Trp2027Cys mutation evolves in Digitaria insularis with cross-resistance to ACCase inhibitors

Journal Pre-proof Trp2027Cys mutation evolves in Digitaria insularis with crossresistance to ACCase inhibitors

Hudson K. Takano, Marcel S.C. Melo, Ramiro F.L. Ovejero, Philip H. Westra, Todd A. Gaines, Franck E. Dayan PII:

S0048-3575(19)30529-2

DOI:

https://doi.org/10.1016/j.pestbp.2019.12.011

Reference:

YPEST 4516

To appear in:

Pesticide Biochemistry and Physiology

Received date:

18 November 2019

Revised date:

23 December 2019

Accepted date:

27 December 2019

Please cite this article as: H.K. Takano, M.S.C. Melo, R.F.L. Ovejero, et al., Trp2027Cys mutation evolves in Digitaria insularis with cross-resistance to ACCase inhibitors, Pesticide Biochemistry and Physiology (2019), https://doi.org/10.1016/ j.pestbp.2019.12.011

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© 2019 Published by Elsevier.

Journal Pre-proof Trp2027Cys mutation evolves in Digitaria insularis with cross-resistance to ACCase inhibitors

Running title: target-site mutation in Digitaria insularis

Hudson K Takanoa , Marcel SC Melob, Ramiro FL Ovejerob, Philip H Westraa , Todd A Gaines a , and

Department of Bioagricultural Sciences and Pest Management, Colorado State University, 300

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Franck E Dayana *

Bayer CropScience, 12901 Nações Unidas Ave., São Paulo, SP 04578-000, Brazil.

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W Pitkin St., Fort Collins, CO 80523, USA.

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*Corresponding author: Franck E. Dayan, [email protected] Author email addresses:

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[email protected]; [email protected]; [email protected];

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[email protected]; [email protected]

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Journal Pre-proof ABSTRACT Sourgrass (Digitaria insularis) is one of the most problematic weeds in South America because glyphosate resistance is widespread across most crop production regions. Acetyl coenzyme A carboxylase (ACCase)-inhibiting herbicides have been intensively used to manage D. insularis, which substantially increased selection pressure for this class of herbicides. We confirmed

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resistance to ACCase herbicides in a D. insularis population from Brazil and characterized its molecular basis. Resistant plants showed high level of resistance to haloxyfop (resistance factor,

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RF=613-fold), low level of resistance to pinoxaden (RF=3.6-fold), and no resistance to

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clethodim. A target-site mutation, Trp2027Cys, was found in the ACCase sequence from

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resistant plants. A protein homology model shows that the Trp2027Cys mutation is near the

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herbicide-binding pocket formed between two ACCase chains, and is predicted to obstruct the access of aryloxyphenoxypropionates (FOP) herbicides to the binding site. A qPCR-based single

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nucleotide polymorphism genotyping method was validated to discriminate susceptible (wild-

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type Trp2027) and resistant (mutant Cys2027) alleles. All resistant plants were homozygous for

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the mutation and the assay could be used for early detection of resistance in D. insularis field samples with suspected resistance to ACCase inhibitors. Keywords: target-site resistance; protein homology modeling; aryloxyphenoxypropionates, cyclohexanediones, and phenylpyrazoline.

1 INTRODUCTION Digitaria insularis (L.) Fedde (common name: sourgrass) is one of the major grass weeds of annual and perennial crops in South America (Lopez Ovejero et al., 2017). It belongs to the

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Journal Pre-proof Poaceae family with C4 photosynthetic metabolism, although seedlings typically have a slow growth rate (Kissman and Groth, 1999; Machado et al., 2006). D. insularis plants can reproduce either asexually by rhizome formation or sexually, producing up to 40,000 hairy seeds plant-1 year-1 that can be dispersed by animals, insects, and wind (Gemelli et al., 2012). Once dispersed, these seeds can germinate under a wide range of temperature and light intensity,

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contributing to the species dissemination (Mendonça et al., 2014). When D. insularis plants form clumps in the field, they become strong competitors for resources and their management

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is even more complex due to the formation of rhizomes (Zobiole et al., 2016). Yield losses due

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to D. insularis interference can reach up to 80% with 8 plants m-2 in soybean (Braz et al., 2019;

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Gazziero et al., 2019).

