Introduction
Palmer amaranth is one of the most troublesome summer annual weeds in most agronomic and non-crop production systems across southern, midwestern, and U.S. Great Plains regions (Aulakh et al. Reference Aulakh, Price, Enloe, van Santen, Wehtje and Patterson2012, Reference Aulakh, Price, Enloe, Wehtje and Patterson2013, Reference Aulakh, Chahal, Kumar, Price and Guillard2021; Bensch et al. Reference Bensch, Horak and Peterson2003; Chahal et al. Reference Chahal, Varanasi, Jugulum and Jhala2017; Crow et al. Reference Crow, Steckel, Mueller and Hayes2016; Grichar Reference Grichar1997: Meyers et al. Reference Meyers, Jennings, Schultheis and Monks2010; Mohseni-Moghadam et al. Reference Mohseni-Moghadam, Schroeder, Heerema and Ashigh2013b; Norsworthy et al. Reference Norsworthy, Oliveira, Jha, Malik, Buckelew, Jennings and Monks2008b; Price et al. Reference Price, Wayne Reeves and Patterson2006, Reference Price, Balkcom, Culpepper, Kelton, Nichols and Schomberg2011; Smith et al. Reference Smith, Baker and Steele2000). An extended emergence period, C4 photosynthetic pathway, high water-use efficiency, dioecious nature (separate male and female plants) of sexual reproduction, prolific seed production (100,000 to 1,000,000 seeds per plant), and a tendency to evolve herbicide resistance are the salient traits for rapid invasion and spread of Palmer amaranth into new regions (Burke et al. Reference Burke, Schroeder, Thomas and Wilcut2007; Ehleringer Reference Ehleringer1983; Horak and Loughin Reference Horak and Loughin2000; Keeley et al. Reference Keeley, Carter and Thullen1987; Ward et al. Reference Ward, Webster and Steckel2013).
Glyphosate was commercialized in 1974 and was a highly efficacious postemergence (POST) herbicide for controlling Palmer amaranth (Corbett et al. Reference Corbett, Askew, Thomas and Wilcut2004; Culpepper and York Reference Culpepper and York1998; Parker et al. Reference Parker, York and Jordan2005). Glyphosate targets the 5-enolypyruvyl-shikimate-3-phosphate synthase (EPSPS) enzyme in the shikimic acid pathway of plants and microorganisms (della-Cioppa et al. Reference Bauer, Klein, Shah, Fraley and Kishore1986). The disruption of this pathway prevents the production of essential aromatic amino acids, including phenylalanine, tryptophan, tyrosine, and other important secondary metabolites that eventually lead to plant death (Duke and Powles Reference Dukes and Powles2008). Commercialization of glyphosate-resistant (GR) crops in the mid-1990s and its rapid adoption resulted in almost exclusive reliance on glyphosate for broad-spectrum weed control (Norsworthy et al. Reference Norsworthy, Smith, Scott and Gbur2007). Due to the high effectiveness and relatively low cost of glyphosate-based weed control in GR crops, glyphosate eventually replaced the use of pre-plant incorporated (PPI), preemergence, selective POST, and post-directed (PD) herbicides and greatly increased the selection of GR weed biotypes (Young Reference Young2006). Within two decades of commercialization of GR crops, several weed species, including Palmer amaranth, were reported with resistance to glyphosate. First, a GR Palmer amaranth biotype was discovered in Macon County, GA, in 2004 (Culpepper et al. Reference Culpepper, Grey, Vencill, Kichler, Webster, Brown, York, Davis and Hanna2006). Currently, GR Palmer amaranth biotypes have been confirmed in 30 U.S. states (Heap Reference Heap2024). Some GR Palmer amaranth biotypes required 115 times higher glyphosate rate than