PPO-inhibitor Resistance Research Articles

Resistance to protoporphyrinogen oxidase inhibitors in giant ragweed (Ambrosia trifida).

Giant ragweed (Ambrosia trifida L.) is one of the most troublesome weed species in corn (Zea mays L.) and soybean [Glycine max (L.) Merr.] cropping systems. Following numerous reports in 2018 of suspected herbicide resistance in several Ambrosia trifida populations from Wisconsin, our objective was to characterize the response of these accessions to acetolactate synthase (ALS), enolpyruvyl shikimate phosphate synthase (EPSPS), and protoporphyrinogen oxidase (PPO) inhibitors applied POST. Four accessions (AT1, AT4, AT6, and AT10) exhibited ≥ 50% plant survival after exposure to the cloransulam 3× rate. Two accessions (AT8 and AT10) and one accession (AT2) exhibited ≥ 50% plant survival after exposure to glyphosate and fomesafen 1× rates, respectively. The AT10 accession exhibited multiple resistance to cloransulam and glyphosate. The AT12 accession was 28.8-fold resistant to fomesafen and 3.7-fold resistant to lactofen. A codon change in PPX2 conferring a R98L substitution was identified as the most likely mechanism conferring PPO-inhibitor resistance. To our knowledge, this is the first confirmed case of PPO-inhibitor resistance in Ambrosia trifida globally and we identified the genetic mutation likely conferring resistance. Proactive and diversified integrated weed management strategies are of paramount importance for sustainable long-term Ambrosia trifida management. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A New V361A Mutation in Amaranthus palmeri PPX2 Associated with PPO-Inhibiting Herbicide Resistance.

Weeds resistant to PPO-inhibiting herbicides threaten the profitability of crop producers relying on this chemistry. In Amaranthus palmeri, mutations at G210 (∆G210) and R128 (R128G/M) of the PPX2 gene were reported to confer PPO-inhibitor resistance. Here, A. palmeri samples from nine states in America, having survived a field application of a PPO-inhibitor, were genotyped to determine the prevalence of these mutations. Less than 5% of the 1828 A. palmeri plants screened contained the ∆G210 mutation. Of the plants lacking ∆G210, a R128 substitution was only found in a single plant. An A. palmeri population from Alabama without mutations at G210 or R128 had a resistance ratio of 3.1 to 3.5 for fomesafen. Of the candidate PPX2 mutations identified in this population, only V361A conferred resistance to lactofen and fomesafen in a transformed bacterial strain. This is the first report of the V361A substitution of PPX2 conferred PPO-inhibiting herbicide resistance in any plant species. Future molecular screens of PPO-inhibitor resistance in A. palmeri and other species should encompass the V361A mutation of PPX2 to avoid false-negative results.

Discovery, mode of action, resistance mechanisms, and plan of action for sustainable use of Group 14 herbicides

AbstractProtoporphyrinogen 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.

Distribution and validation of genotypic and phenotypic glyphosate and PPO-inhibitor resistance in Palmer amaranth (Amaranthus palmeri) from southwestern Nebraska – ERRATUM