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Most D. insularis populations infesting annual crops in South America are glyphosate resistant (GR) (Lopez Ovejero et al., 2017). The first case of GR in this species was reported in

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Paraguay in 2005 (Heap, 2019). Since then, GR populations spread to other countries such as

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Brazil and Argentina through either seed dispersion (e.g., machinery transportation and wind)

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or independent selection in response to intense glyphosate usage (Takano et al., 2018). This is due to the fact that more than 90% of total soybean area in South America is planted with transgenic GR soybean varieties and glyphosate is applied at least three times a year (Peterson et al., 2018). As an alternative postemergence site of action, Group A/1 or acetyl-CoA carboxylase (ACCase)-inhibiting herbicides have been extensively used to control GR D. insularis in South American soybean fields (Gemelli et al., 2012; Takano et al., 2020). Three groups of ACCase inhibitors are available in the market: aryloxyphenoxypropionates (FOPs), cyclohexanediones

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Journal Pre-proof (DIMs), and phenylpyrazoline (DEN) (Dayan et al., 2019). These herbicides inhibit fatty acid biosynthesis in plants and cause the death of monocot weeds due to the presence of a sensitive eukaryotic ACCase version, and crop selectivity is probably the reason why these herbicides are well-adopted by farmers (Dayan et al., 2019; Takano et al., 2020). Interestingly, ACCaseinhibiting herbicides are used in tank-mix with glyphosate due to increased weed control and

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spectrum compared to the products applied individually, even on GR populations (Gemelli et al., 2013).

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To date, 48 species have evolved resistance to ACCase inhibitors (Heap, 2019). The

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enzyme is comprised of three functional domains: biotin-carboxyl carrier protein (BCCP), biotin

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carboxylase (BC), and carboxyltransferase (CT) (Ohlrogge and Browse, 1995). In most cases,

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point mutations in one of these domains can confer resistance to these herbicides. Residues Ile1781, Trp1999, Trp2027, Ile2041, Asp2078, Cys2088 and Gly2096, present in the CT domain

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of the plastidic ACCase gene, have been associated with cross-resistance to ACCase herbicides

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in several grass weed species (Beckie and Tardif, 2012; Délye, 2005; Kaundun, 2014; Liu et al.,

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2007; Powles and Yu, 2010; Takano et al., 2020). Some mutations cause resistance only to FOPs whereas others can cause resistance to all classes (FOP, DIM and DEN). In addition to target-site mutations, enhanced herbicide metabolism can sometimes confer resistance to ACCase inhibitors, especially for FOPs (Délye et al., 2010; Han et al., 2016; Iwakami et al., 2019; Yu et al., 2013). We investigated cross-resistance to ACCase-inhibiting herbicides evolved in a D. insularis population from Brazil. We focused on target-site mutations as they are usually involved in the

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Journal Pre-proof mechanism of resistance to ACCase inhibitors. A molecular marker for early detection of resistance is also provided in this research.

2 MATERIAL AND METHODS 2.1 Plant Material and Growth

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Seeds from a D. insularis population with putative resistance to ACCase inhibitors were collected from a soybean-corn-cotton rotation field located in Primavera do Leste, Mato Grosso

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State, Brazil (15.52 °S and 54.40 °W). ACCase-inhibiting herbicides have been sprayed

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continuously in this field for selective postemergence control of grasses in soybean and cotton

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until they were replaced by glyphosate after transgenic GR crops were launched. Consequently,

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the same D. insularis population had been confirmed as GR in previous research (Lopez Ovejero et al., 2017). In the past years, ACCase herbicides have been intensively used once again with at

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least two applications per year. A known susceptible population was collected from Paulínia,

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São Paulo State, Brazil (22.69 °S and 47.14 °W), with no usage history of ACCase-inhibiting