susceptible biotypes to achieve 50% control (Norsworthy et al. Reference Norsworthy, Griffith, Scott, Smith and Oliver2008a; Steckel et al. Reference Steckel, Main, Ellis and Mueller2008). Currently, resistance to 10 different herbicide sites of action (SOAs) has been identified in Palmer amaranth biotypes across the United States (Heap Reference Heap2024), including inhibitors of acetolactate synthase (ALS; categorized as a Group 2 herbicide by the Weed Science Society of America [WSSA]), microtubule assembly (WSSA Group 3), photosystem II (PS II; WSSA Groups 5 and 6), EPSPS (WSSA Group 9), glutamine synthetase (WSSA Group 10), protoporphyrinogen oxidase (WSSA Group 14), very long-chain fatty acid elongase (WSSA Group 15), 4-hydroxyphenylpyruvate dioxygenase (WSSA Group 27), and synthetic auxins (WSSA Group 4) (Carvalho-Moore et al. Reference Carvalho-Moore, Norsworthy, González-Torralva, Hwang, Patel, Barber, Butts and McElroy2022; Chahal et al. Reference Chahal, Varanasi, Jugulum and Jhala2017; Culpepper et al. Reference Culpepper, Grey, Vencill, Kichler, Webster, Brown, York, Davis and Hanna2006; Foster and Steckel Reference Foster and Steckel2022; Gossett et al. Reference Gossett, Murdock and Toler1992; Heap Reference Heap2024; Jhala et al. Reference Jhala, Sandell, Rana, Kruger and Knezevic2014; Kouame et al. Reference Kouame, Bertucci, Savin, Bararpour, Steckel, Butts, Willett, Machado and Roma-Burgos2022; Kumar et al. Reference Kumar, Liu, Boyer and Stahlman2019, Reference Kumar, Liu and Stahlman2020; Nakka et al. Reference Nakka, Thompson, Peterson and Jugulam2017; Priess et al. Reference Priess, Norsworthy, Godara, Mauromoustakos, Butts, Roberts and Barber2022; Salas et al. Reference Salas, Burgos, Tranel, Singh, Glasgow, Scott and Nichols2016; Sprague et al. Reference Sprague, Stoller, Wax and Horak1997). Furthermore, Palmer amaranth biotypes resistant to multiple herbicide SOAs are present in several corn (Zea mays L.), cotton (Gossypium hirsutum L.), soybean (Glycine max L. Merr.), and vegetable production systems in the United States (Aulakh et al. Reference Aulakh, Chahal, Kumar, Price and Guillard2021; Heap Reference Heap2024; Kouame et al. Reference Kouame, Bertucci, Savin, Bararpour, Steckel, Butts, Willett, Machado and Roma-Burgos2022; Kumar et al. Reference Kumar, Liu, Boyer and Stahlman2019, Reference Kumar, Liu and Stahlman2020).
Weed species have evolved multiple mechanisms conferring glyphosate resistance (Chatham et al. Reference Chatham, Bradley, Kruger, Martin, Owen, Peterson, Mithila and Tranel2015a; Dinelli et al. Reference Dinelli, Marotti, Bonetti, Catizone, Urbano and Barnes2008; Perez-Jones et al. Reference Perez-Jones, Park, Polge, Colquhoun and Mallory-Smith2007; Shaner et al. Reference Shaner, Lindenmeyer and Ostlie2011; Simarmata and Penner Reference Simarmata and Penner2008; Wiersma et al. Reference Wiersma, Gaines, Preston, Hamilton, Giacomini, Buell, Leach and Westra2015). Most commonly reported glyphosate resistance mechanisms include target site mutation in the EPSPS gene (Baerson et al. Reference Baerson, Rodriguez, Tran, Feng, Viest and Dill2002; Kaundun et al. Reference Kaundun, Dale, Zelaya, Dinelli, Marotti, McIndoe and Cairns2011; Perez-Jones et al. Reference Perez-Jones, Park, Polge, Colquhoun and Mallory-Smith2007; Wakelin and Preston Reference Wakelin and Preston2006; Yu et al. Reference Yu, Cairns and Powles2007), reduced absorption and translocation (Dinelli et al. Reference Dinelli, Marotti, Bonetti, Catizone, Urbano