Failure to control Palmer amaranth with glyphosate and protoporphyrinogen IX oxidase (PPO)-inhibitor herbicides was reported across southwestern Nebraska in 2017. The objectives of this study were to 1) confirm and 2) validate glyphosate and PPO-inhibitor (fomesafen and lactofen) resistance in 51 Palmer amaranth accessions from southwestern Nebraska using genotypic and whole-plant phenotypic assay correlations and cluster analysis, and 3) determine which agronomic practices might be influencing glyphosate resistance in Palmer amaranth accessions in that location. Based on genotypic assay, 88% of 51 accessions contained at least one individual with amplification (>2 copies) of the 5-enolypyruvyl-shikimate-3-phosphate synthase (EPSPS) gene, which confers glyphosate resistance; and/or a mutation in the PPX2 gene, either ΔG210 or R128G, which endows PPO-inhibitor resistance in Palmer amaranth. Cluster analysis and high correlation (0.83) between genotypic and phenotypic assays demonstrated that EPSPS gene amplification is the main glyphosate resistance mechanism in Palmer amaranth accessions from southwestern Nebraska. In contrast, there was poor association between genotypic and phenotypic responses for PPO-inhibitor resistance, which was attributed to segregation for PPO-inhibitor resistance within these accessions and/or the methodology that was adopted herein. Genotypic assays can expedite the process of confirming known glyphosate and PPO-inhibitor resistance mechanisms in Palmer amaranth from southwestern Nebraska and other locations. Phenotypic assays are also a robust method for confirming glyphosate resistance but not necessarily PPO-inhibitor resistance in Palmer amaranth. Moreover, random forest analysis of glyphosate resistance in Palmer amaranth indicated that EPSPS gene amplification, county, and current and previous crops are the main factors influencing glyphosate resistance within that geographic area. Most glyphosate-susceptible Palmer amaranth accessions were found in a few counties in areas with high crop diversity. Results presented here confirm the spread of glyphosate resistance and PPO-inhibitor resistance in Palmer amaranth accessions from southwestern Nebraska and demonstrate that less diverse cropping systems are an important driver of herbicide resistance evolution in Palmer amaranth.Nomenclature: fomesafen; glyphosate; lactofen; Palmer amaranth, Amaranthus palmeri S. Watson

Distribution and validation of genotypic and phenotypic glyphosate and PPO-inhibitor resistance in Palmer amaranth (Amaranthus palmeri) from southwestern Nebraska

AbstractFailure to control Palmer amaranth with glyphosate and protoporphyrinogen IX oxidase (PPO)-inhibitor herbicides was reported across southwestern Nebraska in 2017. The objectives of this study were to 1) confirm and 2) validate glyphosate and PPO-inhibitor (fomesafen and lactofen) resistance in 51 Palmer amaranth accessions from southwestern Nebraska using genotypic and whole-plant phenotypic assay correlations and cluster analysis, and 3) determine which agronomic practices might be influencing glyphosate resistance in Palmer amaranth accessions in that location. Based on genotypic assay, 88% of 51 accessions contained at least one individual with amplification (>2 copies) of the 5-enolypyruvyl-shikimate-3-phosphate synthase (EPSPS) gene, which confers glyphosate resistance; and/or a mutation in the PPX2 gene, either ΔG210 or R128G, which endows PPO-inhibitor resistance in Palmer amaranth. Cluster analysis and high correlation (0.83) between genotypic and phenotypic assays demonstrated that EPSPS gene amplification is the main glyphosate resistance mechanism in Palmer amaranth accessions from southwestern Nebraska. In contrast, there was poor association between genotypic and phenotypic responses for PPO-inhibitor resistance, which was attributed to segregation for PPO-inhibitor resistance within these accessions and/or the methodology that was adopted herein. Genotypic assays can expedite the process of confirming known glyphosate and PPO-inhibitor resistance mechanisms in Palmer amaranth from southwestern Nebraska and other locations. Phenotypic assays are also a robust method for confirming glyphosate resistance but not necessarily PPO-inhibitor resistance in Palmer amaranth. Moreover, random forest analysis of glyphosate resistance in Palmer amaranth indicated that EPSPS gene amplification, county, and current and previous crops are the main factors influencing glyphosate resistance within that geographic area. Most glyphosate-susceptible Palmer amaranth accessions were found in a few counties in areas with high crop diversity. Results presented here confirm the spread of glyphosate resistance and PPO-inhibitor resistance in Palmer amaranth accessions from southwestern Nebraska and demonstrate that less diverse cropping systems are an important driver of herbicide resistance evolution in Palmer amaranth.

