Introduction
The evolution of herbicide-resistant weed populations represents a growing challenge for both contemporary and future agriculturalists. The repetitive applications of herbicides with the same active ingredient or mode of action have catalyzed this biological phenomenon (Evans et al. Reference Evans, Tranel, Hager, Schutte, Wu, Chatham and Davis2016; Neve et al. Reference Neve, Busi, Renton and Vila-Aiub2014). Such selection pressure has culminated in the selection of resistance to at least one herbicide in 272 weed species globally (Heap Reference Heap2024). While herbicides are not the sole form of selection pressure in modern agriculture, their utilization precipitates more rapid changes, and the resultant weed resistance is more readily apparent (Coble and Schroeder Reference Coble and Schroeder2016).
The genus Amaranthus encompasses multiple species, some of which are significant agricultural weeds (Gonçalves-Netto et al. Reference Gonçalves-Netto, Nicolai, Carvalho, Borgato and Christoffoleti2016). In Brazil, approximately 10 Amaranthus species are recognized as agriculturally significant. To date, seven instances of herbicide resistance within Amaranthus species have been documented in Brazil, involving redroot pigweed (Amaranthus retroflexus L.), slender amaranth (Amaranthus viridis L.), Palmer amaranth (Amaranthus palmeri S. Watson), and smooth pigweed (Amaranthus hybridus L.) (Heap Reference Heap2024). Amaranthus hybridus is a notably competitive weed for summer crops in South America and is characterized as an annual, erect, herbaceous species with glabrous stems, petiolate ovate leaves with sharp apices, terminal panicles forming inflorescences, and hermaphrodite flowers (Perotti et al. Reference Perotti, Larran, Palmieri, Martinatto, Alvarez, Tuesca and Permingeat2019). This species utilizes the C4 photosynthetic pathway (Ferreira et al. Reference Ferreira, Procópio, Silva, Silva and Rufino2003), exhibits high fecundity and seed longevity (Faccini and Vitta Reference Faccini and Vitta2005), demonstrates emergence fluxes across varying periods of the crop cycles (Brighenti and Oliveira Reference Brighenti, Oliveira, Inoue, Oliveira, Mendes and Constantin2015), and, as a result, is becoming increasingly challenging to manage within cropping systems (Butts et al. Reference Butts, Norsworthy, Kruger, Sandell, Young, Steckel, Loux, Bradley, Conley, Stoltenberg, Arriaga and Davis2016).
In Argentina, A. hybridus has been reported to exhibit resistance to acetolactate synthase (ALS) inhibitors (Tuesca and Nisensohn Reference Tuesca and Nisensohn2001), auxins (Dellaferrera et al. Reference Dellaferrera, Cortés, Panigo, De Preado, Christoffoleti and Perreta2018), and glyphosate (García et al. Reference García, Palma-Bautista, Rojano-Delgado, Bracamonte, Portugal, Alcantara-De-La-Cruz and De Prado2019; Perotti et al. Reference Perotti, Larran, Palmieri, Martinatto, Alvarez, Tuesca and Permingeat2019), with some mechanisms of resistance to these herbicides having been elucidated. For instance, the mechanism conferring glyphosate resistance involves a triple substitution (TAP-IVS: Thr-102-Ile, Ala-103-Val, and Pro-106-Ser) in the EPSPS protein (García et al. Reference García, Palma-Bautista, Rojano-Delgado, Bracamonte, Portugal, Alcantara-De-La-Cruz and De Prado2019; Perotti et al. Reference Perotti, Larran, Palmieri, Martinatto, Alvarez, Tuesca and Permingeat2019; Sulzbach et al. Reference Sulzbach, Turra, Cutti, Kroth, Tranel, Merotto and Markus2024). In the case of ALS-inhibitor resistance, the predominant molecular mechanism resistance identified is target-site mutation. To date, 28 amino acid substitutions at 8 positions within the ALS gene have been documented to confer ALS-inhibitor resistance (Tranel et al. Reference Tranel, Wright and Heap2023). Biotypes of A. hybridus found in Argentina that are resistant to ALS-inhibiting herbicides exhibit a specific site mutation documented as substitutions Trp-574-Leu and Asp-376-Glu in the ALS amino acid sequence (Larran et al. Reference Larran, Lorenzetti, Tuesca, Perotti and Permingeat2018).