and Barnes2008; Lorraine-Colwill et al. Reference Lorraine-Colwill, Powles, Hawkes, Hollinshead, Warner and Preston2003; Wakelin et al. Reference Wakelin, Lorraine-Colwill and Preston2004; Yu et al. Reference Yu, Cairns and Powles2007), enhanced sequestration (Ge et al. Reference Ge, d’Avignon, Ackerman and Sammons2010), and EPSPS gene amplification (Chahal et al. Reference Chahal, Varanasi, Jugulum and Jhala2017; Chatham et al. Reference Chatham, Wu, Riggins, Hager, Young, Roskamp and Tranel2015b; Gaines et al. Reference Gaines, Zhang, Wang, Bukun, Chisholm, Shaner, Nissen, Patzoldt, Tranel, Culpepper, Grey, Webster, Vencill, Sammons, Jiang, Preston, Leach and Westra2010; Kumar et al. Reference Kumar, Jha, Giacomini, Westra and Westra2015). A GR Palmer amaranth biotype with >100 EPSPS gene copies has been reported from Georgia (Gaines et al. Reference Gaines, Zhang, Wang, Bukun, Chisholm, Shaner, Nissen, Patzoldt, Tranel, Culpepper, Grey, Webster, Vencill, Sammons, Jiang, Preston, Leach and Westra2010). Furthermore, increased EPSPS gene copies have also been reported in GR Palmer amaranth biotypes from Mississippi (Ribeiro et al. Reference Ribeiro, Pan, Duke, Nandula, Baldwin, Shaw and Dayan2014), Nebraska (Chahal et al. Reference Chahal, Varanasi, Jugulum and Jhala2017), and New Mexico (Mohseni-Moghadam et al. Reference Mohseni-Moghadam, Schroeder and Ashigh2013a).
GR Palmer amaranth has recently been reported in Connecticut (Aulakh et al. Reference Aulakh, Chahal, Kumar, Price and Guillard2021). However, the mechanism of glyphosate resistance has not been characterized in that biotype. Thus, the main objectives of this research were 1) to determine the glyphosate resistance levels in GR Palmer amaranth biotype from Connecticut when treated at three different plant heights, and 2) to determine whether one or more target site–based mechanisms confers glyphosate resistance in the Connecticut biotype.
Materials and Methods
Plant Material
A confirmed GR Palmer amaranth biotype (CT-Res) from Hartford County, CT (41.93°N, 72.53°W), was investigated. In 2019, the GR plants that survived 6,720 g ae ha−1 of glyphosate (MADDOG®; Loveland Products, Inc., Loveland, CO) in the previously reported whole-plant dose-response bioassay (Aulakh et al. Reference Aulakh, Chahal, Kumar, Price and Guillard2021) were allowed to open-pollinate to develop an “OP1” population. Seeds from female plants were harvested, cleaned thoroughly using a vertical air column blower, and stored in airtight polyethylene bags at 4 C until further testing. In 2022, seedlings from the “OP1” population were treated again with glyphosate (6,720 g ae ha−1), and the survivors were allowed to open pollinate to produce the “OP2” seeds. Seeds from “OP2” female plants were harvested, cleaned, and stored in airtight polyethylene bags at 4 C until further testing. A known glyphosate-susceptible biotype (KS-Sus) from the Kansas State University Agricultural Research Center near Hays, KS (38°50N, 99°18W), was used in the whole-plant dose response bioassays. Previous dose-response experiments confirmed that KS-Sus was highly susceptible to glyphosate with an ED90 value of 424 g ae ha−1 (Aulakh et al. Reference Aulakh, Chahal, Kumar, Price and Guillard2021). Another known glyphosate susceptible biotype (AL-Sus) acquired from the E.V. Smith Research Center near Shorter, AL (32°26N, 85°56W), and the campus of Auburn University was used to determine the underlying target site–based mechanisms of glyphosate resistance.
