Target-site is the main mechanism of resistance to ALS -inhibitor herbicides in a rice flatsedge population from Southern Brazil

: Background: Overuse of ALS-inhibiting herbicides in rice paddy fields has resulted in the selection of Cyperus iria L. (rice flatsedge) with a high level of resistance to this herbicide group. Objective: This study aimed to identify mutations endowing ALS resistance (target-site resistance) and the involvement of a metabolic-mediated resistance mechanism of C. iria to ALS herbicides. Methods: Dose-response experiments were performed to estimate GR 50 values (GR 50 denotes the rate at with a herbicide reduces growth by 50%). Experiments I and II were conducted in a greenhouse, with a factorial arrangement comprised of two C. iria populations, CYPIR-S and CYPIR-R (with and without malathion treatment), which were susceptible and resistant to ALS-inhibiting herbicides, respectively; ALS-inhibiting herbicides (bispyribac-sodium, imazapyr + imazapic, penoxsulam, and pyrazosulfuron-ethyl); and herbicide doses above and below the maximum field rates. Genomic DNA was extracted from CYPIR-S and CYPIR-R for partial sequencing of the ALS gene. Results: The GR 50 values of CYPIR-R were 400 to > 4,000 times higher than those of CYPIR-S, indicating a high level of resistance to all herbicides evaluated. Tests with bispyribac-sodium plus malathion showed that metabolism might be involved. However, dose-response curves demonstrated that a specific mutation in the ALS gene is the main resistance mechanism. DNA sequencing electropherogram analysis for CYPIR-R showed different nucleotide changes at Trp 574 in the first [thymine (T) to adenine (A)] and second bases [guanine (G) to T], which can result in changes from tryptophan (TGG) to arginine (AGG), to leucine (TTG), and/or to methionine (ATG), respectively. Conclusions: Target-site


Introduction
Rice flatsedge (Cyperus iria L.) is an annual plant with a short cycle, tolerant to flooding (Mann et al., 2007), and can coexist with rice crops throughout the growing period (Nunes et al., 2018).This weed is a polyploid species, and some cytotypes have 14x (tetradecaploid) and 16x (hexadecaploid) genomes with n = 56, and n = 64, respectively (Bir et al., 1992).Its polyploid genome causes complex effects of the enlarged copies of each genome, which increases genetic diversity and versatility in many environmental conditions (Fox et al., 2020).Sedges can cause reduction of up to 90% in rice yields (Agostinetto et al., 2016).Among them, C. iria is one of the main troublesome species (Ulguim et al., 2019), requiring control measures.
Acetolactate synthase (ALS) inhibitors are the main herbicides used to control sedges in paddy fields (Schaedler et al., 2013), especially after the introduction of the Clearfield ® Production System.ALS is the crucial enzyme in the biosynthetic pathway of essential branched-chain amino acids (isoleucine, valine, and leucine), and its inhibition by herbicides causes mortality of most sensitive plants (Duggleby et al., 2008).However, the selection pressure imposed by the constant use of herbicides inhibiting the same target site is decisive in the evolution of herbicide resistance (Norsworthy et al., 2012).This begins with the survival of individuals within a population that are not killed by the recommended rate of the herbicides.
ALS is vulnerable to single nucleotide polymorphisms (SNPs), in which a single nucleotide change resulting in a non-synonymous alteration can cause amino acid substitution, altering the enzyme's structure and preventing herbicide binding (Ntoanidou et al., 2016).Apart from target-site point mutations, which can lead to cross-resistance, Non-Target-Site Resistance (NTSR), along with accelerated metabolism, is also a prominent mechanism related to herbicide resistance (Riar et al., 2012;Iwakami et al., 2013;Busi et al., 2017).Cytochrome P 450 monooxygenase proteins (P 450 ) can metabolize ALS herbicides, reducing the amount of herbicide reaching the site of action (Iwakami et al., 2013).When this NTSR is involved, the resistance factor that controls the resistant biotype is lower (< 10-fold) than some mutations at ALS target sites that confer high levels of resistance (Heap, Duke, 2017).Thus, in vivo assays are required to determine the dose to control (C 50 ) or cause growth reduction (GR 50 ) by 50% in relation to the resistant and sensitive biotypes (Yu, Powles, 2014).
TSR, and in particular a Trp -574-Leu mutation in the ALS gene, has previously been identified as the mechanism conferring high cross-resistance to ALSinhibiting herbicides in C. iria in the middle-southern USA (Riar et al., 2015).In addition, no evidence of enhanced metabolism was found.In another study, a precursor to the current one, Chiapinotto et al. (2017) also reported high cross-resistance in a C. iria population, indicating the presence of a possible mutation in the ALS gene.However, mutations in some species of the Cyperaceae family are not easily identifiable, requiring sequencing analysis (Scarabel et al., 2010;Tehranchian et al., 2014).Furthermore, TSR and NTSR mechanisms can sometimes coexist (Brosnan et al., 2016), in which case it is essential to implement alternative management strategies.This study aimed: 1) to evaluate herbicide metabolism as a mechanism of resistance to ALS inhibitors in a C. iria population in southern Brazil with confirmed resistance to this herbicide group; and 2) to partially sequence the ALS gene, to further understand the underlying mechanisms of C. iria resistance to ALS inhibitors.
In October 2018, CYPIR-R and CYPIR-S seeds were placed in 0.05 L plastic pots containing a commercial substrate for germination.When the seedlings were at the one-leaf stage, they were transplanted into 0.5 L pots (two plants per pot) containing sieved soil classified as "haplic planosol" (identified as a silty loam soil type according to texture).When the plants reached 3-4 leaves, herbicides plants were cut close to the ground and placed in a forcedair oven at 60 °C until they reached a constant weight to determine the shoot dry weight (SDW).

