Sulfonylurea resistance in Amaranthus hybridus from southern Brazil

ABSTRACT

Amaranthus hybridus is a C4 broadleaf species widely spread across Brazilian agricultural territory. Recently, several herbicide resistance reports have been documented in southern Brazil, including the reports for enolpyruvilshikymate-3-phosphate (EPSPS)- and acetolactate-synthase (ALS)- inhibitors. The objective of this study was to confirm the existence of an ALS resistant (R) A. hybridus population from Paraná state. Dose-response experiments were conducted with R and a known susceptible (S) population with herbicides from three different chemical groups of ALS inhibitors. Biomass relative to untreated control was quantified and GR₅₀ (dose for 50% of biomass reduction), GR₉₀ (dose for 90% of biomass reduction) and resistant index (RI) were calculated based on non-linear regression analysis. The R population was 6.9-fold resistant to chlorimuron-ethyl and 6.5-fold resistant to metsulfuron-ethyl (sulfonylureas - SUL). Additionally, the recommended rates from each herbicide was not sufficient to reach 90% control to R based on GR₉₀ parameter estimation. There was no resistance to imazethapyr (imidazolinone - IMI) and cloransulan-methyl (triazolopyrimidine - TRI) due to the low doses of GR₉₀ and non-significant RIs. The R A. hybridus population investigated was resistant to ALS inhibitors chlorimuron-ethyl and metsulfuron-ethyl (SUL), but susceptible to IMI and TRI herbicides.

Keywords:
chlorimuron-ethyl; metsulfuron-ethyl; acetolactate synthase; smooth pigweed

INTRODUCTION

Acetolactate-synthase inhibiting (ALS)-herbicides have been globally commercialized since the 1960’s (Garcia et al., 2017). More than 50 different molecules are classified in five chemical groups: imidazolinones (IMI), sulfonylureas (SUL), triazolopyrimidines (TRI), pyrimidinethiobenzoates (PIR) and sulfonylamides (ST). These products are used in low doses, have low toxicity to mammals, are broad spectrum herbicides and selective to many crops due to the high number of molecules available in the market (Tranel & Wright, 2002).

Specially involving target site mutations at ALS gene, the frequency of ALS-resistant individuals in a weed community is usually high compared to other mechanisms of action (Preston & Powles, 2002). Despite that, ALS-inhibitors are still important tool in burndown programs and in weed management systems including glyphosate-resistant species for major crops such as soybeans (Santos et al., 2016; Zobiole et al., 2018).

Although metabolism or reduced translocation may participate in non-target site mechanisms for ALS resistance in some weeds, target site mutations are certainly the most frequent mechanisms of resistance found in the nature (Murphy & Tranel, 2019). By now, eight amino acid positions in ALS gene may harbor nucleotide changes that reduce the affinity of ALS herbicides to the enzyme: Ala122, Pro197, Ala205, Arg376, Asn377, Trp574, Ser653, and Gly654. Among them, many amino acid substitutions can confer distinct levels and patterns of ALS resistance (see ALS mutation database, Tranel et al., 2020).

One of the most relevant weeds from Amaranthus genus is Amaranthus hybridus. It is a C4 broadleaf species that can grow as much as 120 cm and produces more than 250.000 seeds per plant growing without competition during the season as evaluated by Sellers et al. (2003) in Missouri. Hence, A. hybridus has demonstrated to be a strong competitor for resources with several crops, such as soybean, cotton, and sunflower (Carvalho et al., 2008; Soares et al., 2019). The concern about this species has increased in the last few years due to the reports of selection of resistant populations, specially to 5-enolpyruvylshikimate-3-phosphate (EPSPS)-, ALS-inhibitors, and synthetic auxins in Argentina (Dellaferrera et al., 2018; García et al., 2019; Perotti et al., 2019) or to EPSPS- and ALS- inhibitors in Brazil (Heap, 2021).

Currently, herbicide resistant A. hybridus populations are spread across cereal fields in southern Brazil, mainly in soybean areas in Rio Grande do Sul and in Campos Gerais region in Paraná. This study aimed to characterize an ALS-resistant A. hybridus population from Campos Gerais (Ventania, PR).