Effect of Plant Height on Glyphosate Resistance Levels
Whole-plant dose-response bioassays were conducted in the summer of 2023 in a greenhouse at the Connecticut Agricultural Experiment Station, Windsor, CT, to determine the response of CT-Res (“OP2”) Palmer amaranth biotype to glyphosate at three different plant heights (10, 20, and 30 cm). Seeds of both CT-Res (“OP2”) and KS-Sus biotypes were planted in square plastic pots (10 × 10 × 12 cm) containing planting media (Pro-Mix Premium All Purpose®; Quakertown, PA). Pro-Mix Premium All Purpose contains Canadian sphagnum peat moss (80% to 90%), peat humus, perlite, limestone, and mycorrhizae PTB297 technology. Palmer amaranth plants were thinned to one plant per pot at 7 d after emergence. The experiment was arranged in a randomized complete block (blocked by biotype) design with a 9 × 2 × 3 factorial arrangement of treatments. The three factors were 1) nine glyphosate rates: 0×, 0.125×, 0.25×, 0.5×, 1×, 2×, 4×, 8×, and 16×, where 1× is the field-use rate of glyphosate (840 g ae ha−1); 2) two Palmer amaranth biotypes: CT-Res and KS-Sus; and 3) three plant heights: 10, 20, and 30 cm. Each factorial treatment combination was replicated six times (one plant per pot), and the experiment was repeated twice. The greenhouse was maintained at 30/26 C day/night temperatures with a 16-h photoperiod supplemented by overhead sodium halide lamps with light intensity of 450 µ mol s−1. Plants were watered with an overhead sprinkler system as needed to avoid the moisture stress and maintain good growth. Palmer amaranth seedlings were treated with glyphosate (MADDOG®; Loveland Products, Inc., Loveland, CO), and each glyphosate treatment was prepared in distilled water mixed with a nonionic surfactant (Induce; Helena Chemical Co., Collierville, TN) at 0.25% vol/vol. Glyphosate treatments were applied with a compressed CO2 backpack sprayer through a single TeeJet AI8002VS flat-fan spray nozzle (Spraying Systems Co., Wheaton, IL) calibrated to deliver 187 L ha−1 spray volume at 207 kPa and 3.5 km h−1. Plants were harvested at 21 d after treatment (DAT), and shoot fresh weight was determined. The fresh weights were then converted into percent biomass reduction compared with the nontreated control (Wortman Reference Wortman2014) as shown in Equation 1:
$${\rm{Biomass}}\;{\rm{reduction}}\;\left( \% \right) = {{\left( {\overline C - \left. B \right)} \right.} \over {\overline C}} \times 100$$
where
$\overline C$
is the mean fresh weight biomass of the nontreated control and B is the biomass of an individual treated plant.
Statistical Analysis
Due to nonsignificant interaction (P = 0.324) of treatment-by-run, data on fresh shoot biomass reduction (%) of both CT-Res and KS-Sus Palmer amaranth biotypes were averaged across two runs. A three-parameter log-logistic model (Eq. 2) was fitted on biomass reduction using the drc package in R software (R Foundation for Statistical Computing, Vienna, Austria) (Knezevic et al. Reference Knezevic, Streibig and Ritz2007):
$$Y\; = {\rm{ }}{d \over {1 + \exp \;[b(logx - {\rm{ }}loge)]}}$$
where Y is the percent fresh shoot biomass reduction, x is the herbicide rate, d is the upper limit, e is the GR50 value (amount of glyphosate needed for 50% reduction in fresh shoot biomass), and b represents the relative slope around the parameter “e”. The level of resistance was calculated by dividing the GR90 value (amount of glyphosate needed for 90% reduction in fresh shoot biomass) of the resistant biotype (CT-Res) by that of the susceptible biotype (KS-Sus) for the corresponding plant height.
Mechanism(s) of Glyphosate Resistance
Genomic DNA Isolation
The AL-Sus plants were grown using the same planting medium and greenhouse conditions previously mentioned in the whole-plant dose-response bioassays. Fresh leaf tissue was collected from the nontreated AL-Sus plants (two plants) and the CT-Res plants (six plants) that survived 6,720 g ae ha−1 of glyphosate in the 2023 dose-response bioassay. The harvested leaf tissue (100 mg) was immediately flash-frozen in liquid nitrogen (−195.79 C) and stored at −80 C for genomic DNA (gDNA) isolation and extraction. The gDNA extraction was performed with the Wizard® Genomic DNA purification kit (Promega Corporation. Madison, WI) protocol for plant tissue. Quantification of extracted DNA was performed with a Nanodrop™ One C (Thermo Fisher Scientific, Waltham, MA).