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herbicides. Seeds were germinated in 0.3 dm3 volume-pots filled with soil (SunGro Horticulture, Agawam, MA 01001) and one plant per pot was grown until the four-leaf growth stage in the greenhouse. Environmental conditions were set up for 25/20 °C day/night, 75% relative humidity, and 12 h light day−1 with natural sunlight or 500 μmol m−1 s −1 PAR for cloudy days. 2.2 Whole-plant dose-response and cross-resistance characterization Three separate experiments were conducted twice in a 2 x 8 factorial design with three replications. Factor A consisted of two populations (susceptible and putative resistant), whereas Factor B consisted of eight herbicide doses corresponding to 0, 1/8, 1/4, 1/2, 1, 2, 4,

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Journal Pre-proof and 8 times the recommended field rate. Each experiment included a different herbicide: clethodim (1X = 108 g ai ha -1), haloxyfop (1X = 60 g ai ha -1), or pinoxaden (1X = 60 g ai ha -1). All treatments included 0.5% (v/v) methylated seed oil. Plants were sprayed with a commercial chamber track sprayer (DeVries Manufacturing, Inc., Hollandale, MN 56045) equipped with an 8002EVS single even, flat-fan nozzle (TeeJet; Spraying Systems Co., Denver, CO 80207)

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calibrated to deliver 187 L ha −1 spray solution at the level of the plant canopy. Control was visually assessed at 21 days after treatment using a scale in which 0% corresponds to no injury

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and 100% to plant death (Burgos et al., 2013). Model assumptions were satisfied, and data

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were subjected to ANOVA. A four-parameter logistic regression model was fit to the data and

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LD50 (Lethal Dose to cause 50% injury) values were generated to calculate the resistance factor

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(RF = LD50 resistant/LD50 susceptible) (Burgos et al., 2013; Ritz et al., 2015). Graphs were generated with Prism 8 software (GraphPad, San Diego, CA 92108).

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In a separate experiment, different herbicides from the three groups (FOP, DIM and

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DEN) were tested to characterize cross-resistance or susceptibility patterns in each biotype

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(susceptible and putative resistant). Plant growth, spraying conditions, and plant response evaluation were identical to the dose-response experiments described above. Herbicide treatments were tested at the recommended field rate (Table 1) in three replications per biotype and the experiment was repeated. Normality and homogeneity of variances were checked. Data from the two experiment replications were pooled, means were subjected to ANOVA and compared by Tukey test (p<0.05). 2.3 ACCase gene sequencing

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Journal Pre-proof Total genomic DNA was extracted from 30 resistant plants and 15 susceptible plants. Leaf tissue (50 mg) was ground with liquid nitrogen and DNA was extracted with DNeasy plant mini kit (Qiagen, Germantown, MD 20874). Total DNA was quantified using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA USA 02451). Two pairs of universal ACCase primers previously reported (Délye et al., 2011) were used to amplify ACCase gene

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sequence in D. insularis: ACCase F1 (5’-CAGCITGATTCCCAIGAGCGITC-3’), ACCase R1 (5’CTCTCAGCATAGCACTCGATGCGATCTGGGTTTATCTTGATA-3’), ACCase F2 (5’-

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CAGCCTGATTCCCACGAGCGGTCTGTTCCTCGTCCAGGGCAAGTTTG-3’), and ACCase R2 (5’-

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CCATGCAITCTTIGAGITCCTCTGA-3’). PCR was performed in 25 L final volume using Econotaq

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Plus Green 2X (Lucigen, Middleton, WI 53562) with 12.5 L Master Mix, 2.0 L primer mix (0.2

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M final concentration of each primer), 9.5 L ultrapure water and 1.0 L DNA template at 10

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ng L-1. PCR cycling settings were: one cycle at 95 °C for 2 min, 35 cycles at 94 °C for 30 s, 58 °C for 30 s, 72 °C for 60 s, and final extension at 72 °C for 10 min. PCR product was run in 0.7%

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agarose gel to verify fragment size (data not shown) and samples were sent for purification and

