the number of waterhemp plants surviving the herbicide application. Thus, the selection for resistance can be delayed with the addition of a herbicide from an alternative site of action by postponing and/or reducing waterhemp emergence. These data strongly suggest that soil-residual PPO-inhibiting herbicides can influence the FOR in a field population, placing even greater importance on the implementation of best management practices such as full herbicide use rates and using herbicides from multiple sites of action to mitigate the risk of selecting for herbicide-resistant weed biotypes.

Studies were conducted to determine acifluorfen and lactofen absorption, translocation, and metabolism in protox-inhibiting herbicide-susceptible and -resistant common waterhemp. Acifluorfen and lactofen absorption was similar in both biotypes. Herbicide absorption was 12% in both susceptible and resistant common waterhemp 6 h after treatment (HAT). Absorption increased to 32 and 42% in susceptible and resistant plants, respectively, at 72 HAT. Translocation was similar in both biotypes for both herbicides. Herbicide translocation out of the treated leaf ranged between 5 and 15%. In a separate study, resistant common waterhemp plants were treated with acifluorfen or lactofen, alone or with tridiphane. Acifluorfen or lactofen injury to resistant common waterhemp was not altered with the addition of tridiphane. Treatments of 14C-acifluorfen or -lactofen on susceptible and resistant common waterhemp resulted in similar lactofen metabolism in both biotypes, but acifluorfen was not metabolized in either biotype within 24 HAT. This data indicate that differences in herbicide absorption, translocation, or metabolism are not the mechanism of common waterhemp resistance to protox-inhibiting herbicides.

Field studies were conducted to determine the effect of emergence timing on the fitness of the next generation as represented by seed mass, maturation, and afterripening of common waterhemp cohorts. Five natural cohorts were documented both in 2009 and 2010. Different maternal environments resulting from varied cohort emergence timings did not influence seed maturation time and seed mass, but had an inconsistent effect on seed afterripening. Here are our major findings. (1) Waterhemp cohorts needed similar amounts of time to generate viable seeds (20 to 27 d after flower initiation) and the seeds produced were of similar size (2.0 to 2.35 g), and (2) waterhemp has strong primary dormancy that may be released within 4 mo during the afterripening process, depending on the dormancy level. Seeds produced by later cohorts were more sensitive to the afterripening period, suggesting more flexibility in life strategy. Seeds from the 2009 cohorts had similar afterripening patterns; newly harvested seeds had strong primary dormancy (<10% germination), which was gradually released during dry storage and reached the maximum germination (>80%) rate 4 mo after harvest (MAH). However, germination then dropped to 40% 6 and 8 MAH, suggesting the induction of secondary seed dormancy. Strong primary dormancy at harvest for 2010 seeds was sustained in dry afterripening, perhaps because of higher dormancy level, which was the result of less-favorable parental environments brought by 10 to 30 times higher population densities and 2.5 to 5 times higher accumulative precipitation than in 2009 (see Wu and Owen 2014). We also tested the soil seed-bank seed population densities for each waterhemp cohort and found that early cohorts greatly influenced the seed population densities at the soil surface level and the turnover rate of the soil seed bank. Results from this research will provide insights into better management of waterhemp, targeting a better understanding of the seed bank.