ALS Gene Sequencing
Genomic DNA (gDNA) was extracted from 100 mg of leaf tissue from six plants of CYPIR-R and one plant of CYPIR-S, using the Wizard Genomic DNA Purification Kit (Promega)measured by DeNovix (Wilmington, Delaware).Polymerase Chain Reactions (PCR) were performed in 25.0 µL samples containing 2.0 µL of template DNA (approximately 40 ng), 5.0 µL of 5X GoTaq buffer, 0.2 µL of 5 U µL -1 GoTaq G2 Hot Start Polymerase (Promega), 0.5 µL of 10 mM dNTPs, 0.5 µL of each forward and reverse primer at 10 µM, 1.5 µL of 25 mM MgCl 2 , and 14.8 µL of nuclease-free water.The PCR cycling was performed with a T100 Thermal Cycler (Bio-Rad) and was composed of an initial denaturing step of 95 °C for 2 min, followed of 40 cycles at 95 °C for 30 s, 53 °C for 30 s, and 72 °C for 30 s, with a final extension step at 72 °C for 5 min.Amplified fragment size was verified on 1% agarose gel using a 100 base pairs (bp) molecular weight (ladder, Sinapse).
were applied.Malathion treatment (1,000 g a.i.ha -1 ) was applied 30 minutes before the herbicides (Zhang et al., 2017).All chemical treatments were applied with a CO 2 pressurized sprayer equipped with flat fan nozzles (XR 110.015 model, 0.5 m apart).The application was performed at 250 kPa working pressure, delivering 150 L ha -1 of carrier volume.Nozzles were positioned at 0.5 m from the top of the leaves.During application the average temperature was 18.7 °C, relative humidity (RH) was 68.2%, and wind speed was 2.52 km h -1 (monitored with a digital Kestrel ® 4500 Weather Meter).
In February 2019, the experiment was repeated, following the same factorial scheme and procedures.In order to obtain a better fit of the dose-response curves, and mainly the GR 50 value of the herbicide imazapyr + imazapic, herbicide doses were adjusted for the CYPIR-R to 0, 1,2,4,8,16,32,64,128 and 256x for all herbicides.The average temperature was 20.5 °C, RH was 82.3%, and average wind speed was 5.04 Km h -1 .
In each experiment, a visual evaluation of control was performed 28 days after treatment (DAT), based on a percentage scale, where 0% indicated no effect and 100% denoted total weed death (data not shown).The remaining Table 2 -Estimated parameters b, d, GR 50 (g a.i.ha -1 ) and resistance factor (RF) 1 by the non-linear mixed-effect equation2 , based on shoot dry weight (% of the untreated control), for resistant (CYPIR-R with and without malathion treatment) and sensitive (CYPIR-S with and without malathion treatment) Cyperus iria biotype, 28 days after herbicides treatment between-assay variability using the metadrm function, with the arguments cid2 (as a factor identifying individual curves) and ind (as a factor containing the between-curve grouping of the data).
Y = 1 + exp {b(log(x) -log(e))} d (Equation 1) where Y is the SDW reduction (%); e is the GR 50 value (the dose that causes 50% SDW reduction); d is the upper limit; and b indicates the slope around e (Ritz et al., 2019).
The metadrm (using drm and summary functions) estimated the parameters and their standard errors, where p-values (p < 0.05) indicated significance of the parameters.GR 50 and the resistance factors (RF = GR 50 CYPIR-R/ GR 50 CYPIR-S) were estimated by the ED and EDcomp functions (95% confidence intervals).We fit a joint model based on the entire dataset including both experiments to create the figures and to test the lack of fit of the model (when not significant, the regression analysis was accepted as describing the variation of data similar to ANOVA).
In the absence of herbicides, treatment with malathion alone did not reduce the SDW compared to the untreated control, either for the CYPIR-R or CYPIR-S biotypes (data not shown), which is consistent with the results of Zhao et al. (2017).The GR 50 values for CYPIR-S + malathion decreased only for imazapyr + imazapic in The previously published forward F22 (TGTTCCAACTCAGGTGCAGA) and reverse R22 (ATCATATCCTTGAATGCTCCTC) primers (Riar et al., 2015) were used for amplification of a 1,055 bp ALS fragment (Figure 1).PCR products were purified (ExoSAP-IT™ Express reagent -Thermo Fisher), 10X diluted, and 1 µL was used in the sequencing reactions.These reactions were performed with the BigDye cycle sequencing terminator kit (Thermo Fisher), and sequencing was performed with an Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher).The partial ALS sequences of CYPIR-S and CYPIR-R were uploaded to the GenBank of the National Center for Biotechnology Information (NCBI) under accession numbers OP503617 and OP503618, respectively.All sequence electropherograms were analyzed and aligned with the gene sequences encoding the ALS enzyme of Arabidopsis thaliana L. (X51514) and Cyperus brevifolius (Rottb.)(AB719979.1),using the open-source bioinformatics software UGENE (Unipro, 2012).
A sequence of 966 bp was amplified in resistant and sensitive samples.All seven samples from CYPIR-R and CYPIR-S plants were in alignment with the gene encoding the ALS enzyme in A. thaliana, indicating that sequencing covered the amino acids (Aas) from 390 to 599, where the domains β (Aas 281-451) and γ (Aas 463-639) are partially found (McCourt et al., 2006).Within these domains is a conserved region named B [Aas 573-576], covering the position Trp 574 , where mutations related to resistance to ALS-inhibiting herbicides occur (Riar et al., 2015).