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sequencing (Genewiz, South Plainfield, NJ 07080). In total, amplification products from six plants were sequenced using the same primers used for amplification. 2.4 Homology modeling of D. insularis ACCase The segments of D. insularis ACCase sequence where the Trp2027 mutation is located were aligned to the sequence of wheat ACCase using EMBOSS Needle (Li et al., 2015). This information was used to build a homology model of D. insularis ACCase by introducing the single nucleotide polymorphism (SNP) from D. insularis on a preexisting wheat ACCase structure available to our laboratory. The homology modeling pipeline was similar to that reported

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Journal Pre-proof previously to model the binding of glufosinate on a resistant form of glutamine synthetase from Lolium perenne (Brunharo et al., 2019). Briefly, the model was refined using GROMACS (version 2018.3) (Abraham et al., 2015; Hess et al., 2008) on a 24-Core Intel® Xeon® 5600 series processors workstation. Steric clashes or inappropriate geometries were corrected through molecular dynamics simulation and evaluated using MolProbity (Chen et al., 2010; Davis et al.,

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2007) as described before. The coordinates for haloxyfop bound to the carboxyltransferase (CT) domain of yeast ACCase were obtained from the crystal structure 1UYS (Zhang et al., 2004)

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available from the RCSB protein data bank (www.rcsb.org). Proteins and ligand interactions

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were visualized using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger,

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LLC) (DeLano, 2002).

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2.5 Single nucleotide polymorphism (SNP) genotyping assay A Kompetitive Allele Specific PCR (KASP) assay was performed to genotype a mutation

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(Trp2027Cys) in the ACCase sequence from a larger number of D. insularis samples. Two

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forward primers that were identical except for the SNP responsible for the amino acid

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substitution (bold) were designed. Additional nucleotides were attached to the 5’ end of these primers, which were specific for the HEX (Resistant) and FAM (Susceptible) labeled oligo (underlined) in the KASP Master Mix (LGC Genomics, Beverly, MA 01915). The susceptible forward primer was 5’-GAAGGTGACCAAGTTCATGCTTTGCCTCTCTTCATCCTTGCTAACTGG-3’ and the resistant forward primer 5’GAAGGTCGGAGTCAACGGATTTTGCCTCTCTTCATCCTTGCTAACTGC-3’. The universal reverse primer was designed to match both susceptible and resistant sequences (3’ACAGATCTCTTTGTCCACCGGAG-5’). The reaction mix contained KASP Master Mix, both forward

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Journal Pre-proof primers (0.2 μM final concentration each), universal reverse primer (0.4 μM final concentration), and sample DNA (40 ng) in each well of a 96-well plate. In total, 30 resistant and 24 susceptible plants were assayed. Ultrapure water was used as non-template control (NTC). PCR conditions were 94 °C for 15 min, followed by 10 cycles of 94 °C for 20 s, 59 to 54 °C for 60 s (0.5 °C decrease per cycle); followed by 35 cycles of 94 °C for 20 s, and 55 °C for 60 s, taking an

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endpoint fluorescence reading by cooling the plate to 30 °C for 10 s and reading the plate in both the HEX and FAM fluorescent channels. Samples were plotted in a scatter plot as

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percentage of fluorescence and genotyping assignment was conducted based on linear

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software 3.5.1 (R Core Team, Vienna, Austria).

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discriminant analysis (Patterson et al., 2017; Takano et al., 2019), which was performed in R

3 RESULTS AND DISCUSSION

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3.1 Whole-plant dose-response and cross-resistance characterization

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The population identified as resistant to ACCase-inhibiting herbicides had high level

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resistance to haloxyfop (Resistance Factor, RF = 613-fold) (Figure 1A) with ED50 values of 3.8 and 2,332 g ha-1 for susceptible and resistant plants, respectively (Figure 1B). The resistant population also showed low level resistance to pinoxaden (RF = 3.6-fold), and ED50 values were 16 and 58 g ha -1 for susceptible and resistant plants, respectively (Figure 1B). However, these two populations were both susceptible to clethodim (Figure 1C). The ED 50 values for susceptible and resistant populations were 4.3 and 4.4 g ha-1 of clethodim, respectively. In addition to clethodim (108 g ha-1), other DIM herbicides such as sethoxydim (230 g ha1