Sequencing of EPSPS Thr102 and Pro106 Codons
The conserved region of the EPSPS gene encompassing Pro106 and Thr102 codons was amplified for the CT-Res and AL-Sus biotypes by polymerase chain reaction (PCR). The primers used in this experiment were obtained from EPSPS genomic sequences available on the National Center for Biotechnology Information database under accession MT025716.1. The primer set previously identified for Palmer amaranth EPSPS sequence (200 base pairs [bp]) was used (Gaines et al. Reference Gaines, Zhang, Wang, Bukun, Chisholm, Shaner, Nissen, Patzoldt, Tranel, Culpepper, Grey, Webster, Vencill, Sammons, Jiang, Preston, Leach and Westra2010; Whaley et al. Reference Whaley, Wilson and Westwood2006): (forward) EPSF1, 5′-ATG TTG GAC GCT CTC AGA ACT CTT-3′ GGT; (reverse) EPSR8, 5′-TGA ATT TCC TCC AGC AAC GGC AA-3′. The PCR was performed with the DreamTaq Green PCR Master Mix (2×) (Thermo Fisher Scientific) using the following thermocycle conditions: an initial denaturation at 95 C for 1 min; 40 denaturation cycles at 95 C for 30 s, primer annealing at 52 C for 30 s, and extension at 72 C for 3 min. A final extension at 72 C for 10 min was included. Amplicons were visualized with electrophoresis (1% agarose). The amplicons were extracted from agarose gels with the Wizard® SV Gel and PCR Clean-Up System (Promega) and quantified spectrophotometrically as previously described. Samples were sent for Sanger sequencing at the Genomics Core Facility at the Pennsylvania State Huck Institute of Life Sciences. Sequencing primers were used to cover all single nucleotide polymorphisms (SNPs) known to confer glyphosate resistance (Heap Reference Heap2024). Sequencing primers for EPSPS were EPSF1 and EPSPR8. Sequencing results were aligned and visually analyzed using Geneoius Prime software (Biomatters Inc., Boston, MA). The EPSPS sequence of the CT-Res biotype was aligned to a reference AL-Sus biotype EPSPS sequence to determine substitutions at Pro106 or Thr102 codons.
EPSPS Genomic Copy Number
Genomic DNA was used to quantify the number of copies of the EPSPS gene in CT-Res plants relative to the ALS gene (housekeeping gene) with a real-time PCR (Quantum Studio 5; Thermo Fisher) and the Power Track™ SYBR™ Green Master Mix protocol (Thermo Fisher). Primers for the housekeeping gene were: (forward) ALSF2, 5′-GCT GCT GAA GGC TAC GCT-3′ and (reverse) ALSFR2, 5′-GCG GGA CTG AGT CAA GAA GTG-3′ for ALS amplification. EPSPS amplification primers were: (forward) ECC_EPSPS_F1, 5′-CCA GAC CAA ATA CTT TCG GA-3′ and (reverse) ECC_EPSPS_R2, 5′CGG TAT GCT TAG AGG TGA AA-3′ (Gaines et al. Reference Gaines, Zhang, Wang, Bukun, Chisholm, Shaner, Nissen, Patzoldt, Tranel, Culpepper, Grey, Webster, Vencill, Sammons, Jiang, Preston, Leach and Westra2010). Three technical replicates and negative controls were also included. The real-time PCR conditions were as follows: enzyme activation at 95 C for 2 min, 40 cycles of denaturation at 95 C for 15 s, and 40 cycles of annealing and extension at 60 C for 1 min. A melt curve was produced to evaluate the specificity of the primers by setting the following conditions: First, a ramp rate of 1.6 C s−1 increases the temperature gradually up to 95 C, holding it for 15 s. A second ramp rate 1.6 C s−1 up to 60 C for 1 min was included, followed by a final dissociation step with a ramp rate of 0.075 C s−1 up to 95 C for 15 s. The 2−ΔΔCt method was used to quantify copy number variation of the EPSPS gene relative to the ALS gene. The EPSPS gene copies in CT-Res plants were assessed relative to a known glyphosate-susceptible biotype (AL-Sus, a calibrator sample). Data analysis was performed using R studio software by calculating the mean fold change per sample and further applying the least squares means comparison using the emmeans (Lenth Reference Lenth2022) package. Means comparison were performed using the multcomp (Hothorn et al. Reference Hothorn, Bretz and Westfall2008) package (α = 0.05), and data were plotted using the ggplot2 (Wickham Reference Wickham, Gentleman, Hornik and Parmigiani2016) package.