The weed management decisions made by growers significantly influence the selection of herbicide-resistant weeds within their fields. When poor weed control is observed, growers often respond by increasing herbicide doses, thereby intensifying the selection pressure for resistance (Bracamonte et al. Reference Bracamonte, Fernández-Moreno, Barro and De Prado2016; González-Torralva et al. Reference González-Torralva, Gil-Humanes, Barro, Brants and De Prado2012). However, resistance can also be spread from one field to another through field machinery operations, high seed production by weeds, and seed crop contaminants, affecting regions previously unaffected by resistance (Penckowski et al. Reference Penckowski, Maschietto, Borsato, Adegas, Moreira, Bianchi, Rizzardi, Oliveira and Dornelles2020). Resistance monitoring studies, aimed at understanding the frequency and distribution of resistant weeds in agricultural areas, serve as a crucial tool for increasing awareness about herbicide resistance and aiding in the adoption of integrated management strategies at the regional level (Schultz et al. Reference Schultz, Chatham, Riggins, Tranel and Bradley2015).
Recently, field crop extension scientists and farmers have observed diminished control of Amaranthus spp. with glyphosate, chlorimuron, and imazethapyr herbicides in Brazil. Consequently, the primary objective of this study was to quantify the resistance levels to glyphosate, chlorimuron, and imazethapyr in four Amaranthus populations from southern Brazil and to investigate the mechanism underlying resistance to glyphosate and ALS inhibitors. Additionally, this study aimed to determine the frequency of shifts in sensitivity to glyphosate and chlorimuron in Brazil.
Materials and Methods
Plant Material and Growth
Seeds from two plants from two Amaranthus populations with putative resistance to glyphosate, chlorimuron, and imazethapyr were collected from separate soybean [Glycine max (L.) Merr.] fields in Brazil. Additionally, seeds from two reference populations known to be sensitive were obtained; these populations had no history of exposure to glyphosate or ALS-inhibiting herbicides (Table 1).
Table 1. Identification and location of the Amaranthus hybridus populations studied
Plants from the putatively resistant population were treated with glyphosate (1,000 g e.a ha−1, Roundup WG®, 720 g e.a kg−1, Monsanto from Brazil LTDA, 1100 Domingo Jorge Road, São Paulo, SP 047779-900, Brazil), chlorimuron (20 g e.a ha−1, Classic WG®, 250 g e.a kg−1, Du Pont from Brazil S.A, 506 Alameda Itapecuru Road, Barueri, SP 06454-080, Brazil), and imazethapyr (100 g e.a ha−1, Pivot SL®, 100 g e.a L−1, 5756 Industrial Domingos Giomi District, Indaiatuba, SP 13347-390, Brazil). Surviving plants were maintained in the greenhouse for seed production. Seeds from Amaranthus populations were germinated in 0.3-L pots filled with a commercial substrate (BASE substratos, Basaplant tubetes). Plants were potted individually and grown in a greenhouse at 32 C/25 C (day/night) under a 16-h photoperiod until they reached the 4-leaf stage.
Whole-Plant Dose–Response Curve
The dose–response curve was plotted for two generations of Amaranthus (G1 and G2). The experimental design was completely randomized in a 6 by 12 factorial scheme for glyphosate and a 5 by 9 factorial design for chlorimuron and imazethapyr. The first factor was the Amaranthus population (Table 1) and the second factor was herbicide. For Herbicide dosages were: glyphosate (Roundup WG®, 720 g kg−1) at 0, 45, 90, 180, 360, 720, 1,440, 2,880, 5,760, 11,520, 23,040, and 46,080 g ha−1; chlorimuron (Classic WG®, 250 g kg−1) at 0, 2.5, 5.0, 10, 20, 40, 80, 160, and 320 g ha−1; and imazethapyr (Pivot SL® 100 g L−1) at 0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 g ha−1. Each combination of population and dose was replicated four times. The application was performed in a spray chamber equipped with flat-fan nozzles (XR110.02, TeeJet®, Cotia, São Paulo, Brazil) calibrated to deliver 200 L ha−1 at 200 kPa pressure.
Aboveground biomass of each plant was harvested at 21 d after treatment (DAT) and oven-dried for 3 d at 60 C for biomass (dry weight) determination. The biomass reduction data were converted into percent biomass reduction relative to the nontreated control plants (Wortman Reference Wortman2014) using Equation 1:
$${\rm{Biomass\;reduction}}\;\left( \% \right) = {{{\left( {C - B\;} \right)}}\over{C }} \times 100$$
where C represents the mean biomass of the four nontreated control replicates, and B represents the biomass of an individually treated experimental plant.