) and tepraloxydim (100 g ha-1) were also effective controlling the resistant D. insularis

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Journal Pre-proof population at 21 DAT (Table 2; Figure 2). On the other hand, fenoxaprop, haloxyfop, and quizalofop provided less than 31% control of the resistant population when sprayed at their labeled rate. Pinoxaden (60 g ha -1) provided 63% control on the resistant population. All herbicides sprayed at their recommended rate controlled the susceptible population. It is important to mention that plants were sprayed at the initial stage of development (four leaves)

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as those originating from rhizomes are more difficult to control (Gemelli et al., 2012). Clethodim is currently the most used ACCase herbicide in soybean fields of Brazil

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(Takano et al., 2020). This is because it generally provides higher levels of control on GR D.

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insularis, compared to other ACCase-inhibiting herbicides (Gemelli et al., 2013; Zobiole et al.,

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2016). Best management practices such as the use of cover crops and herbicide rotation and

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mixture have been shown as effective tools for D. insularis control (Marochi et al., 2018). These

insularis.

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strategies should be used to prevent the evolution of cross-resistance to DIM herbicides in D.

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3.2 ACCase gene sequencing and homology modeling of D. insularis ACCase

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The two sets of primers generated two fragments with 280-bp (F1+R1) and 360-bp (F2+R2) in length. The first fragment includes residues Ile1781 and Trp1999, while the second covers Trp2027, Ile2041, Asp2078, Cys2088 and Gly2096. A nonsynonymous single nucleotide polymorphism (SNP) mutation in the third nucleotide of the amino acid codon at position 2027 was found in the ACCase sequence from the resistant D. insularis population (Figure 3). This SNP results in an amino acid substitution from tryptophan (Trp) to cysteine (Cys) at position 2027 (Trp2027Cys). No other nonsynonymous polymorphisms were found in the ACCase gene of D. insularis, indicating that the two biotypes only differ for the Trp2027Cys mutation. In D.

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Journal Pre-proof insularis, the Trp2027 residue is located near the cavity formed between the two ACCase chains (Figure 4). Tryptophan is a hydrophobic non-polar residue whereas cysteine is a hydrophilic polar residue. The change at position 2027 causes a replacement of a mostly buried aromatic residue (tryptophan) with a buried thiol group in the mutant (cysteine) (Délye et al., 2005). The mutation Trp2027Cys probably affects the lipophilic properties of the binding site in ACCase,

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negatively affecting the interaction of haloxyfop with the enzyme, thus imparting resistance to some of the ACCase-inhibiting herbicide classes. This is consistent with in vitro enzymatic data

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of ACCase purified from blackgrass (Alopecurus myosuroides Huds.) in response to fenoxaprop,

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diclofop, clodinafop and haloxyfop. Herbicide doses required to inhibit 50% ACCase activity (I 50)

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were up to 361 times higher for Trp2027Cys mutant than those for the wild-type ACCase (Délye

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et al., 2005). Similar to our findings, this mutation did not provide any resistance to clethodim and cycloxydim at the enzyme level.

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Residue Trp2027 is well conserved across several homomeric ACCase sequences from

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multiple species (Délye, 2005). The cross-resistance pattern observed for the resistant D.

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insularis population is consistent with the type of mutation evolved in this species. Similar to D. insularis, wild oat (Avena fatua) populations from Canada harboring the Trp2027Cys mutation showed high levels of resistance to FOP and possibly low level of resistance to DEN (Beckie et al., 2012). In contrast, other types of mutations such as Asp2078Gly in ACCase can confer broader types of cross-resistance to FOP, DIM and DEN (McCullough et al., 2016; Osuna et al., 2012). In Lolium spp., resistant populations evolved different mutations: Ile1781Leu conferring dominant resistance to pinoxaden, clodinafop, haloxyfop, sethoxydim and clethodim; Ile2041Asn/Val with dominant or partially dominant resistance to FOPs but no substantial