Results and Discussion
Effect of Plant Height on Glyphosate Resistance Levels
The estimated rates of glyphosate required for a 50% reduction in shoot fresh weight (GR50) of 10-, 20-, and 30-cm-tall CT-Res biotype were 5,138, 6,908, and 13,221 g ae ha−1, respectively (Table 1, Figure 1, A, B, and C). In contrast, the corresponding GR50 values for 10-, 20-, and 30-cm-tall KS-Sus biotype were 74, 108, and 247 g ae ha−1, respectively. The reduction in shoot fresh weight of the CT-Res biotype with 840 g ae ha−1 of glyphosate was below 10%, regardless of the plant height at the time of treatment. Glyphosate rates estimated for a 90% reduction in shoot fresh weight (GR90) were 18,056, 29,942, and 100,716 g ae ha−1 for the CT-Res biotype plants treated at heights of 10, 20, and 30 cm, respectively (Table 1). Complete control of the CT-Res biotype was not achieved even at the highest rate of glyphosate (13,340 g ae ha−1) tested in the dose-response bioassay. Similar GR90 values have previously been reported for 10-cm-tall GR Palmer amaranth biotypes in Nebraska and Arkansas (Chahal et al. Reference Chahal, Varanasi, Jugulum and Jhala2017; Norsworthy et al. Reference Norsworthy, Oliveira, Jha, Malik, Buckelew, Jennings and Monks2008b). On the contrary, the KS-Sus plants up to 20 cm tall were at least 90% controlled with 840 g ae ha−1 of glyphosate. However, the GR90 value was much higher (2,251 g ae ha−1) for 30-cm-tall KS-Sus plants. Several researchers found large differences in GR50 and GR90 values of susceptible and GR Palmer amaranth biotypes (Norsworthy et al. Reference Norsworthy, Griffith, Scott, Smith and Oliver2008a; Sosnoskie et al. Reference Sosnoskie, Kichler, Wallace and Culpepper2011; York Reference York2007). A GR Palmer amaranth biotype from Arkansas had an I50 value of 2,800 g ae ha−1 compared with 35 ae ha−1 for the susceptible biotype (Norsworthy et al. Reference Norsworthy, Griffith, Scott, Smith and Oliver2008a). Sosnoskie et al. (Reference Sosnoskie, Kichler, Wallace and Culpepper2011) reported 50% control of the glyphosate-susceptible and GR biotypes with glyphosate rates of 91 and 103 g ae ha−1, respectively. In the same study, ≥90% reduction in fresh weight was observed with glyphosate at 197 g ae ha−1 and 2,363 g ae ha−1 for the susceptible and GR Palmer amaranth biotypes, respectively. Several GR Palmer amaranth biotypes from North Carolina had I50 values between 180 g ae ha−1 and 360 g ae ha−1, compared with 89 g ae ha−1 for the local glyphosate-susceptible biotype (York Reference York2007).
Regression parameter estimates based on shoot fresh weight (% of nontreated) of a glyphosate-resistant Palmer amaranth population from Connecticut and a glyphosate-susceptible population from Kansas 21 d after treatment with various glyphosate doses a,b
a Abbreviations: b, the relative slope around the GR50 value; d the upper limit of biomass reduction; CT-Res, glyphosate-resistant Palmer amaranth biotype from Enfield, CT; KS-Sus, susceptible Palmer amaranth biotype from Hays, KS; GR50, the effective dose (g ae ha-1) of glyphosate needed for 50% fresh shoot weight reduction (% of nontreated); GR90, the effective dose (g ae ha-1) of glyphosate needed for 90% fresh shoot weight reduction (% of nontreated); R/S, resistance index (estimated as a ratio of GR50 of a CT-Res to GR50 of the KS-Sus Palmer amaranth biotype).
b Data were obtained from a greenhouse study conducted at the Connecticut Agricultural Experiment Station, Windsor, CT.