Statistical Analysis
The dose-response SDW data are reported as percentage in relation to the SDW of the untreated control, and were evaluated using the R program (R Core Team, 2019).The medrc and drc packages were used to create dose-response curves with the non-linear mixedeffects model, assuming a three-parameter log-logistic equation with a separate set of b, d, and e parameters for each of the biotypes (Equation 1).Individual assay effects were included on these parameters to model the ALS gene ALS protein comparison with CYPIR-S (Figure 2b).Similar results were obtained by Owen et al. (2012).Furthermore, malathion reduced the dose for CYPIR-R control by bispyribac-sodium.The GR 50 value for CYPIR-R was 117 g a.i.ha -1 , while the value for CYPIR-R + malathion was 51 g a.i.ha -1 (Table 2; Figure 2a).The dose-response curve indicated a high and broad cross-resistance of CYPIR-R to ALS-inhibiting herbicides belonging to the chemical groups of SU, IMI, triazolopyrimidine (TP -type 2), and PB.Similar results were reported by Chiapinotto et al. (2017).Malathion was used as an indicator to detect metabolic resistance in the resistant biotype (Riar et al., 2012) and may or may not have an effect on the sensitive biotype (Owen et al., 2012;Zhao et al., 2017).Malathion is an organophosphate insecticide used as an indicator of enhanced metabolism in NTSR (Wang et al., 2018), since it inhibits P 450 activity (Riar et al., 2012;Iwakami et al., 2013).Malathion's chemical structure has a phosphorus atom covalently linked to sulfur or oxygen, and reduces herbicide doses due to competition in P 450 -mediated reactions and oxidative desulfurization (Busi et al., 2017).
Adding malathion to bispyribac-sodium, imazethapyr and penoxsulam in Echinochloa crus-galli increased the efficiency of the control of the resistant biotypes (Riar et al., 2012).In Echinochloa glabrescens, pretreatment with malathion also reduced the control dose of the penoxsulam-resistant biotype (Yan et al., 2019).In the present study, malathion reduced the control dose of bispyribac-sodium, indicating that its metabolism might be involved.However, studies are needed to assess whether there is overexpression of the transcription of genes coding for P 450 enzymes and/or the identification of herbicide metabolites (Yasuor et al., 2009;Iwakami et al., 2013).The absence of plant growth reduction with application of the other herbicides plus malathion could have been due to the presence of P 450 isozymes (Riar et al., 2015).However, when only enhanced metabolism is involved, the resistance factor for controlling the resistant population is lower (<10fold) than some mutations at the ALS target site that confer high levels of resistance (Heap, Duke, 2017).