Data were analyzed using ANOVA, and a nonlinear logistic regression model was fit using Equation 2:
$$y = {{a}\over{{\left[ {1 + \left( {{x}\over{b}} \right)c} \right]}}}$$
In this equation,
$$y$$
represents the control,
$$x$$
represents the herbicide dose (g ha−1),
$$a$$
represents the maximum value,
$$b$$
represents the dose providing 50% response (GR50), and
$$c$$
represents the curve slope around
$$b$$
. The RF was calculated as the ratio of the GD50 values among the putative resistant and sensitive populations.
DNA Extraction
Leaf tissue from herbicide-treated greenhouse-grown plant (4 plants per tested population) was utilized for DNA extraction. Genomic DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega) adhering to the manufacturer’s protocol (Promega Corporation, Madison, Wisconsin, USA). DNA quantification was conducted using the Denovix instrument, and DNA integrity was assessed on a 1% agarose gel (DeNovix , Wilmington, Delaware, USA). All DNA samples were diluted to a concentration of 50 ng µl−1, and a 2.0 µl aliquot was used for PCR amplification.
Amaranthus Species Identification
The species of the four populations (BR18Asp050, BR21Asp206, BR18Asp051, and BR21Asp205) were identified using three molecular approaches. The first approach was based on the methodology developed by Wright et al. (Reference Wright, Molin and Nandula2016). Briefly, the methodology is based on a PCR assay using the amplification of a region on intron 1 of the EPSPS gene, which considers seven Amaranthus species: A. palmeri, spiny amaranth (Amaranthus spinosus L.), waterhemp [Amaranthus tuberculatus (Moq.) Sauer], A. retroflexus, A. hybridus, prostrate pigweed (Amaranthus blitoides S. Watson), and A. viridis. For this approach, positive controls were used for A. palmeri, A. spinosus, A. hybridus, and A. viridis. The second approach was based on Sanger sequencing of PCR fragments amplified from the internal transcribed spacer (ITS) region as reported previously (Murphy and Tranel Reference Murphy and Tranel2018; Sulzbach et al. Reference Sulzbach, Turra, Cutti, Kroth, Tranel, Merotto and Markus2024). And the third approach was based on Sanger sequencing of the EPSPS intron 1 region (Wright et al. Reference Wright, Molin and Nandula2016). All primers and PCR conditions were performed as reported in the cited publications.
EPSPS Sequencing
The presence of the TAP-IVS triple substitution in the samples was verified using a PCR-based assay (Mathioni et al. Reference Mathioni, Oliveira, Lemes, Ozorio and Rosa2022), and a 195-bp fragment of the EPSPS gene was screened for novel mutations through amplification and sequencing. The primer set used was EPSF1 (5′-ATGTTGGACGCTCTCAGAACTCTTGGT-3′) and EPSR8 (5′-TGAATTTCCTCCAGCAACGGCAA-3′), reported by Gaines et al. (Reference Gaines, Zhang, Wang, Bukun, Chisholm, Shaner and Westra2010). PCR reactions were carried out in a 25-µl volume containing 100 ng of DNA and the following reagents: 200 µM dNTPs, 1.5 mM MgCl2, 200 nM primers, and 1 unit of GoTaq G2 Hot Start DNA Polymerase (Promega Corporation, Madison, Wisconsin, USA). PCR cycling conditions were as follows: 95 C for 2 min, 40 cycles of 95 C for 30 s, 58 C for 30 s, and 72 C for 30 s; final extension was performed at 72 C for 5 min. The amplification and expected size of amplicons were verified on 1% agarose gels. PCR products were cleaned before sequencing using the ExoSAP-IT Express (Thermo Fisher) following the manufacturer’s instructions (Thermo Fisher® Waltham, Massachusetts, USA). Sequencing reactions were prepared using the BigDye Terminator v. 3.1 chemistry (Thermo Fisher) and sequenced on a genetic analyzer sequencing instrument (AB 3500, Applied BioSystems, Thermo Fisher® Waltham, Massachusetts, USA). The EPSPS sequences from A. hybridus (MH482844.1, MH482843.1) were used for analysis, and alignment was performed with the UGene (v. 1.45.0, Unipro) software (Okonechnikov et al. Reference Okonechnikov, Golosova and Fursov2012).
