Target Site-Based Resistance to ALS Inhibitors, Glyphosate, and PPO Inhibitors in an Amaranthus palmeri 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, argi-nine-to-glutamine, tryptophan-to-leucine, and serine-to-asparagine replace-ments, 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.


Introduction
The collective attributes of glyphosate herbicide, from its systemic action to its nonselective, wide range of postemergence activity, has contributed to its broad appeal throughout the world in both crop and noncrop lands since its commercialization in 1974. With the introduction of glyphosate-resistant (GR) crops in the mid-1990s, glyphosate was used selectively and predominantly for weed control in GR crops without concern for crop injury. The widespread adoption of GR crops around the world has led to overuse of the herbicide and reduced crop rotation, which resulted in the evolution of several GR weed biotypes. As of May 2020, GR populations have been reported for 48 weed species worldwide [1], including Amaranthus palmeri (S.) Wat. (Palmer amaranth).
Before the commercialization of GR crops, acetolactate synthase (ALS) inhibiting herbicides were used extensively for weed management in crop and noncrop areas. A major downside to the widespread use of ALS inhibitors has been the rapid and extensive evolution of resistance in several grasses and broadleaf weed populations across the world. For example, within 5 years of introduction of chlorsulfuron, the first ALS inhibitor to be commercialized, Lactuca serriola L. and Kochia scoparia (L.) Shrad populations became resistant [2] [3] [4]. As of May 2020, 165 weed species have been documented to be resistant to one or more ALS inhibitors [1]. Among these resistant weed species are several Amaranthus spp. including A. palmeri.
Resistance to multiple herbicides, such as glyphosate and ALS inhibitors, has been documented in A. palmeri [5]

Plant Growth and Herbicide Treatment Evaluations
Experiments involving herbicide responses on A. palmeri seedlings were per- PA-R plants were generated from a parent a male plant (A. palmeri is dioecious with male and female reproductive organs developing on different plants) using a cloning procedure described before [8]. Briefly, an axillary branch, approximately 3 cm long, was cut from the stem and lateral leaves removed leaving 4 leaves per stalk. The cut end was lightly coated with Rootone rooting hormone (TechPac, Lexington, KY) and placed in moist growth media as above. The plantlets were kept in indirect sunlight for 3 wk, then transplanted into larger pots, and watered and fertilized as described before.
For PRE studies, the soil used in studies on herbicide effects on A. palmeri was a Bosket sandy loam (Bosket sandy loam, fine-loamy, mixed, thermic Mollic Hapludalfs Twenty-five seeds of PA-S were planted at a depth of 0.5 cm and covered with additional soil. Ten PA-R cloned plantlets were transplanted into each pot immediately after herbicide application. Pots were watered instantly after herbicide application to activate the herbicide and as needed thereafter. Emerged PA-S and transplanted PA-R seedlings that remained herbicide injury-free were counted 4 wk after treatment (WAT).
All herbicide treatments were applied using an air-

Molecular Analysis
To check for known target-site resistance mutations in the PPX2 gene, genomic DNA was extracted from eight PPO-inhibitor-resistant and two PPO-inhibitorsensitive A. palmeri samples using a modified CTAB protocol [9]. A Taq-Man-based quantitative PCR approach was used to detect the presence of any ΔG210, R128G, and R128M mutations in PPO2, following previously described protocols [10] [11]. These same ten samples were also checked for known mutations in the EPSPS and ALS genes. EPSPS gene amplification and EPSPS P106S mutation were detected via quantitative PCR and a derived cleaved amplified polymorphic sequences (dCAPS) assay, respectively, following previously described protocols [12]. The ALS gene was amplified using primers specific to the

Statistical Analysis
Data from dose-response and cross-resistance studies were subjected to analysis of variance using the general linear model procedure in SAS 9.4 (SAS Institute, Cary, NC). Data from the two experiments were combined because there were no significant interactions between experiments. Nonlinear regression was used to define a three-parametric power equation y = y 0 + ax b to relating the herbicide dose effects (x) on shoot dry weight (y), where y 0 is an asymptote, a is a constant, and b is the slope of the curve. Equation parameters were calculated with SigmaPlot 12.5 (Systat Software Inc., San Jose, CA). ED 50 (effective dose to achieve 50% control) and GR 50 (dose required to reduce shoot dry weight by 50%) estimates were derived from curves fit in SigmaPlot at 50% control or reduction in shoot dry weight. In the cross-resistance experiment, means were separated using Fisher's protected LSD at P = 0.05.