Arg-128-Leu target-site mutation in PPO2 evolves in wild poinsettia (Euphorbia heterophylla) with cross-resistance to PPO-inhibiting herbicides

AbstractWild poinsettia (Euphorbia heterophylla L.) is a troublesome broadleaf weed in grain production areas in South America. Herbicide resistance to multiple sites of action has been documented in this species, including protoporphyrinogen oxidase (PPO) inhibitors. We investigated the physiological and molecular bases for PPO-inhibitor resistance in a E. heterophylla population (RPPO) from Southern Brazil. Whole-plant dose–response experiments revealed a cross-resistance profile to three different chemical groups of PPO inhibitors. Based on dose–response parameters, RPPO was resistant to lactofen (47.7-fold), saflufenacil (8.6-fold), and pyraflufen-ethyl (3.5-fold). Twenty-four hours after lactofen treatment (120 g ha−1) POST, RPPO accumulated 27 times less protoporphyrin than the susceptible population (SPPO). In addition, RPPO generated 5 and 4.5 times less hydrogen peroxide and superoxide than SPPO, respectively. The chloroplast PPO (PPO1) sequences were identical between the two populations, whereas 35 single-nucleotide polymorphisms were found for the mitochondrial PPO (PPO2). Based on protein homology modeling, the Arg-128-Leu (homologous to Arg-98-Leu in common ragweed [Ambrosia artemisiifolia L.] was the only one located near the catalytic site, also in a conserved region of PPO2. The cytochrome P450 monooxygenase inhibitor malathion did not reverse resistance to lactofen in RPPO, and both populations showed similar levels of PPO1 and PPO2 expression, suggesting that metabolic resistance and PPO overexpression are unlikely. This is the first report of an Arg-128-Leu mutation in PPO2 conferring cross-resistance to PPO inhibitors in E. heterophylla.

Target Site-Based Resistance to ALS Inhibitors, Glyphosate, and PPO Inhibitors in an <i>Amaranthus palmeri</i> Accession from Mississippi

Extensive acceptance of glyphosate-resistant (GR) row crops coupled with the simultaneous increase in glyphosate usage has sped the evolution of glyphosate resistance in economically important weeds. GR Amaranthus palmeri populations are widespread across the state with some exhibiting multiple resistance to acetolactate synthase (ALS) inhibiting herbicides such as pyrithiobac. A GR and ALS inhibitor-resistant accession was also resistant to the protoporphyrinogen oxidase (PPO) inhibiting herbicide fomesafen. The PPO inhibitor resistance profile and multiple herbicide resistance mechanisms in this accession were investigated. In addition to fomesafen, resistance to postemergence applications of acifluorfen, lactofen, carfentrazone, and sulfentrazone was confirmed. There was no resistance to preemergence application of fomesafen, flumioxazin, or oxyfluorfen. Molecular analysis of the ALS gene indicated the presence of point mutations leading to single nucleotide substitutions at codons 197, 377, 574, and 653, resulting in proline-to-serine, arginine-to-glutamine, tryptophan-to-leucine, and serine-to-asparagine replacements, respectively. The resistant accession contained up to 87-fold more copies of the EPSPS gene compared to a susceptible accession. A mutation leading to a deletion of glycine at codon 210 (ΔG210) of PPO2 gene was also detected. These results indicate that the mechanism of resistance in the Palmer amaranth accession is target-site based, i.e., altered target site for ALS and PPO inhibitor resistance and gene amplification for glyphosate resistance.

Comparing responses of sensitive and resistant populations of Palmer amaranth (Amaranthus palmeri) and waterhemp (Amaranthus tuberculatus var. rudis) to PPO inhibitors