ALS Sequencing
The ALS gene was amplified with primers designed for this study based on the GenBank ALS sequence MH036304.1 for A. hybridus: Ahy-ALS-F1 (5′-ATGGCGTCCACTTCTTCAAACC-3′), Ahy-ALS-F2 (5′-GACCTGTGCTGTATACTGGAG-3′), Ahy-ALS-R1 (5′-CTCCAGTATACAGCACAGGTC-3′), Ahy-ALS-R2 (5′-CTTACAACCGCATCGCCCTTCG-3′), and Ahy-ALS-R3 (5′- CTAATAAGCCCTTCTTCCATCACC-3′). These primers amplify fragments containing all the known plant ALS mutations associated with resistance. The PCR reactions were performed in 25 µl with 100 ng of DNA and the following reagents: 200 µM dNTPs, 1.5 mM MgCl2, 200 nM primers, and 1 unit of GoTaq G2 Hot Start DNA Polymerase. PCR cycling conditions were as follows: 95 C for 3 min, 40 cycles of 95 C for 30 s, 58 C for 30 s, and 72 C for 90 s; final extension was performed at 72 C for 5 min. The amplification and expected size of amplicons were verified on 1% agarose gels. PCR products were cleaned before sequencing using the ExoSAP-IT Express (Thermo Fisher) following the manufacturer’s instructions. Sequencing reactions were prepared using the BigDye Terminator v. 3.1 chemistry (Thermo Fisher) and sequenced on a genetic analyzer sequencing instrument (AB 3500, Applied BioSystems). The ALS sequences from A. hybridus (MH036304.1 and MH036306.1) were used for analysis, and alignment was performed with the UGene (v. 1.45.0, Unipro) software (Okonechnikov et al. Reference Okonechnikov, Golosova and Fursov2012).
Frequency of Shifts in Sensitivity to Glyphosate and Chlorimuron in Amaranthus Populations across Brazil
Seed collection was carried out in areas where Amaranthus plants exhibited survival after herbicide application. A total of 740 samples were collected over a period of 4 yr (2018 to 2021). Seeds were obtained from agricultural fields located in 266 municipalities across eight Brazilian states. The seed collection methodology adhered to the protocol suggested by Burgos et al. (Reference Burgos, Tranel, Streibig, Davis, Shaner, Norsworthy and Ritz2013). Upon identification of a crop site with herbicide application failures, seeds were randomly collected from a minimum of 10 mature plants and subsequently combined to form a single composite sample. Each seed sample was catalogued with site-specific information, including geographic coordinates, state, and municipality.
In greenhouse experiments, the seeds from all Amaranthus populations were germinated and planted in 1-L pots filled with a commercial substrate (Base Industria de Insumos Agrícolas Ltda, Arthur Nogueira, São Paulo, Brazil); here, one pot contained eight plants. Each combination of population and herbicide was replicated three times (24 plants tested per population) in a completely randomized design. A single discriminatory dose was utilized to determine the shift in sensitivity to glyphosate and chlorimuron, using the recommended field doses of 720 and 20 g ha−1 for glyphosate and chlorimuron, respectively. Control plants, including both glyphosate- and chlorimuron is sensitive (BR18Asp050) and glyphosate- and chlorimuron is resistant (BR18Asp051), were included as reference standards in all assays.
Glyphosate and chlorimuron treatments were administered when the plants reached the 4- to 6-leaf stage. The application was performed in a spray chamber equipped with flat-fan nozzles calibrated to deliver 200 L ha−1 at 200 kPa pressure. Plant mortality was evaluated at 21 DAT using the scale described in Table 2. After each population was classified based on the shift sensitivity criteria, the data were grouped by class (Table 2). Maps of the frequency and dispersion of Amaranthus populations were created for each herbicide using TIBCO Spotfire 10.3.1 Analyst® (TIBCO Spotfire®, Palo Alto, CA, USA). In the maps, color-code classification was used to identify each sample (Table 2).