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Journal Pre-proof resistance to DIMs and moderate resistance to DEN; and Cys2088Arg endowing partially dominant resistance to clodinafop, sethoxydim and pinoxaden (Scarabel et al., 2011). These observations suggest that even though all three herbicide groups (FOP, DIM and DEN) target the same enzyme, their binding sites are not exactly the same. While residues Ile1781 and Asp2078 are involved in sensitivity to both DIM and FOP herbicides, residues Trp2027, Ile2041

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and Gly2096 are associated with sensitivity to FOP and possibly DEN (Beckie and Tardif, 2012; Délye, 2005; Kaundun, 2014; Liu et al., 2007; Powles and Yu, 2010; Takano et al., 2020).

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Therefore, different mutations can rise in response to the selection pressure exerted in diverse

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weed populations. The continuous use of clethodim and other graminicides targeting ACCase to

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manage D. insularis in Brazil could potentially lead to the evolution of other types of mutations

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conferring broader resistance to multiple classes of ACCase (Takano et al., 2020). 3.3 Single nucleotide polymorphism (SNP) genotyping assay

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The SNP genotyping assay clearly discriminated individuals from susceptible (TGG) and

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resistant (TGC) nucleotides for the Trp2027 codon (Figure 5). All plants tested (n=24) from the

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susceptible population were homozygous for the susceptible allele (TGG/TGG), whereas resistant plants (n=30) were homozygous for the resistant allele (TGC/TGC). No heterozygous plants (TGG/TGC) were detected among all tested plants. The absence of heterozygous plants is possibly resulting from how these plants evolved resistance in the field by several years of selection pressure with ACCase inhibitors. In addition, self-pollination is predominant in D. insularis, which contributes to the fixation of beneficial mutations in the population under selection pressure (Melo et al., 2015; Ng et al., 2004). Other types of quantitative real-time PCR (qPCR)-based genotyping assays have been used to identify fluazifop resistance in itchgrass

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Journal Pre-proof (Rottboellia cochinchinensis) populations (Barrantes-Santamaría et al., 2018). The robustness of our method demonstrates how this assay could be used to survey a large number of samples in a short period of time. This would allow early identification of resistance in fields with suspected evolution of resistance to ACCase inhibitors. In addition to target-site mutations, we cannot rule out the presence of herbicide

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metabolism as an additional resistance mechanism. A wild oat (A. fatua) population with lowlevel of resistance to pinoxaden exibited increased sensitivity to the herbicide in the presence

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of malathion (a cytochrome P450 inhibitor), suggesting possible involvement of herbicide

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metabolism (Beckie et al., 2012). Thus, further research into herbicide metabolism in D.

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insularis is needed.

4 CONCLUSION

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Cross-resistance to ACCase inhibitors (FOP and DEN, but not DIM) was confirmed in a D.

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insularis population from Brazil. Resistant plants were homozygous for a Trp2027Cys mutation

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in the carboxyltransferase (CT) domain of ACCase, which has previously conferred FOP/DEN resistance in other grass weed species. Our protein homology model shows that residue Trp2027 is located in the proximity of the binding site for haloxyfop on D. insularis ACCase, likely affecting herbicide inhibition. A qPCR-based genotyping assay was validated for rapid detection of resistant plants in field populations.

Funding: This work was partially supported by the USDA National Institute of Food and Agriculture, Hatch project COL00783, accession number 1016207 (to T.G.) and Hatch Project

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Journal Pre-proof COL00785, accession number 1016591 (to F.D.), to the Colorado State University Agricultural

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Experiment Station.

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Journal Pre-proof Table 1. Active ingredient, commercial product, manufacturer, concentrations and recommended field rate for each herbicide tested on Digitaria insularis populations. Chemical

Active

group1

ingredient

(g ai ha -1)

Clethodim

108

Select

240

Arysta

Sethoxydim

230

Poast

184

BASF

Tepraloxydim

100

Aramo

Fenoxaprop

110

Haloxyfop

DENs

Manufacturer

(g L-1)

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BASF

Podium EW

110

Bayer

60

Verdict R

120

Corteva

Quizalofop

100

Targa

50

Arysta

Pinoxaden

60

Axial

50

Syngenta

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200

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FOPs: aryloxyphenoxypropionates; DIMs: cyclohexanediones; DEN: phenylpyrazoline.