AbstractResistance to protoporphyrinogen oxidase (PPO) inhibitors was first observed in waterhemp in 2001 and was conferred by the deletion of a glycine residue at the 210th position (ΔGly-210) of the PPO enzyme. PPO-inhibitor resistance in Palmer amaranth was first observed in 2011, 10 years later. The objectives of this study were to directly compare PPO inhibitor responses in plants of both species with or without the ΔGly-210 mutation. Using greenhouse experiments, early (EPOST) and late (LPOST) postemergence dose responses using lactofen and fomesafen, and preemergence (PRE) dose responses using fomesafen and flumioxazin, were obtained for a sensitive and resistant population each of waterhemp and Palmer amaranth. An additional spray study confirmed each sensitive population used in the dose responses was representative of its respective species, with regards to PPO-inhibitor sensitivity. When treated at either POST timing, Palmer amaranth was more tolerant than waterhemp, and the ΔGly-210 mutation provided greater resistance in Palmer amaranth (48-fold to >3,440-fold, depending on timing and herbicide) than in waterhemp (31-fold to 123-fold). The level of tolerance in Palmer amaranth was striking; the sensitive Palmer amaranth population treated LPOST survived as well or better than the resistant waterhemp population treated EPOST. With PRE applications, response differences both between species and between resistant and sensitive populations generally were less pronounced, relative to POST applications. Collectively, this research indicates Palmer amaranth tolerance to POST-applied PPO inhibitors could have initially slowed (relative to waterhemp) evolution of resistance to these herbicides, and resistant and sensitive populations of both species are more likely to be effectively controlled with PRE rather than POST applications.

Protoporphyrinogen oxidase (PPO) inhibitor–resistant waterhemp (Amaranthus tuberculatus) from Nebraska is multiple herbicide resistant: confirmation, mechanism of resistance, and management

AbstractA waterhemp [Amaranthus tuberculatus(Moq.) J. D. Sauer] biotype (designated as “NER”) collected from a soybean [Glycine max(L.) Merr.] production field in eastern Nebraska survived the POST application of fomesafen at the labeled rate. The objectives of this study were to (1) quantify the level of resistance to protoporphyrinogen oxidase (PPO) inhibitors (acifluorfen, fomesafen, and lactofen) applied POST, (2) determine the mechanism of PPO-inhibitor resistance in the NER biotype, (3) determine whether NER possessed multiple resistance to acetolactate synthase (ALS)-, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-, and photosystem II (PSII)-inhibiting herbicides, and (4) control NER with POST soybean herbicides. A whole-plant dose–response bioassay revealed that the NER biotype was 4- to 6-fold resistant to PPO-inhibiting herbicides depending on the known susceptible biotype (S1 or S2) used for comparison. A Kompetitive Allele Specific PCR (KASP™) assay was developed and performed for rapid and robust detection of the ΔG210 mutation (deletion of a codon) in thePPX2Lgene. All samples of the NER biotype tested positive for the ΔG210 mutation. Dose–response bioassays confirmed that the NER biotype was resistant to three additional herbicide sites of action. Chlorimuron and imazethapyr, both ALS inhibitors, applied at 32X the labeled rate resulted in <80% reduction in the aboveground biomass of the NER biotype. The same biotype was 3- and 7-fold resistant to glyphosate (EPSPS inhibitor) and atrazine (PSII inhibitor), respectively. Glufosinate, 2,4-D choline plus glyphosate, and dicamba were the only soybean POST herbicides that controlled NER effectively (≥92% aboveground biomass reduction).Amaranthus tuberculatusis the first confirmed weed species in Nebraska to evolve resistance to four distinct herbicide sites of action, leaving no POST herbicide choice for effective control in glyphosate-resistant and conventional (non-transgenic) soybean.

Carfentrazone-ethyl resistance in an Amaranthus tuberculatus population is not mediated by amino acid alterations in the PPO2 protein.