Table 2. Color code for shifts in sensitivity to glyphosate and chlorimuron in Amaranthus populations at 21 d after treatment (DAT)
Results and Discussion
Amaranthus Species Identification
The four populations (BR18Asp050, BR21Asp206, BR18Asp051, and BR21Asp205) used in the plant dose–response curve were analyzed using the three approaches described in “ Materials and Methods.” When the PCR-based approach described by Wright et al. (Reference Wright, Molin and Nandula2016) was used, the four populations presented positive bands (positive amplification) for the primers AW473+AW483 and AW471+AW482 (Wright et al. Reference Wright, Molin and Nandula2016), which amplified fragments of A. hybridus and A. retroflexus, respectively. These EPSPS intron 1 fragments were Sanger sequenced, resulting in a 1,053-bp sequence that was analyzed and compared with the A. hybridus (KT833344) and A. retroflexus (KT833348) sequences. A total of 18 single-nucleotide polymorphisms (SNPs) were observed among these two species, and the analyzed populations showed 13 SNPs identical to A. hybridus. When the ITS sequences for the four biotypes were analyzed, the alignment rendered identical (100% similarity) sequences among the four biotypes with 100% similarity to A. hybridus AMACHY-S (Sulzbach et al. Reference Sulzbach, Turra, Cutti, Kroth, Tranel, Merotto and Markus2024) and 99.5% similarity to a sample identified as A. retroflexus (GenBank accession no. KF493795). Taking all the results together, we conclude that the four populations in this study belong to the A. hybridus species.
Whole-Plant Dose–Response Curve
Dose–response analysis indicated a reduction in biomass correlating with increased herbicide dose. However, significant differences were observed among populations. Notably, some populations exhibited high-level resistance to glyphosate (Figure 1A). Resistance factors, based on the GD50, were 34 and 129 for BR18Asp051 and BR21Asp205, respectively (Table 3). The sensitive populations displayed GD50 values of 4.20 and 29.7 g ha−1 for BR18Asp050 and BR21Asp206, respectively. Even the highest dose tested in this study (32×) failed to effectively control the BR18Asp051 and BR21Asp205 populations.
Figure 1. Glyphosate biomass reduction (A), chlorimuron biomass reduction (B), and imazethapyr biomass reduction (C) responses in BR18Asp050, BR21Asp206, BR18Asp051, and BR21Asp205 populations of Amaranthus hybridus at 21 d after treatment (DAT).
Table 3. Glyphosate, chlorimuron, and imazethapyr doses required for biomass reduction (GR) of 50% in the BR18Asp050, BR21Asp206, BR18Asp051, and BR21Asp205 populations at 21 d after treatment, and resistance factors (RF) for Amaranthus hybridus populations
a Amaranthus putative resistant: BR18Asp051 and BR21Asp205; susceptible: BR18Asp050 and BR21Asp205.
b Herbicide dose (g ha−1) to control 50% of the variable (GD50); lower/upper confidence interval (CI) of the variable e; RF, resistance factor (resistant/susceptible).
* Statistically significant at P < 0.05.
The initial report of glyphosate-resistant A. hybridus in Argentina emerged in 2013, with subsequent studies from the same region indicating high levels of resistance (RF = 314) (Heap Reference Heap2024; Perotti et al. Reference Perotti, Larran, Palmieri, Martinatto, Alvarez, Tuesca and Permingeat2019). Another study in Argentina reported a resistance factor of 125 for A. hybridus (García et al. Reference García, Palma-Bautista, Vazquez-Garcia, Rojano-Delgado, Osuna, Torra and De Prado2020), while Resende et al. (Reference Resende, Christoffoleti, Netto, Presoto, Nicolai, Maschietto, Borsato and Penckowski2022) identified lower resistance factors (13, 14, and 15) in the same A. hybridus populations from Paraná State in Brazil. The resistance factor in this study surpassed those previously cited; however, BR19Asp050 was the most sensitive population with a GD50 of 4.20, whereas other studies reported higher GD50, values: 66.6 g e.a ha−1 (Perotti et al. Reference Perotti, Larran, Palmieri, Martinatto, Alvarez, Tuesca and Permingeat2019), 17.7 g e.a ha−1 (García et al. Reference García, Palma-Bautista, Vazquez-Garcia, Rojano-Delgado, Osuna, Torra and De Prado2020), and 227.63 g e.a ha−1 (Resende et al. Reference Resende, Christoffoleti, Netto, Presoto, Nicolai, Maschietto, Borsato and Penckowski2022).
Glyphosate was first introduced commercially in the early 1970s; despite its widespread use, resistant weeds were not reported until 1996 (Powles et al. Reference Powles, Lorraine-Colwill, Dellow and Preston1998). Currently, glyphosate-resistant weeds impact hundreds of millions of acres in the United States and other countries, such as Argentina and Brazil, that have adopted genetically engineered crops (Bain et al. Reference Bain, Selfa, Dandachi and Velardi2017). In Brazil, 11 glyphosate-resistant species have been reported, leading to crop yield losses, complicating management, and increasing weed control costs.