ALS gene sequencing
Alterations in the Trp 574 position were detected in three samples of the CYPIR-R biotype (Figure 3).DNA electropherogram analysis of the three samples of CYPIR-R showed the presence of double peaks at position Trp 574 .This overlap demonstrates changes in the first [thymine (T) to adenine (A)] and/or second nucleotide bases [guanine (G) to T].Thus, different nucleotide changes are present at position 574 of the ALS gene and can result in changes from tryptophan (TGG) to arginine (AGG), to leucine (TTG), and/or to methionine (ATG) -Trp -574-Arg, Trp -574- Leu, and Trp -574-Met, respectively (Figure 4).However, this study did not distinguish whether the mutation found was an accumulation of the single nucleotide substitution codons of AGG and TTG, or an accumulation of the double nucleotide substitution codon ATG in the wild-type codon TGG. C. iria may be a hexadecaploid species (Bir et al., 1992).Thus, even using cloning techniques (Lamego et al., 2009), precise determination of allelic variants that endow resistance is very difficult.
High doses of ALS-inhibiting herbicides applied in rice fields tend to select weed plants with mechanisms that confer elevated resistance (Ulguim et al., 2019).High cross-resistance (> 10-fold), like the CYPIR-R, is a result of mutations at the site of action (TSR) (Yu, Powles, 2014).The herbicides do not bind directly to the active site of the ALS enzyme, but are able to block access to the substrate (Duggleby et al., 2008).The substitution of amino acids in conserved regions maintains the enzyme's functionality without pleiotropic effects but compromises the binding of herbicides, conferring high cross-resistance (Yu, Powles, 2014).
In some species of the Cyperaceae family, including the Cyperus genus, mutations are not easily identifiable due to the level of ploidy, the occurrence of additional genes, or the presence of alleles (Scarabel et al., 2010;Tehranchian et al., 2014;Riar et al., 2015).In C. esculentus, no mutation was identified in DNA sequencing (Tehranchian et al., 2014).However, in the electropherogram analysis, double peaks were observed at the second base of codon Trp 574 , indicating a change from G to T, changing tryptophan to leucine.The results showed heterozygosity or gene duplication (Tehranchian et al., 2014).Similar results were found in polyploid Echinochloa crus-galli (Panozzo et al., 2013), Poa annua (McElroy et al., 2013) and Alopecurus japonicus (Zhao et al., 2019).Thus, at the position of the mutant nucleotides, there are double peaks in the resistant biotype but single ones in the sensitive biotype.Double peaks were observed in the first and second nucleotide bases of CYPIR-R.A similar result was found in glyphosate-resistant polyploid E. colona plants with double nucleotide peaks at the EPSPS gene, indicating that mutations can occur (e.g., Pro -106-Leu, Pro -106-Thr and Pro -106- Ile) (Han et al., 2016).Considering the complexity of the C. iria genome, reported as a polyploid species with biotypes showing 14x and 16x genomes with n = 56, and n = 64 (Bir et al., 1992), that variation in chromosome number may be associated with the differences among biotypes, intraspecific variation (Bir et al., 1992), and natural hybridization among C. iria and other Cyperus species (Chozin, Yasuda, 1991).Also, many Cyperus species have been reported to have cross-pollination reproduction (Ihenetu et al., 2021) and agmatoploidy that occurs in species that have so-called holocentric chromosomes in which chromosomal breaks are recurrent (Arias et al., 2011).The C. iria genome has not been sequenced yet.Thus, there is no information related to copy number of the ALS gene in the genome.Its polyploid genome leads to the complex effects of the enlarged copies of each genome, which improves genetic diversity and versatility in many environmental conditions (Fox et al., 2020).
Polyploid species can accumulate more than one mutation at the same position.In Capsella bursa-pastoris (L.) Medik., two mutations at position 197 (Pro -197-Ser and Pro -197-His) were identified (Wang et al., 2019).According to the level of ploidy, certain species have a variable number of ALS genes and may have mutations in different positions.In Schoenoplectus mucronatus, three genes encoding ALS were identified.Mutations that confer resistance were related to ALS1, with alterations in Pro -197-His and Trp -574-Leu (Scarabel et al., 2010).Similar results were also found in S. juncoides, with two genes coding for ALS, each responsible for a mutation associated with resistance (Yamato et al., 2013).Thus, it is not possible to rule out the presence of mutations in positions not covered by the CYPIR-R sequencing, corroborating the results found in this study.