Glyphosate dose-response curves for 10-cm (A), 20-cm (B), and 30-cm (C) tall CT-Res and KS-Sus biotypes. CT-Res, resistant Palmer amaranth biotype found in Hartford County, Connecticut; KS-Sus, Palmer amaranth biotype collected from Kansas State University Agricultural Research Center near Hays, KS. Percent reduction in the shoot fresh biomass was calculated using Equation 1 in the text (Wortman Reference Wortman2014). A three-parameter log-logistic model was fitted on biomass reduction using Equation 2 in the text (Knezevic et al. Reference Knezevic, Streibig and Ritz2007) using the drc package (R statistical software; R Foundation for Statistical Computing, Vienna, Austria); AL-Sus1AL-Sus2CT-Res1CT-Res2CT-Res3CT-Res4CT-Res5CT-Res6AL-Sus1AL-Sus2CT-Res1CT-Res2CT-Res3CT-Res4CT-Res5CT-Res6.
In this study, the CT-Res Palmer amaranth biotype exhibited 69-fold, 64-fold, and 54-fold resistance to glyphosate when plants were treated at heights of 10, 20, and 30 cm, respectively (Table 1). Aulakh et al. (Reference Aulakh, Chahal, Kumar, Price and Guillard2021) reported 10-fold resistance to glyphosate in the same CT-Res Palmer amaranth biotype compared with the KS-Sus biotype. However, it is important to note that whole-plant dose-response bioassay in an earlier study was conducted on GR Palmer amaranth plants propagated from a field-collected segregating biotype. In the current dose-response study, test plants were grown from OP2 seeds of plants that survived 6,720 g ae ha−1 of glyphosate herbicide. Similar levels of glyphosate resistance have also been reported for GR Palmer amaranth from Kansas, Mississippi, and Nebraska (Chahal et al. Reference Chahal, Varanasi, Jugulum and Jhala2017; Kumar et al. Reference Kumar, Liu, Boyer and Stahlman2019; Kumar et al. Reference Kumar, Liu and Stahlman2020; Nandula et al. Reference Nandula, Reddy, Kroger, Poston, Rimando, Duke, Bond and Ribeiro2012).
EPSPS Gene Sequencing
The point mutations at the Pro106 (amino acid substitution from proline to serine, threonine, alanine, or leucine) and Thr102 (amino acid substitution from threonine to isoleucine) codons in the EPSPS gene have previously been reported to confer glyphosate resistance in some GR weed species (Sammons and Gaines Reference Sammons and Gaines2014; Yu et al. Reference Yu, Jalaludin, Han, Chen, Sammons and Powles2015). However, the sequence analysis of the EPSPS gene revealed no point mutations at the Pro106 and Thr102 residues in the CT-Res Palmer amaranth plants (Figure 2). These results rule out the possibility of a point mutation at the Pro106 or Thr102 codons in the EPSPS gene for a possible mechanism of glyphosate resistance in the CT-Res biotype. Lack of target-site mutations conferring glyphosate resistance has also previously been reported in GR kochia [Kochia scoparia (L.) Schrad], Palmer amaranth, and spiny amaranth biotypes (Gaines et al. Reference Gaines, Zhang, Wang, Bukun, Chisholm, Shaner, Nissen, Patzoldt, Tranel, Culpepper, Grey, Webster, Vencill, Sammons, Jiang, Preston, Leach and Westra2010; Kumar et al. Reference Kumar, Jha, Giacomini, Westra and Westra2015; Nandula et al. Reference Nandula, Wright, Molin, Ray, Bond and Eubank2014).
EPSPS gene sequence demonstrating no point mutations at the Pro106 (amino acid substitution from proline to serine, threonine, alanine, or leucine) and Thr102 (amino acid substitution from threonine to isoleucine) codons. AL-Sus1 and AL-Sus2 indicate glyphosate susceptible plants from Alabama; CT-Res1, CT-Res2, CT-Res3, CT-Res4, CT-Res5, and CT-Res6 indicate glyphosate-resistant plants from Connecticut.