For the ALS-inhibiting herbicides, resistance was also observed in the evaluated populations. The resistance factors based on GD50 were 15 and 35 for the BR18Asp051 and BR21Asp205 populations, respectively (Table 3). In the case of the BR18Asp051 population, the highest chlorimuron dose (320 g e.a ha−1) almost achieved complete control of the population, but the BR21Asp205 population did not achieve 80% control with this dose (Figure 1B). The resistance factors for imazethapyr were 6 and 47 for BR18Asp051 and BR21Asp205 populations, respectively (Table 3). Even at the maximum imazethapyr dose, neither population showed effective control (Figure 1C).
Amaranthus hybridus populations from Illinois, USA, exhibited a resistance factor exceeding 152 for an ALS-inhibiting herbicide (Maertens et al. Reference Maertens, Sprague, Tranel and Hines2004). In Argentina, a resistance factor of 38 was observed for A. hybridus (García et al. Reference García, Palma-Bautista, Vazquez-Garcia, Rojano-Delgado, Osuna, Torra and De Prado2020). A Brazilian study detected cross-resistance to ALS-inhibiting herbicides in an A. hybridus population, with resistance factors of 6.9 and 6.5 for chlorimuron and metsulfuron-ethyl, respectively, although, resistance to imazethapyr was not confirmed (Mendes et al. Reference Mendes, Silva, Ferreira and Oliveira2022). Moreover, the recommended rates for each herbicide were insufficient to achieve 90% control of the resistant population based on GD90 parameter estimation (Mendes et al. Reference Mendes, Silva, Ferreira and Oliveira2022).
Since 1980, ALS-inhibiting herbicides have been marketed globally (Garcia et al. Reference Garcia, Nouwens, Lonhienne and Guddat2017). They remain crucial in burndown programs and weed management systems including glyphosate-resistant species for major crops such as soybeans (Santos et al. Reference Santos, Timossi, Lima, Gonçalves and Santana2016; Zobiole et al. Reference Zobiole, Krenchinski, Pereira, Rampazzo, Rubin and Lucio2018). In South America, farmers have relied for decades on the residual herbicide imazethapyr and subsequently glyphosate postemergence for managing Amaranthus species (García et al. Reference García, Palma-Bautista, Vazquez-Garcia, Rojano-Delgado, Osuna, Torra and De Prado2020). However, their continuous applications may have been selected for A. hybridus populations with multiple resistance. In this study, the BR18Asp051 and BR21Asp205 populations were cross-resistant to chlorimuron and imazethapyr (ALS inhibitors), and also exhibited multiple resistance to glyphosate (EPSPS inhibitor) and chlorimuron and imazethapyr (ALS inhibitors).
EPSPS Gene Resistance Mechanism
Sequencing of the 195-bp fragment of the EPSPS gene revealed a triple amino acid substitution in all plants from glyphosate-resistant populations BR18Asp051 and BR21Asp205 (Figure 2). Four nucleotide replacements resulted in codon changes: ACA for ATA (Thr-102-Ile), GCG for GTC (Ala-103-Val), and CCA for TCA (Pro-106-Ser).
Figure 2. Alignment of EPSPS protein fragments showing the TAP-IVS triple substitution (Thr-102-Ile, Ala-103-Val, and Pro-106-Ser) (highlighted). Sequences S (sensitive, highlighted in green) and R (resistant, highlighted in yellow) are the reference sequences (GenBank accession nos. AXN57302.1 and AXN57301.1).
Simultaneously, substitutions at positions 102 and 106 (TIPS) were previously identified in species such as goosegrass [Eleusine indica (L.) Gaertn.] (Yu et al. Reference Yu, Jalaludin, Han, Chen, Sammons and Powles2015), hairy beggarticks (Bidens pilosa L.) (Alcántara De La Cruz et al. Reference Alcántara De La Cruz, Fernández-Moreno, Ozuna, Rojano-Delgado, Cruz-Hipolito, Dominguez-Valenzuela and De Prado2016), and greater beggarticks (Bidens sulbalternans DC.) (Takano et al. Reference Takano, Fernandes, Adegas, Oliveira, Westra, Gaines and Dayan2020). Recent studies by Perotti et al. (Reference Perotti, Larran, Palmieri, Martinatto, Alvarez, Tuesca and Permingeat2019) and García et al. (Reference García, Palma-Bautista, Vazquez-Garcia, Rojano-Delgado, Osuna, Torra and De Prado2020) also reported this triple substitution (TAP-IVS) in A. hybridus from Argentina correlating with a high resistance factor, akin to the finding of this study.