To date, the only known mechanism conferring protoporphyrinogen IX oxidase (PPO)-inhibitor resistance in waterhemp (Amaranthus tuberculatus) is a glycine deletion in PPO2 (ΔG210), which results in cross-resistance to foliar PPO-inhibiting herbicides. However, a metabolism-based, HPPD-inhibitor resistant waterhemp population from Illinois (named SIR) was suspected of having a non-target site resistance (NTSR) mechanism due to its resistance to carfentrazone-ethyl (CE) but sensitivity to diphenylethers (DPEs). In greenhouse experiments, SIR sustained less injury than two PPO inhibitor-sensitive populations (WCS and SEN) after applying a field-use rate of CE, and after initial rapid necrosis, regrowth of SIR plants was comparable to a known PPO inhibitor-resistant population (ACR) possessing the ΔG210 mutation. Dose-response analysis determined 50% growth reduction rates in CE-resistant (SIR and ACR) and sensitive (SEN) waterhemp populations, which showed SIR was 30-fold resistant compared to SEN and two-fold more resistant than ACR. Deduced amino acid sequences derived from SIR PPX2 partial cDNAs did not contain the ΔG210 mutation found in ACR or other target-site mutations that confer PPO-inhibitor resistance previously reported in Palmer amaranth (Amaranthus palmeri). Although several SIR cDNAs contained amino acid substitutions, none were uniform among samples. Additionally, SIR plants treated with malathion and CE showed a significant reduction in biomass accumulation compared to CE alone. These results indicate robust CE resistance in SIR is not mediated by amino acid changes in the PPO2 protein, but instead resistance may be conferred through a NTSR mechanism such as enhanced herbicide metabolism.

Distribution of PPX2 Mutations Conferring PPO-Inhibitor Resistance in Palmer Amaranth Populations of Tennessee

AbstractProtoporphyrinogen IX oxidase (PPO)–inhibiting herbicides (WSSA Group 14) have been used in agronomic row crops for over 50 yr. Broadleaf weeds, including glyphosate-resistant Palmer amaranth, have been controlled by this herbicide site of action PRE and POST. Recently, Palmer amaranth populations were reported resistant to PPO inhibitors in 2011 in Arkansas, in 2015 in Tennessee, and in 2016 in Illinois. Historically, the mechanism for this resistance involves the deletion of a glycine at position 210 (ΔG210) in a PPO enzyme encoded by the PPX2 gene; however, the ΔG210 deletion did not explain all PPO inhibitor–resistant Palmer amaranth in Tennessee populations. Recently, two new mutations within PPX2 (R128G, R128M) that confer resistance to PPO inhibitors were identified in Palmer amaranth. Therefore, research is needed to document the presence and distribution of the three known mutations that confer PPO inhibitor resistance in Tennessee. In 2017, a survey was conducted in 18 fields with Palmer amaranth to determine whether resistance existed and the prevalence of each known mutation in each field. Fomesafen was applied at 265 g ai ha–1 to Palmer amaranth infestations within each field to select for resistant weeds for later analysis. Where resistance was described (70% of surviving plants), the ΔG210 mutation was detected in 47% of resistant plants. The R128G mutation accounted for 42% of resistance, similar to the frequency of the ΔG210 mutation. The R128M mutation was less frequent than the other two mutations, accounting for only 10% of the resistance. All mutations detected in this study were heterozygous. Additionally, no more than one of the three PPX2 mutations were detected in an individual surviving plant. Similar to previous research, about 70% of PPO resistance was accounted for by these three known mutations, leaving about 30% of resistance not characterized in Tennessee populations. Survivors not showing the three known PPO mutations suggest that other resistance mechanisms are present.

Competitiveness of Herbicide-Resistant Waterhemp (Amaranthus tuberculatus) with Soybean