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1

Concentration

Product

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FOPs

Commercial

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DIMs

1X Field Rate

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Journal Pre-proof Table 2. Visual control (%) of different ACCase-inhibiting herbicides on susceptible and resistant Digitaria insularis populations. Population2 Treatment1

Rate (g ha -1)

108

100 a

100 a

Sethoxydim

230

100 a

100 a

Tepraloxydim

100

100 a

100 a

Fenoxaprop

110

100 a

26 c

Haloxyfop

60

100 a

30 c

Quizalofop

100

100 a

Pinoxaden

60

100 a

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30 c

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0

63 b 0

All treatments were sprayed with 0.25% (v/v) methylated seed oil. 2Means followed by

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Untreated check

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Clethodim

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Susceptible Resistant

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different letter in column are significantly different by Tukey test (p<0.05).

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Journal Pre-proof Figure Legends Figure 1. Dose response for haloxyfop (A), pinoxaden (B), and clethodim (C) in susceptible (blue circles) and resistant (red squares) Digitaria insularis populations. Lethal dose for 50% control (LD50) and resistant factor (RF) were 3.8 and 2,332 (RF=613), 16 and 58 (RF=3.6) g ha -1 , and 4.3 and 4.4 (RF=1) for haloxyfop, pinoxaden and clethodim, and for susceptible and resistant

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populations, respectively. Figure 2. Cross-resistance pattern in Digitaria insularis resistant to ACCase inhibitors. Unt:

); Quiz: quizalofop (75 g ha -1); Pino: pinoxaden (60 g ha-1).

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1

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untreated; Seth: sethoxydim (230 g ha -1); Clet: clethodim (108 g ha -1); Halo: haloxyfop (60 g ha -

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Figure 3. Alignment of ACCase sequences from susceptible (S) and resistant (R) Digitaria

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insularis populations. Bold residues are positions where mutations have evolved in other species affecting herbicide binding to the enzyme. Resistant plants harbored a mutation (G-to-

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C) at the third nucleotide of the codon at position 2027, resulting in an amino acid substitution

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shown).

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(Trp2027Cys). Neither population had sequence variation for residue Ile1781 (sequence not

Figure 4. Modeling of Digitaria insularis acetyl-CoA carboxylase (ACCase). Carboxyltransferase, CT, domain of ACCase dimer showing chains A and B in blue and gold, respectively (A). A closer view of the interface between chains A and B highlighting the position of haloxyfop within the catalytic domain (B). Residue Trp2027 is located within the protein and cannot be seen on the surface (C). The mutation is in the proximity of haloxyfop, suggesting that this amino acid substitution is altering the architecture of the binding domain.

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Journal Pre-proof Figure 5. Single nucleotide polymorphism genotyping for the G-to-C substitution resulting in a Trp2027Cys mutation in Digitaria insularis. The y-axis corresponds to fluorescence measures for the Fluor HEX probe for the G allele, and the x-axis corresponds to the Fluor FAM probe for the C allele. The three clusters correspond to genotypes: susceptible (GG-homozygous), resistant

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(CC-homozygous), and negative control.

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Journal Pre-proof References

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Journal Pre-proof Trp2027Cys mutation evolves in Digitaria insularis with cross-resistance to ACCase inhibitors

Highlights Digitaria insularis is a troublesome glyphosate resistant weed that has now evolved resistance to ACCase inhibitors, particularly FOP and DEN herbicides. A target site mutation (Trp2027Cys) was present only in resistant plants. This mutation is known to cause resistance to FOP herbicides in other weed species.

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We generated a protein homology model for D. insularis ACCase, demonstrating that the Trp2027Cys mutation is near the binding site of haloxyfop, between two ACCase chains.

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We also validated a qPCR-based molecular marker for early detection of resistance in fields under high selection pressure for ACCase resistance.

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