The resistance to glyphosate in the analyzed populations is mainly caused by the triple mutations in the EPSPS gene; however, other resistance mechanisms cannot be ruled out, such an increase in the number of EPSPS gene copies, an increase in gene expression, change in herbicide absorption and translocation, and metabolism. Amaranthus hybridus biotypes from Argentina showed reduced glyphosate translocation and signs of EPSPS gene amplification; however, these mechanisms seem to have made minor contributions to herbicide resistance (García et al. Reference García, Palma-Bautista, Rojano-Delgado, Bracamonte, Portugal, Alcantara-De-La-Cruz and De Prado2019). On the other hand, a higher copy number of the EPSPS gene was identified in some biotypes of this species in Argentina and Brazil (García et al. Reference García, Palma-Bautista, Vazquez-Garcia, Rojano-Delgado, Osuna, Torra and De Prado2020; Sulzbach et al. Reference Sulzbach, Turra, Cutti, Kroth, Tranel, Merotto and Markus2024); however, the resistant plants showed expression levels similar to those of the susceptible individuals (Perotti et al. Reference Perotti, Larran, Palmieri, Martinatto, Alvarez, Tuesca and Permingeat2019).
ALS Gene Resistance Mechanism
The sequencing analysis of the ALS gene identified only a point mutation at position 574 in the ALS gene when comparing resistant populations BR18Asp051 and BR21Asp205 with the ALS–inhibitor susceptible populations BR18Asp050 and BR21Asp206. No other mutation was observed in these two populations. This mutation, a T to G nucleotide change, resulted in a codon shift from TTG (leucine) to TGG (tryptophan) (Figure 3). The same substitution was reported in an A. hybridus population from Argentina, conferring cross-resistance to ALS-inhibiting herbicides (Larran et al. Reference Larran, Lorenzetti, Tuesca, Perotti and Permingeat2018)..
Figure 3. Alignment of Amaranthus hybridus ALS protein fragments showing the Trp-574-Leu (W574L) substitution (highlighted). Sensitive sequence with the tryptophan amino acid highlighted in green, and resistant sequence with the leucine amino acid highlighted in yellow for the reference sequences (GenBank accession nos. MH036304.1 and MH036306.1).
Mutations conferring resistance to ALS inhibitors in various weeds species can occur at eight positions within the ALS genes (Tranel et al. Reference Tranel, Wright and Heap2023). Cross-resistance patterns among ALS-inhibitor classes vary depending on the specific amino acid position and substitution (Shaner Reference Shaner1999). For instance, resistance to IMI herbicide is often due to an Ala-122 or Ser-653 substitution, which also confers a lower level of resistance to SU herbicides (Bernasconi et al. Reference Bernasconi, Woodworth, Rosen, Subramanian and Siehl2016; Tranel et al. Reference Tranel, Wright and Heap2023). Conversely, a Pro-197 substitution typically results in SU herbicide resistance with low or no cross-resistance to IMI herbicides (Guttieri et al. Reference Guttieri, Eberlein, Mallory-Smith, Till and Hofman1992). Cross-resistance usually involves an Ala-205 or Trp-574 substitution, with the latter conferring higher resistance levels (García et al. Reference García, Palma-Bautista, Vazquez-Garcia, Rojano-Delgado, Osuna, Torra and De Prado2020).
The Trp-574-Leu mutation identified in the BR18Asp051 and BR21Asp205 populations is associated with high level of resistance to SU and IMI herbicides. In Illinois, USA, the same mutation in A. hybridus was linked to substantial resistance to imazamox and thifensulfuron (Maertens et al. Reference Maertens, Sprague, Tranel and Hines2004).
Frequency of Shifts in Sensitivity to Glyphosate and Chlorimuron in Amaranthus spp. Populations across Brazil
Overall, 88% of the Brazilian populations were deemed sensitive to glyphosate, with mortality rates between 90% and 100%. Additionally, 2% of populations exhibited mortality rates between 20% and 89% (yellow classification), distributed across nine municipalities. A mortality rate between 0% and 19% (red classification) was observed in 10% of the total population, spread over 30 different municipalities. Shifts in sensitivity to glyphosate were noted in four states: Rio Grande do Sul, Paraná, São Paulo, and Santa Catarina. (Figures 4A and 5A).