Fomesafen Dose Response
Response of PA-R and PA-S plants to fomesafen dose is represented in Figure 1.
The ED 50 values of PA-R and PA-S for fomesafen were 3.30 and 0.06 kg·ha −1 indicating that the PA-R accession is 55-fold more resistant to the herbicide compared to PA-S. This level is higher than the 6-to 21-fold resistance reported in certain A. palmeri populations from Arkansas populations [9].

Glyphosate Dose Response
Response of PA-R and PA-S plants to glyphosate dose is represented in Figure 2.  This was most likely due to the reduction in shoot dry weight being 0%, 0%, and 33% of the nontreated control with pyrithiobac, imazaquin, and trifloxysulfuron, respectively, even at the highest rates applied. Since the GR 50 value estimates for the PA-R accession would seemingly lie outside the dose range, it would be more accurate to report R/S ratios as greater than a certain value based on the highest tested dose. This approach is frequently followed when resistance to a herbicide has already been widely reported and resistance is confirmed in a study followed by additional research and analysis. Therefore, the reported R/S ratios were based on extrapolated data and constrained to fit within the accuracy of their estimation. A similar procedure was used in an earlier report [13], where R/S values for ALS-inhibitor resistanr A. palmeri and A. spinosus L. (spiny amaranth) were calculated based on inferred data. Thus, the R/S values of the PA-R accession were >72, >933, and >78 for pyrithiobac, imazaquin, and trifloxysulfuron, respectively.

Cross Resistance to PPO Inhibitors
Both PA-R and PA-S accessions were controlled 100% by flumioxazin, fomesafen and oxyfluorfen, all applied PRE, thereby indicating lack of any cross-resistance in the PA-R accession (Table 1). A PRE dose-response study that includes doses less and more than the respective herbicide doses used for flumioxazin, fomesafen, and oxyfluorfen would provide a better understanding of resistance to herbicides applied PRE. Several PPO inhibiting herbicides applied POST were ineffective in controlling the PA-R accession, except saflufenacil (Table 1). Acifluorfen, lactofen, carfentrazone, and sulfentrazone provided 63%, 6%, 52%, and 18% control, respectively, of PA-R plants, while saflufenacil provided 95% control. PA-S plants were completely controlled by all herbicides evaluated. The above results indicate the PA-R accession is cross-resistant to selected herbicides applied POST, but not to some when treated PRE.    R2  GCA  CCC  GCT  GAT  CGA  TGG  AGC  GGC   R3  GCA  CCC  GCT  GAT  CGA/CGT  TGG  AGC/AAC  GGC   R6  GCA  CCC  GCT  GAT  CGA  TGG  AGC  GGC   R7  GCA  CCC  GCT  GAT  CGA  TGG/TTG  AGC  GGC   R8  GCA  CCC  GCT  GAT  CGA  TGG  AGC  GGC   R9  GCA  CCC  GCT  GAT  CGA  TGG  AGC/AAC  GGC   R10  GCA  CCC/TCC  GCT  GAT  CGA/CAA  TGG  AGC  GGC   S1  GCA  CCC  GCT  GAT  CGA  TGG  AGC  GGC   S2  GCA  CCC  GCT  GAT  CGA  TGG  AGC  GGC/

Conclusion
Growers in Mississippi and across the United States must implement short-and long-term integrated herbicide resistance management practices comprising chemical, mechanical, and cultural strategies to combat multiple herbicide resistant A. palmeri populations such as the one reported here. Short-term control methods may include targeted spraying of resistant plants with drones equipped with precision sprayers. Long-term practices could include implementation of cover crops, crop rotation, modified row spacing, and remote sensing-hyperspectral imaging technologies.