Conclusions
TSR was involved in the high and broad cross-resistance of the Cyperus iria population, due to nucleotide changes that can result in an amino acid substitutions at the Trp 574 of ALS gene.This is the first report of the resistance mechanism to ALS-inhibiting herbicides in a C. iria population occurring in paddy rice fields in Brazil.Practices such as reducing selection pressure, using herbicides with different mechanisms of action or herbicide mixtures, as well as crop rotation are recommended for management of the species.

Figure 1 -
Figure 1 -Strategy for partial sequencing of the coding gene for the ALS enzyme, with forward (F22) and reverse (R22) primers used to amplify and sequence the enzyme in Cyperus iria L. biotypes sensitive and resistant to ALS-inhibiting herbicides.Scheme adapted from Riar et al. (2015)

Figure 2 -
Figure 2 -Average of observed and adjusted values of shoot dry weight (% relative to untreated control) of resistant (with and without malathion pretreatment -∆ CYPIR-R + Malathion and • CYPIR-R, respectively) and sensitive (with and without malathion pretreatment -+ CYPIR-S and x CYPIR-S + Malathion, respectively) Cyperus iria L. biotypes, at 28 days after treatment with bispyribac-sodium (a), imazapyr + imazapic (b), penoxsulam (c) and pyrazosulfuron-ethyl (d) -by the joint model based on the entire dataset.The horizontal bar represents the 95% confidence interval to obtain 50% shoot dry weight reduction (GR 50 )

Figure 4 -
Figure 4 -Resistant and sensitive Cyperus iria L DNA electropherogram for the ALS gene.The overlapping of peaks in CYPIR-R at position Trp 574 shows changes in the first (thymine to adenine) and in the second (guanine to thymine) nucleotide base, that is, changing tryptophan (TGG) to leucine (TTG), to arginine (AGG), and/or to methionine (ATG)