AbstractWaterhemp [Amaranthus tuberculatus(Moq.) J. D. Sauer] is a troublesome weed occurring in cropping systems throughout the U.S. Midwest with an ability to rapidly evolve herbicide resistance that could be associated with competitive disadvantages. Little research has investigated the competitiveness of differentA. tuberculatuspopulations under similar environmental conditions. The objectives of this study were to evaluate: (1) the interspecific competitiveness of three herbicide-resistantA. tuberculatuspopulations (2,4-D and atrazine resistant [2A-R], glyphosate and protoporphyrinogen oxidase [PPO]-inhibitor resistant [GP-R], and 2,4-D, atrazine, glyphosate, and PPO-inhibitor susceptible [2AGP-S]) with soybean [Glycine max(L.) Merr.]; and (2) the density-dependent response of eachA. tuberculatuspopulation within a constant soybean population in a greenhouse environment.Amaranthus tuberculatuscompetitiveness with soybean was evaluated across five target weed densities of 0, 2, 4, 8, and 16 plants pot−1(equivalent to 0, 20, 40, 80, and 160 plants m−2) with 3 soybean plants pot−1(equivalent to 300,000 plants ha−1). At the R1 soybean harvest time, no difference in soybean biomass was observed acrossA. tuberculatuspopulations. AtA. tuberculatusdensities <8 plants pot−1, the 2AGP-S population had the greatest biomass and stem diameter per plant. At the R7 harvest time, the 2AGP-S population caused the greatest loss in soybean biomass and number of pods compared with the other populations at densities of <16 plants pot−1. The 2AGP-S population had greater early-season biomass accumulation and stem diameter compared with the otherA. tuberculatuspopulations, which resulted in greater late-season reduction in soybean biomass and number of pods. This research indicates there may be evidence of interspecific competitive fitness cost associated with the evolution of 2,4-D, atrazine, glyphosate, and PPO-inhibitor resistance inA. tuberculatus. Focus should be placed on effectively using cultural weed management practices to enhance crop competitiveness, especially early in the season, to increase suppression of herbicide-resistantA. tuberculatus.

Annual Bluegrass (Poa annua) Biotypes Exhibit Differential Levels of Susceptibility and Biochemical Responses to Protoporphyrinogen Oxidase Inhibitors

AbstractAn annual bluegrass (Poa annuaL.) biotype with limited susceptibility to POST flumioxazin applications was identified in Georgia. The objectives of this research were to quantify tolerance levels of this biotype (R-biotype) to protoporphyrinogen oxidase (PPO) inhibitors and characterize physiological responses to flumioxazin. In dose–response experiments on 3- to 5-tiller plants, flumioxazin and sulfentrazone rates required to reduce dry-shoot biomass 50% from the nontreated were >14.5 and 10.4 times greater for the R-biotype, as compared with a susceptible (S)-biotype, respectively. Establishment of the R-biotype from seed was completely controlled by PRE applications of flumioxazin and oxadiazon, similar to the S-biotype. Tank mixtures of chlorpyrifos with flumioxazin did not enhance biomass reductions of the R-biotype, suggesting that tolerance levels may not be related to cytochrome P450–associated metabolism. In laboratory experiments, the R-biotype averaged 27% less electrolyte leakage, as compared with the S-biotype, after flumioxazin treatments. Lipid peroxidation in the R-biotype, as measured by malondialdehyde levels, averaged 25% less than the S-biotype at 72 h after broadcast flumioxazin treatments at 280 and 560 g ha−1. The tolerance to POST applications of PPO inhibitors in thisP. annuabiotype is associated with less lipid peroxidation and electrolyte leakage as compared with the S-biotype. These biochemical differences in biotypes may contribute to erratic levels of POST control from flumioxazin and could contribute to PPO-inhibitor resistance.

A Statewide Survey of PPO-Inhibitor Resistance and the Prevalent Target-Site Mechanisms in Palmer amaranth (Amaranthus palmeri) Accessions from Arkansas