Absorption and translocation of three commercial formulations of glyphosate, the isopropylamine salt formulated as Roundup Ultra™ (IPA1) and Roundup UltraMax™ (IPA2) and the diammonium salt formulated as Touchdown™ IQ (DA), were compared in three- to five-leaf velvetleaf, common waterhemp, and pitted morningglory. Absorption of 14C-glyphosate in velvetleaf was not significantly different among the three formulations up to 50 h after treatment (HAT). More absorption of 14C-glyphosate occurred in the IPA1 (26.0%) vs. the IPA2 (17.7%) formulation over 74 h. Of the total 14C-glyphosate absorbed, 20 to 35% was translocated from the treated leaf to the rest of the plant. Initial absorption of 14C-glyphosate was rapid in common waterhemp with the IPA1 (42.7%) and IPA2 (30.7%) formulations; both were higher compared with absorption of the DA formulation (11.5%) by 2 HAT. These differences continued up to 26 HAT, but no differences were evident by 74 HAT. Up to 65% of the 14C-glyphosate absorbed was translocated out of the treated leaf by 74 HAT, with roots the primary sink. Initial absorption of 14C-glyphosate was slow in pitted morningglory compared with the other species. More foliar absorption occurred in plants treated with the DA (13.6%) vs. the IPA2 formulation (4.9%) by 6 HAT. Absorption beyond 26 HAT was not different among the three glyphosate formulations. Translocation of 14C-glyphosate to roots was 27% greater as the DA salt than IPA1 and IPA2 by 74 HAT. The distribution pattern of glyphosate was similar in all species; phosphorimages demonstrated movement both acropetal and basipetal, with accumulation in roots greater than in any other plant parts. An efficacy study parallel to the 14C study showed no difference among the three glyphosate formulations on the species investigated at both 74 HAT and 2 wk after treatment.

The fungal plant pathogen Microsphaeropsis amaranthi is virulent against a number of key weeds in the Amaranthaceae, including common waterhemp, and is under investigation as a bioherbicide. Common waterhemp has become a key weed in midwestern crop production systems and is a good target for a bioherbicide that could be integrated into weed management systems. We investigated the direct effects of a range of chemical herbicides and adjuvants upon conidia of M. amaranthi and found that many herbicides and most adjuvants were strongly inhibitory to germination. On the other hand, M. amaranthi was compatible with a selection of postemergence herbicides commonly used in midwestern weed management systems, including carfentrazone, chloransulam, and imazethapyr. Most glyphosate products suppressed or abolished germination of M. amaranthi conidia, but by testing adjuvants commonly used in glyphosate products and technical-grade glyphosate salts, it was revealed that this inhibition was due to formulation additives and not the active ingredient. When glyphosate and conidia of M. amaranthi were sprayed onto common waterhemp seedlings, the herbicide predisposed plants to infection by M. amaranthi. When M. amaranthi was applied 1 to 3 d after glyphosate, the glyphosate rate required to control common waterhemp was reduced by half. Similar results were observed on clones propagated from a common waterhemp plant resistant to glyphosate. When M. amaranthi was applied to seedlings 2 d before glyphosate, the efficacy of the herbicide was reduced. These findings demonstrate that positive interactions between herbicides and M. amaranthi exist but reveal practical difficulties that may limit the integration of the strategy in the field.

It is generally expected that, in the case of multiple herbicide resistance, different resistance mechanisms within a weed will follow Mendel’s law of independent assortment. Research was conducted to investigate anecdotal observations suggesting that target site–based resistances to inhibitors of acetolactate synthase (ALS) and protoporphyrinogen oxidase (PPO) did not follow independent assortment in common waterhemp. Cosegregation of the two resistances was observed in backcross lines (population sensitive to both herbicides as recurrent parent). Specifically, whereas 52% of backcross plants were resistant to a PPO inhibitor, this percentage increased to 92% when the backcross plants were preselected for resistance to an ALS inhibitor. Molecular marker analysis confirmed that the corresponding genes (ALS and PPX2) were genetically linked. When data from all plants analyzed were pooled, the genetic distance between the two genes was calculated to be 7.5 cM. The two genes were found to be about 195 kb apart in the recently published grain amaranth genome, explaining the observed genetic linkage. There is likely enough recombination that occurs between the linked genes to prevent the linkage from having significant implications in terms of resistance evolution. Nevertheless, documentation of the happenstance linkage between target-site genes for resistance to ALS and PPO inhibitors in waterhemp is a reminder that one should not assume distinct resistance mechanism will independently assort.