Figure 4. Distribution of Amaranthus populations based on glyphosate (A) and chlorimuron (B) sensitivity in Brazil.
Figure 5. Frequency (%) of glyphosate (A) and chlorimuron (B) tested populations and total population (in parenthesis) of Amaranthus for selected states in Brazil.
Most of the populations with altered glyphosate sensitivity were in Rio Grande do Sul, where 273 populations were tested. Of these, 77% were sensitive (green classification), 4% were yellow, and 19% were red (Figure 5A). Yellow and red classifications were found in 7 and 16 municipalities, respectively. In Paraná, of the 200 tested populations, 2.5% were yellow and 6% were red, with these classifications found in 13 and 4 municipalities, respectively. In Santa Catarina and São Paulo, glyphosate sensitivity shifts were detected in two and one locations, respectively (Figure 5A).
Shifts in sensitivity to chlorimuron in Amaranthus spp. are widespread across various Brazilian regions (Figure 4B). Of all samples analyzed, 66% were sensitive to chlorimuron (green classification), 11% yellow, and 23% were red (Figure 5B). The most severe situation was in the midwest, particularly in Mato Grosso state, where only 15% of the population was sensitive to chlorimuron. The first ALS–inhibitor resistant A. hybridus population in Brazil was reported in 2011 in Mato Grosso’s cotton (Gossypium hirsutum L.) fields (Heap Reference Heap2024).
Ten percent of the 740 samples tested nationwide showed control rates below 90% for both glyphosate and chlorimuron (Figure 6). These were in the states of Rio Grande do Sul, Santa Catarina, Pananá, and São Paulo. The populations showing shifts in sensitivity to herbicides may include different Amaranthus species. Therefore, a crucial advancement in understanding Amaranthus resistance in Brazil involves accurate identification of these species.
Figure 6. Frequency distribution (%) and number of populations (in parenthesis) of Amaranthus with single and multiple resistance to glyphosate and chlorimuron for selected states in Brazil.
Resistant A. hybridus plants or seeds may have been introduced to Brazil from other countries, as many producers import forage seeds from neighboring countries, and significant movement of machinery and people occurs between these regions. Another point is that the resistance mechanism observed in this study, for glyphosate (TAP-IVS mutation), has already been found in populations from Argentina (García et al. Reference García, Palma-Bautista, Vazquez-Garcia, Rojano-Delgado, Osuna, Torra and De Prado2020; Larran et al. Reference Larran, Lorenzetti, Tuesca, Perotti and Permingeat2018; Perotti et al. Reference Perotti, Larran, Palmieri, Martinatto, Alvarez, Tuesca and Permingeat2019). The Occurrence of a triple mutation in the same gene is rare, suggesting that the resistant populations are likely due to the dissemination of seeds from resistant populations from Argentina (Sulzbach et al. Reference Sulzbach, Turra, Cutti, Kroth, Tranel, Merotto and Markus2024). Although the long-term use of ALS herbicide in soybeans and cotton could explain the spread of chlorimuron resistance across southern and midwestern Brazil.
In Brazil, the first area with Amaranthus populations exhibiting glyphosate resistance was identified in 2016 (Heap Reference Heap2024); thus, the number and distribution of areas where glyphosate was ineffective in this study are concerning. This suggests that in a short period, Amaranthus species could become a significant challenge for Brazilian agriculture.
In conclusion, the collective findings of this study underscore the need to reevaluate current weed management programs in Brazil. The adoption of preemergence herbicides is still low compared with other countries facing herbicide-resistant A. hybridus (e.g., Argentina and the United States). Diversifying herbicide mode of action, mandating the use of preemergence herbicides, and increasing the use of combined mechanisms may become more crucial strategies. Our data also emphasize the importance of an effective herbicide-resistant monitoring program, which could help prevent weed resistance by focusing efforts on regional issues and increasing stakeholders’ awareness in each area.
Acknowledgments
The authors thank Maria Salete Marchioretto and the Research Herbarium Anchieta/UNISINOS for the morphological species identification and Syngenta Crop Protection Brazilian Team, especially the Resistance Management Laboratory, for all the support.
Funding statement
This research received specific grant funding from Syngenta Crop Protection Brazil.
Competing interests
The authors declare no conflicts of interest.