EPSPS Gene Amplification
The EPSPS gene amplification (increased copy number) has previously been reported in various GR weed biotypes (Chatham et al. Reference Chatham, Wu, Riggins, Hager, Young, Roskamp and Tranel2015b). The qPCR analysis indicated that plants of CT-Res Palmer amaranth biotype had approximately 33 to 111 relative copies of the EPSPS gene (Figure 3). These results are consistent with previously reported GR Palmer amaranth biotypes from Georgia and Mississippi with 33 to 100 EPSPS gene copies (Gaines et al. Reference Gaines, Zhang, Wang, Bukun, Chisholm, Shaner, Nissen, Patzoldt, Tranel, Culpepper, Grey, Webster, Vencill, Sammons, Jiang, Preston, Leach and Westra2010; Ribeiro et al. Reference Ribeiro, Pan, Duke, Nandula, Baldwin, Shaw and Dayan2014). In contrast, GR spiny amaranth (Amaranthus spinosus L.) from Mississippi and GR Italian ryegrass from Arkansas have been reported with 26 to 37 and 15 to 25 relative EPSPS gene copies, respectively (Nandula et al. Reference Nandula, Wright, Molin, Ray, Bond and Eubank2014; Salas et al. Reference Salas, Dayan, Pan, Watson, Dickson, Scott and Burgos2012). Furthermore, lower folds of EPSPS gene amplification (2-fold to 10-fold) have been reported in GR Palmer amaranth biotypes from New Mexico; GR kochia biotypes from Colorado, Montana, and Kansas; and GR tall waterhemp [Amaranthus tuberculatus (Moq.) Sauer] biotypes (Kumar et al. Reference Kumar, Jha, Giacomini, Westra and Westra2015; Lorentz et al. Reference Lorentz, Gaines, Nissen, Westra, Strek, Dehne, Ruiz-Santaella and Beffa2014; Mohseni-Moghadam et al. Reference Mohseni-Moghadam, Schroeder and Ashigh2013a; Wiersma et al. Reference Wiersma, Gaines, Preston, Hamilton, Giacomini, Buell, Leach and Westra2015).
Bar plot of EPSPS gene copy number fold change relative to the ALS gene, obtained with the 2−ΔΔCt method. The same letters indicate no significant difference among biotypes (P = 0.05). Error bars indicate standard deviation. AL-Sus indicates glyphosate-susceptible plants from Alabama; CT-Res1, CT-Res2, CT-Res3, CT-Res4, CT-Res5, and CT-Res6 indicate glyphosate-resistant plants from Connecticut.
Practical Implications
Results from this research suggest that plant height influences glyphosate resistance in GR Palmer amaranth and that the CT-Res Palmer amaranth biotype has evolved high-level (54-fold to 69-fold) resistance to glyphosate compared with the KS-Sus biotype. The molecular test further confirmed that the GR Palmer amaranth from Connecticut has evolved resistance to glyphosate via EPSPS gene amplification by 33-fold to 111-fold compared with the AL-Sus biotype. The current research project did not test any nontarget-based mechanisms (such as alteration in absorption, translocation, sequestration or metabolism) of glyphosate resistance in the CT-Res biotype; therefore, further research should determine whether additional mechanisms of resistance are involved. Nonetheless, the occurrence of GR Palmer amaranth in Connecticut is a serious concern, considering that glyphosate is the most common herbicide used for weed control. These results clearly suggest that effective alternative (other than glyphosate) PRE and POST herbicides (with multiple SOAs) would be needed to control this GR Palmer amaranth biotype. Field surveys are underway to collect more Palmer amaranth biotypes in Connecticut to assess the distribution of GR biotypes. Future studies will evaluate the response of GR Palmer amaranth biotype to alternative PRE and POST herbicides for various cropping systems in Connecticut.
In addition to effective herbicide programs, Connecticut producers should also consider adopting integrated Palmer amaranth control strategies, including cultural practices (such as cover crops, competitive crop rotations/sequences, optimum crop seeding rates and row spacing, etc.), mechanical practices (strategic tillage, electrocution, harvest weed control techniques, etc.), and precision agricultural technologies (drones for weed scouting, precision sprayers, etc.) for managing GR Palmer amaranth seedbanks and its further spread.
Acknowledgements
We thank Mr. Ethan Paine for technical assistance in greenhouse experiments.
Funding statement
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interests
Drs Aulakh, Kumar, Brunharo, Price, and Mr. Veron declare none.
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