Palmer amaranth is one of the most problematic weeds in the midsouthern United States, and the evolution of resistance to protoporphyrinogen oxidase (PPO) inhibitors in biotypes already resistant to glyphosate and acetolactate synthase (ALS) inhibitors is a major cause of concern to soybean and cotton growers in these states. A late-season weed-escape survey was conducted in the major row crop–producing counties (29 counties) to determine the severity of PPO-inhibitor resistance in Arkansas. A total of 227 Palmer amaranth accessions were sprayed with fomesafen at 395 g ha−1to identify putative resistant plants. A TaqMan qPCR assay was used to confirm the presence of the ΔG210 codon deletion or the R128G/M (homologous to R98 mutation in common ragweed) target-site resistance mechanisms in thePPX2gene. Out of the 227 accessions screened, 44 were completely controlled with fomesafen, and 16 had only one or two severely injured plants (≥98% mortality) when compared with the 1986 susceptible check (100% mortality). The remaining 167 accessions were genotypically screened, and 82 (49%) accessions were found to harbor the ΔG210 deletion in thePPX2gene. The R128G was observed in 47 (28%) out of the 167 accessions screened. The mutation R128M, on the other hand was rare, found in only three accessions. About 13% of the accessions were segregating for both the ΔG210 and R128G mutations. Sixteen percent of the tested accessions had mortality ratings <90% and did not test positive for the ΔG210 or the R128G/M resistance mechanisms, indicating that a novel target or non–target site resistance mechanism is likely. Overall, PPO inhibitor–resistant Palmer amaranth is widespread in Arkansas, and the ΔG210 resistance mechanism is especially dominant in the northeast corridor, while the R128G mutation is more prevalent in counties near Memphis, TN.

Two new PPX2 mutations associated with resistance to PPO-inhibiting herbicides in Amaranthus palmeri.

Resistance to herbicides that inhibit protoporphyrinogen oxidase (PPO) is a widespread and growing problem for weed managers across the midwestern and midsouthern United States. In Amaranthus spp., this resistance is known to be conferred by a glycine deletion at the 210th amino acid (ΔG210) in PPO2. Preliminary analysis indicated that the ΔG210 mutation did not fully account for observed resistance to PPO inhibitors in two Amaranthus palmeri populations from Tennessee and one from Arkansas. Sequencing PPX2 cDNA from six resistant plants uncovered two new mutations at the R98 site (R98G and R98M), a site previously found to endow PPO-inhibitor resistance in Ambrosia artemisiifolia. Sequencing of this region from additional plants sprayed with 264 g fomesafen ha-1 showed the presence of one or both R98 mutations in a subset of the resistant plants from all three populations. No plants sensitive to fomesafen contained either mutation. A derived cleaved amplified polymorphic sequence (dCAPS) assay to test for the presence of these mutations in A. palmeri was developed. Two new mutations of PPX2 (R98G, R98M) likely confer resistance to PPO-inhibitors in A. palmeri, and can be rapidly identified using a dCAPS assay. © 2017 Society of Chemical Industry.

Distribution of the ΔG210 Protoporphyrinogen Oxidase Mutation in Illinois Waterhemp (Amaranthus tuberculatus) and an Improved Molecular Method for Detection

Molecular assays are often implemented by weed scientists for detection of herbicide-resistant individuals; however, the utility of these assays can be limited if multiple mechanisms of evolved resistance exist. Waterhemp resistant to protoporphyrinogen oxidase (PPO)– inhibiting herbicides is conferred by a target-site mutation in PPX2L (a gene coding for PPO), resulting in the loss of a glycine at position 210 (ΔG210). This ΔG210 mutation of PPX2L is the only known mechanism responsible for PPO-inhibitor resistance (PPO-R) in waterhemp from five states (Illinois, Indiana, Iowa, Kansas, and Missouri); however, a limited number of populations have been tested, especially in Illinois. To verify the ubiquity of the ΔG210 in PPO-R waterhemp populations in Illinois, a previously published allele-specific PCR (asPCR) was used for the detection of the ΔG210 mutation to associate this mutation with phenotypic resistance in 94 Illinois waterhemp populations. The ΔG210 mutation was detected in all populations displaying phenotypic resistance to lactofen (220 g ai ha−1), indicating the deletion is likely the only mechanism of resistance. With evidence that the ΔG210 mutation dominates PPO-R waterhemp biotypes, molecular detection techniques have considerable utility. Unfortunately, the previously published asPCR is time consuming, very sensitive to PCR conditions, and requires additional steps to eliminate the possibility of false negatives. To overcome these limitations, a streamlined molecular method using the TaqMan® technique was developed, utilizing allele-specific, fluorescent probes for high-throughput, robust discrimination of each allele (resistant and susceptible) at the 210th amino acid position of PPX2L.