MATERIAL AND METHODS

Seeds from a putative resistant population were collected in a field located in Ventania (24°23’12” S, 50°09’27” W). The field has been cultivated with soybean in the summer and cereals (wheat or oat rotation) in the winter for more than ten years. Plants were collected and identified as A. hybridus running an identification key of Amaranthus species proposed by Senna (2015) and Milani et al. (2020). Seeds from different plants that survived to ALS-inhibitors applications were collected and bulked in paper bags and stored at room temperature. Resistant (R) and a known ALS-susceptible population (collected in Maringá, PR - 23°20’56” S, 52°04’26” W) were sown in 200-cell flats filled with potting soil (Horta3^®, MecPlant). Seedlings were transplanted into 1 L-pots containing the same soil and kept in a greenhouse under 10 mm daily irrigation and average temperature of 30 ℃ (day) / 20 ℃ (night); and 60% humidity.

Preliminary single-dose herbicide screenings were performed with post emergence applications of glyphosate at 960 g ae ha^-1 and chlorimuron-ethyl at 20 g ha^-1. Twenty-one days after treatment (DAT), 100% survival was observed for R population after chlorimuron application while all plants died after glyphosate application.

Dose-response experiments were conducted in two A. hybridus populations (resistant - R and susceptible - S) and the experiments were composed by seven herbicide doses and an untreated control (check). Four experiments of dose-response were performed, one for each ALS-inhibitor. For imazethapyr, cloransulam-methyl, and metsulfuron-ethyl, the doses were equivalent to ⅟₁₆ , ⅟₈, ¼, ½, 1, 2, and 4 times the labeled dose for S and ⅟₈, ¼, ½, 1, 2, 4, and 8 times the labeled dose for R. For chlorimuron, R and S plants were treated with ⅟₈, ¼, ½, 1, 2, 4, and 8 times the labeled dose. Labeled doses were chosen based on the recommendation of each herbicide (Rodrigues & Almeida, 2018), as follows: imazethapyr 106 g ha^-1, cloransulam 25.2 g ha^-1, chlorimuron 20 g ha^-1, and metsulfuron 2.4 g ha^-1.

Treatments were sprayed when plants had four fully expanded leaves (approximately 5 cm-tall). Each experimental unit (or replicate) was composed by 1L pot with three plants. The experiments were conducted in a completely randomized design with four replications. One experiment at the same conditions was conducted preliminarily to define a logical range of doses for each herbicide/population (data not shown).

Treatments were applied with a backpack sprayer and a hand boom equipped with three ST 110.015^® nozzle (TeeJet Technologies), delivering 150 L ha^-1 of solution and CO₂-pressurized at 30 psi. Treatments were applied when the air temperature was lower than 30 ℃, humidity higher than 60%, and wind speed under 3 km h^-1.

At 28 DAT, shoots were collected and kept in an oven at 65℃ for three days before biomass of each experimental unit had been quantified. Aboveground biomass data of each replication was expressed in percentage of untreated check for each population (Li et al., 2017; Schwartz-Lazaro et al., 2017). Data were submitted to non-linear regression analysis using the dcr package in R software (Ritz et al., 2015). A three-parameter log-logistic equation was fit:

$Y=\frac{a}{\left[1+{\left(\frac{x}{GR50}\right)}^b\right]}$

Where, Y is the relative biomass (%), a is the asymptote, x is herbicide dose, GR₅₀ is the dose to promote 50% of biomass reduction, and b is the sloop around GR₅₀. These parameters are illustrated in the Table 1. Resistant index (RI) was calculated by GR₅₀ R/GR₅₀ S ratio. GR₅₀ of both populations were compared by t-test (p > 0.05) to identify significant differences between R and S GR₅₀ parameters. The dose to provide efficient control (90%) was estimated through each model adjustment (Rana & Jhala, 2016).

Table 1:
Dose (g ha^-1) for 50% (GR₅₀) and 90% (GR₉₀) of biomass reduction, and resistant index (RI) of acetolactate-synthase-resistant (R) and susceptible (S) Amaranthus hybridus populations

Herbicide	Population	p-value	GR50	RI (R/S) ratio	GR90
Imazethapyr	R	<0.0001	9.7 (±2.6)	2 ns	73.9 (±27.8)
	S	<0.0001	4.7 (±1.1)		10 (±2.5)
Cloransulan-methyl	R	<0.0001	0.03 (±0.07)	0.05 ns	3.3 (±2.1)
	S	<0.0001	0.6 (±0.6)		1.7 (±0.3)
Chlorimuron-ethyl	R	<0.0001	43.5 (±15.2)	6.9 *	> 640
	S	<0.0001	6.3 (±1.4)		18.1 (±3.8)
Metsulfuron-ethyl	R	<0.0001	0.98 (±0.22)	6.5 ***	7.1 (±2.4)
	S	<0.0001	0.15 (±0.02)		0.4 (±0.1)

• Significant by t-test at ^*0.05, ^**0.01, and ^***0.001. ns: non-significant by t-test (p > 0.05).

RESULTS AND DISCUSSION

According to GR₅₀ values, R population demonstrated two times less sensitive to imazethapyr than S (Table 1 and Figure 1a). However, t-test comparing GR₅₀-R and -S was not significant (p-value=0.0833), not confirming resistance to imazethapyr. Besides, a dose of 73.9 g ha^-1 was sufficient to provide 90% control of R population, while the recommended dose is 106 g ha^-1 (Rodrigues & Almeida, 2018). R and S plants demonstrated similar response to cloransulam (Figure 1b). RI value lower than one for R population and the relatively low dose required to provide GR₉₀ (0.03 g ha^-1) evidentially illustrated sensibility to this herbicide (Table 1). Furthermore, the GR₅₀’s values were not different between R and S (p-value = 0.3871 in the t-test).

Figure 1:
Dose-response curves for acetolactate-synthase-resistant (R) and susceptible (S) Amaranthus hybridus populations. (a) imazethapyr, (b) cloransulam-methyl, (c) chlorimuron-ethyl, and (d) metsulfuron-ethyl.

For chlorimuron, GR₅₀ was 43.5 g ha^-1 for R and 6.3 g ha^-1 for S populations, respectively, resulting in an RI of 6.9 (Table 1). Doses higher than the highest dose sprayed in this experiment (640 g ha^-1) are needed to provide GR₉₀, suggesting that the recommended dose is no longer effective, which confirms the resistance to chlorimuron. (Figure 1c). Similar results were found for metsulfuron, another SUL herbicide (Table 1 and Figure 1d). GR₅₀ values resulted in an RI of 6.5, and the GR₉₀ was 7.1 g ha^-1, which is at least three times the labeled dose to control Amaranthus species (Rodrigues & Almeida, 2018).

Recently, ALS resistance in A. hybridus populations was broadly studied in the United States (Maertens et al., 2004; Whaley et al., 2006), Argentina (Larran et al., 2018; Dellaferra et al., 2018; García et al., 2019), and Italy (Milani et al., 2020). For the most populations already investigated, ALS resistance is often associated with cross-resistance to all chemical groups. Whaley et al. (2006) observed four A. hybridus populations resistant to IMI, SUL and PYR, and three of them were also resistant to TRI. Romagnoli et al. (2013) found several biotypes resistant to IMI, SUL, and TRI across central and northern Argentina. Differently, ALS-resistant A. hybridus from this research were considered resistant to SUL but susceptible to IMI and TRI (Table 1).

Herbicides from SUL group play important roles in cereal crop production due to their efficiency in weed control in modalities such as pre- and post-emergence in burndown applications (e.g. chlorimuron), post-emergence in wheat or oats (e.g. metsulfuron), and post-emergence in corn (e.g. nicosulfuron) (Oliveira Neto et al., 2019; Cholette et al., 2019). Therefore, the loss of SUL to control A. hybridus limits the options of herbicide treatments in these crops and crops and make difficult to plan weed control practices. The populations of Amaranthus palmeri resistant to glyphsoate and ALS inhibitors found in Brazil (Küpper et al., 2017) were introduced from Argentina (Alcántara-de-la-Cruz et al., 2020). Because the multiple resistance of A. hybridus to glyphosate and ALS inhibitors from Argentina is governed by multiple mutations in the target enzymes (triple mutation for high glyphosate resistance) (Larran et al., 2018, García et al., 2019), the risks that the populations of A. hybridus with these resistance profiles found in southern Brazil have also been introduced from Argentina is very great (Alcántara-de-la-Cruz et al., 2020).

Different cross-resistance profiles to ALS inhibitors are extremely dependent on the mechanism of resistance involved (Powles & Yu, 2010). Target site mutation from A. hibrydus biotypes was identified in four positions ALS gene so far. Ala122Thr conferred resistance only to IMI herbicides and susceptibility to other groups (Whaley et al., 2007). The remaining mutations documented (Asp376Glu, Trp574Leu, and Ser653Asn) endowed all chemical groups with simultaneous resistance (Whaley et al., 2006; Whaley et al., 2007). Typically, cross-resistance only to SUL herbicides is conferred by mutations at Pro197 (Tranel et al., 2020), which is probably the mechanism of resistance in this population. Further investigation on the mechanism of resistance must be conducted for the R population in future researches.