Distribution of Herbicide Resistances and Molecular Mechanisms Conferring Resistance in Missouri Waterhemp (Amaranthus rudisSauer) Populations

A survey of soybean fields containing waterhemp was conducted just prior to harvest in 2012 to determine the scope and extent of herbicide resistance and multiple herbicide resistances among a sample of Missouri waterhemp populations. Resistance was confirmed to glyphosate and to acetolactate synthase (ALS), protoporphyrinogen oxidase (PPO), photosystem II (PSII), and 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, but not to 2,4-D. Of the 187 populations tested, 186 exhibited resistance to chlorimuron. The proportions of populations with atrazine or glyphosate resistance were similar, with 30 and 29% of the populations surviving the 3× rates. Lactofen resistance was observed in 5% of the populations, whereas mesotrione resistance was only found in 1.6% of the populations. All populations tested were susceptible to 2,4-D at the 3× rate. At least 52% of the waterhemp populations tested exhibited resistance to herbicides from two mechanism of action. Resistance to atrazine plus chlorimuron as well as glyphosate plus chlorimuron was present in 29% of the populations. Three-way resistance, primarily comprised of resistance to atrazine plus chlorimuron plus glyphosate, was present in 11% of the populations. Resistance to herbicides from four mechanisms of action was found in 2% of the populations, and one population exhibited resistance to herbicides from five mechanisms of action. DNA analysis of a subsample of plants revealed that previously documented mechanisms of resistance in waterhemp, including the ΔG210 deletion conferring PPO-inhibitor resistance, the Trp574Leu amino acid substitution conferring ALS-inhibitor resistance, and elevated 5-enolypyruvyl-shikimate-3-phosphate synthase copy number and the Pro106Ser amino acid substitution resulting in glyphosate resistance, explained survival in many, but not all, instances. Atrazine resistance was not explained by the Ser264Gly D1 protein substitution. Overall, results from these experiments indicate that Missouri soybean fields contain waterhemp populations with resistance to glyphosate, ALS-, PPO-, PSII-, and HPPD-inhibiting herbicides, which are some of the most common mechanisms of action currently utilized for the control of this species in corn and soybean production systems. Additionally, these results indicate that slightly more than half of the populations tested exhibit resistance to more than one herbicide mechanisms of action. Managing the current resistance levels in existing populations is of utmost importance. The use of multiple, effective herbicide modes of action, both preemergence and postemergence, and the integration of optimum cultural and mechanical control practices will be vital in the management of Missouri waterhemp populations in the future.

Will theAmaranthus tuberculatusResistance Mechanism to PPO-Inhibiting Herbicides Evolve in OtherAmaranthusSpecies?

Resistance to herbicides that inhibit protoporphyrinogen oxidase (PPO) has been slow to evolve and, to date, is confirmed for only four weed species. Two of these species are members of the genusAmaranthusL. Previous research has demonstrated that PPO-inhibitor resistance inA. tuberculatus(Moq.) Sauer, the first weed to have evolved this type of resistance, involves a unique codon deletion in thePPX2gene. Our hypothesis is thatA. tuberculatusmay have been predisposed to evolving this resistance mechanism due to the presence of a repetitive motif at the mutation site and that lack of this motif in other amaranth species is why PPO-inhibitor resistance has not become more common despite strong herbicide selection pressure. Here we investigate inter- and intraspecific variability of thePPX2gene—specifically exon 9, which includes the mutation site—in ten amaranth species via sequencing and a PCR-RFLP assay. Few polymorphisms were observed in this region of the gene, and intraspecific variation was observed only inA. quitensis. However, sequencing revealed two distinct repeat patterns encompassing the mutation site. Most notably,A. palmeriS. Watson possesses the same repetitive motif found inA. tuberculatus. We thus predict thatA. palmeriwill evolve resistance to PPO inhibitors via the samePPX2codon deletion that evolved inA. tuberculatus.