Once many glyphosate resistant A. hybridus biotypes have been reported in southern Brazil, including the Campos Gerais region (Penkcowski & Maschietto, 2019), understanding ALS resistance is one of the most important keys to implement efficient management strategies for A. hybridus control. This study identified a specific resistance pattern to ALS inhibitors in an A. hybridus population that is susceptible to glyphosate. Additional investigations are necessary in multiple resistant populations to identify resistance or susceptibility to SUL herbicides and other chemical groups of ALS inhibitors. Likewise, integrated weed management practices must be adopted to mitigate multiple resistance evolution, not only to A. hybridus, but also to other Amaranthus species present in Brazilian agricultural areas, such as A. retroflexus and A. palmeri (Francischini et al., 2014; Küpper et al., 2017). These practices should include mode of action rotation, pre-emergent applications, cultural control, mechanical control, and weed border control (Beckie & Harker, 2017).

CONCLUSION

The R A. hybridus population was resistant to ALS inhibitors herbicides chlorimuron-ethyl and metsulfuron-ethyl due to the RI calculated (6.5-fold and 6.9-fold, respectively) and also to the recommended rate for each herbicide no longer be enough to control R plants (GR₉₀ > 640 g ha^-1 for chlorimuron-ethyl and 7.2 g ha^-1 for metsulfuron-ethyl). On the other hand, this population is not resistance to other ALS inhibitors imazethapyr (imidazolinone) and cloransulam-methyl (triazolopyrimidine).

REFERENCES

• Alcántara-de-la-CruzROliveiraGMCarvalhoLBSilvaMFGF2020 Herbicide resistance in Brazil: Status, impacts, and future challenges. IntechOpen, http://dx.doi.org/10.5772/intechopen.91236.
  » https://doi.org/http://dx.doi.org/10.5772/intechopen.91236
• BeckieHJHarkerKN2017 Our top 10 herbicide-resistant weed management practices. Pest Management Science, 73:1045-1042
• CarvalhoSJPDLópez-OvejeroRFChristoffoletiPJ2008 Crescimento e desenvolvimento de cinco espécies de plantas daninhas do gênero Amaranthus. Bragantia, 67:317-326
• CholetteTBSoltaniNHookerDCRobinsonDESikkemaPH2019 Suppression of annual ryegrass in corn with nicosulfuron. Weed Technology, 33:173-177
• DellaferreraICortésEPanigoEDe PradoRChristoffoletiPPerretaPerretaMM2018 First report of Amaranthus hybridus with multiple resistance to 2,4-D, dicamba, and glyphosate. Agronomy, 10.3390/agronomy8080140.
  » https://doi.org/10.3390/agronomy8080140
• FrancischiniACConstantinJOliveira JrRSSantosGFranchiniLHMBiffeDF2014 Resistance of Amaranthus retroflexus to acetolactate synthase inhibitor herbicides in Brazil. Planta Daninha, 32:437-446
• GarciaMDNouwensALonhienneTGGuddatLW2017 Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families. Proceedings of the National Academy of Sciences, 114:1091-1100
• GarcíaMJPalma-BautistaCRojano-DelgadoAMBracamonteEPortugalJAlcántara-de la CruzRDe PradoR2019 The triple amino acid substitution TAP-IVS in the EPSPS gene confers high glyphosate resistance to the superweed Amaranthus hybridus. International Journal of Molecular Sciences, 20:2396
• HeapIM2021 International herbicide-resistant weeds database. Available at: <Available at: www.weedscience.org >. Accessed on: April 6th, 2021.
  » www.weedscience.org
• KüpperABorgatoEAPattersonELNettoAGNicolaiMCarvalhoSJChristoffoletiPJ2017 Multiple resistance to glyphosate and acetolactate synthase inhibitors in Palmer amaranth (Amaranthus palmeri) identified in Brazil. Weed Science, 65:317-326
• LarranASLorenzettiFTuescaDPerottiVEPermingeatHR2018 Molecular mechanisms endowing cross-resistance to ALS-inhibiting herbicides in Amaranthus hybridus from Argentina. Plant Molecular Biology Reporter, 36:907-912
• LiJLiMGaoXFangF2017 A novel amino acid substitution Trp574Arg in acetolactate synthase (ALS) confers broad resistance to ALS‐inhibiting herbicides in crabgrass (Digitaria sanguinalis). Pest Management Science , 73:2538-2543
• MaertensKDSpragueCLTranelPJHinesRA2004 Amaranthus hybridus populations resistant to triazine and acetolactate synthase‐inhibiting herbicides. Weed Research, 44:21-26
• MilaniAScarabelLSattinM2020 A family affair: resistance mechanism and alternative control of three Amaranthus species resistant to acetolactate synthase inhibitors in Italy. Pest Management Science , 76:1205-1213
• MurphyBPTranelPJ2019 Target-site mutations conferring herbicide resistance. Plants, 8:10
• Oliveira NetoAMConstantinJOliveira JúniorRSGuerraNBlainskiEAlmeida DanH2019 Management of Sumatran fleabane after maize harvest in the fallow period shorter than 60 days. Communications in Plant Sciences, 9:53-58
• PenkcowskiLHMaschiettoEHG2019 Suspeita de Amaranthus hybridus resistente a glyphosate no Paraná. Available at: <Available at: https://maissoja.com.br/suspeita-de-amaranthus-hybridus-resistente-ao-herbicida-glyphosate-no-parana/ >. Accessed on: October 2nd, 2020.
  » https://maissoja.com.br/suspeita-de-amaranthus-hybridus-resistente-ao-herbicida-glyphosate-no-parana/
• PerottiVELarranASPalmieriVEMartinattoAKAlvarezCETuescaDPermingeatHR2019 A novel triple amino acid substitution in the EPSPS found in a high‐level glyphosate‐resistant Amaranthus hybridus population from Argentina. Pest management science, 75:1242-1251
• PowlesSBYuQ2010 Evolution in action: plants resistant to herbicides. Annual Review of Plant Biology, 61:317-347
• PrestonCPowlesSB2002 Evolution of herbicide resistance in weeds: initial frequency of target site-based resistance to acetolactate synthase-inhibiting herbicides in Lolium rigidum. Heredity, 88:08-13
• RanaNJhalaAJ2016 Confirmation of glyphosate-and acetolactate synthase (ALS)-inhibitor-resistant kochia (Kochia scoparia) in Nebraska. Journal of Agricultural Sciences, 8:54-62
• RitzCBatyFStreibigJCGerhardD2015 Dose-response analysis using R. PloS ONE, 10:e0146021
• RodriguesBNAlmeidaFS2018 Guia de herbicidas. 7ª ed. Londrina, Independent Production. 764p
• RomagnoliMVTuescaDPermingeatHR2013 Characterization of Amaranthus quitensis resistance to three families of herbicides. Ecología Austral, 23:119-125
• SantosTTMTimossiPCLimaSFGonçalvesDCSantanaMV2016 Associação dos herbicidas diclosulam e glyphosate na dessecação visando o controle residual de plantas daninhas na cultura da soja. Revista Brasileira de Herbicidas, 15:138-147
• Schwartz-LazaroLMNorsworthyJKScottRCBarberLT2017 Resistance of two Arkansas Palmer amaranth populations to multiple herbicide sites of action. Crop Protection, 96:158-163
• SellersBASmedaRJJohnsonWGKendigJAEllersieckMR2003 Comparative growth of six Amaranthus species in Missouri. Weed Science , 51:329-333
• SennaLR2015 Identificação de espécies de plantas daninhas do gênero Amaranthus L. (Amaranthaceae Juss.) no Brasil. In: Inoue MH, Oliveira Jr RS, Mendes KF & Constantin J (Eds.) Manejo de Amaranthus. São Carlos, RiMa Editora. p. 01-20
• SoaresMMFreitasCDMOliveiraFSMesquitaHCSilvaTSSilvaDV2019 Efeitos da competição e do déficit hídrico sobre o crescimento de girassol e plantas daninhas. Revista Caatinga, 32:318-328
• TranelPJWrightTR2002 Resistance of weeds to ALS-inhibiting herbicides: what have we learned? Weed Science , 50:700-712
• TranelPJWrightTRHeapIM2020 Mutations in herbicide-resistant weeds to ALS inhibitors. Available at: <Available at: http://www.weedscience.com/ >. Accessed on: October 5th, 2020.
  » http://www.weedscience.com/
• WhaleyCMWilsonHPWestwoodJH2006 ALS resistance in several smooth pigweed (Amaranthus hybridus) biotypes. Weed Science , 54:828-832
• WhaleyCMWilsonHPWestwoodJH2007 A new mutation in plant Als confers resistance to five classes of Als-inhibiting herbicides. Weed Science , 55:83-90
• ZobioleLHSKrenchinskiFHPereiraGRRampazzoPERubinRSLucioFR2018 Management programs to control Conyza spp. in pre-soybean sowing applications. Planta Daninha , 36:10

Publication Dates

• Publication in this collection
  13 June 2022
• Date of issue
  May-Jun 2022

History

• Received
  29 Oct 2020
• Accepted
  18 June 2021