Mechanisms of Lolium rigidum multiple resistance to ALS- and ACCase-inhibiting herbicides and their impact on plant fitness

Journal Pre-proof Mechanisms of Lolium rigidum multiple resistance to ALS- and ACCase-inhibiting herbicides and their impact on plant fitness

E. Anthimidou, S. Ntoanidou, P. Madesis, I. Eleftherohorinos PII:

S0048-3575(19)30528-0

DOI:

https://doi.org/10.1016/j.pestbp.2019.12.010

Reference:

YPEST 4515

To appear in:

Pesticide Biochemistry and Physiology

Received date:

11 November 2019

Revised date:

27 December 2019

Accepted date:

27 December 2019

Please cite this article as: E. Anthimidou, S. Ntoanidou, P. Madesis, et al., Mechanisms of Lolium rigidum multiple resistance to ALS- and ACCase-inhibiting herbicides and their impact on plant fitness, Pesticide Biochemistry and Physiology (2019), https://doi.org/ 10.1016/j.pestbp.2019.12.010

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© 2019 Published by Elsevier.

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Running title: Lolium rigidum multiple resistance to ALS-and ACCase-inhibitors

Mechanisms of Lolium rigidum multiple resistance to ALS- and ACCaseinhibiting herbicides and their impact on plant fitness E. Anthimidoua , S. Ntoanidoua , P. Madesis b, I. Eleftherohorinos a

Aristotle University of Thessaloniki, School of Agriculture, Thessaloniki, Greece

f

a

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b

Institute of Applied Biosciences-CERTH, 6th Km. Charilaou-Thermi Road,

Eleftherohorinos,

Laboratory

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Correspondence: Ilias

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Thessaloniki

of Agronomy,

School of

Agriculture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece. Tel:

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++30 2310 998629; Fax:++30 2310 998634; E-mail: [email protected]

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ABSTRACT Three putative resistant (R1, R2, R3) and one susceptible (S) Lolium rigidum populations originating from Greece were studied for resistance to ALS and ACCase inhibiting herbicides, using whole plant, sequencing of als and accase gene, and in vitro ALS activity assays. The S and two R (R1, R2) populations were also evaluated for fitness in competition with wheat. The whole plant assay indicated unsatisfactory control of the R populations with mesosulfuron-methyl + iodosulfuron-methyl or pinoxaden application, whereas sequencing of the als gene revealed that all ALSresistant individuals had a Pro-197 substitution by Leu, Glu, Ser, Ala, Thr, or Gln. In

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addition, the accase gene of all pinoxaden resistant individuals had an Ile-2041 substitution by Asn or Thr. Furthermore, sequencing of the individuals surviving mesosulfuron-methyl + iodosulfuron-methyl or pinoxaden treatment revealed co-

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existence of point mutations in the accase or als genes, respectively, demonstrating

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multiple resistance. The in vitro activity of the ALS enzyme confirmed that resistance to mesosulfuron-methyl + iodosulfuron-methyl was due altered target-site. The

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recorded higher vigor and greater competitive ability of S population against wheat as compared with that of the R populations suggests an associated fitness cost with

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multiple resistance.

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fitness cost

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Keywords: Lolium rigidum; multiple resistance; ALS mutation; ACCase mutation;

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1. Introduction Lolium rigidum Gaud. (rigid ryegrass) is a diploid (2n=14) and obligated cross pollinated species with a gametophytically self-incompatibility system (Spoor and McCraw, 1984). It possesses a combination of biological factors such as prolific seed production, seed viability, pollen movement, high degree of genetic variability and high phenotypic plasticity, which enables it to adapt to adverse environmental conditions and enhances rapid evolution of resistance to most of the herbicides used (Manalil, 2014). Weeds are the most important biotic factor affecting agricultural production and

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causing yield losses in various crops worldwide. Herbicides are the most efficient tools to control weeds and to avoid or reduce crop yield losses (Matzrafi et al., 2017), but their intensive use imposed a strong selective pressure that has led to the evolution

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of many resistant populations globally (Heap, 2019). These resistant populations are

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severe threat to the sustainability of intensive cropping systems and endanger food security for the ever-increasing world population. Among weed species with

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resistance, L. rigidum is particularly troublesome, because of its tendency to developed cross- and multiple resistance to a wide array of herbicides due to its

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outcrossing nature (Heap, 2019).

Acetolactate synthase (ALS) and acetyl-coenzyme A carboxylase (ACCase)

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inhibitors have long been used to provide effective control of L. rigidum in winter

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cereals. The ALS-inhibiting herbicides comprise the largest site of action group with 57 active ingredients and by far are the most broadly used herbicides in agriculture since they were first introduced in 1982. These herbicides inhibit the key enzyme ALS in the biosynthetic pathway of branched chain amino acids valine, leucine and isoleucine that are considered essential for plant growth. The strong selection pressure of these herbicides due to their high activity (active at very low rates) has led to the evolution of 162 resistant weed species (Heap, 2019). Among the most common weed species in winter cereals that have evolved resistance to these herbicides are corn poppy (Papaver rhoeas) (Kaloumenos et al., 2011), wild mustard (Sinapis arvensis) (Ntoanidou et.al., 2017), and L. rigidum (Kaloumenos et al., 2012; Kotoula-Syka et al., 2000; Yu et al., 2008). ACCase-inhibiting herbicides are commonly used

worldwide to

selectively

control grass weeds in a variety of crops. These herbicides inhibit plastidic ACCase enzyme, a key enzyme for fatty acid synthesis in all plants. However, their repeated

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and extensive use for many years has led to the evolution of herbicide resistant populations in many important grass weed species such as black grass (Alopecurus myosuroides) (Délye et al., 2005; Petit et al., 2010), wild oat (Avena fatua) (CruzHipolito et al., 2011), sterile oat (Avena sterilis) (Papapanagiotou et al., 2012; 2015) and L. rigidum (Scarabel et al., 2011). Until now there have been reported 48 grass weed species with resistance to these herbicides (Heap, 2019). Herbicide resistance includes target-site-based resistance (TSR) and non-targetsite-based

resistance

(NTSR)

mechanisms.

TSR

is

the

result

of structural

modification, or over-expression of a specific gene encoding for the target protein

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(Délye et al., 2013; Matzrafi et al., 2017). The most common and important TSR mechanism involves one or more mutations in the DNA encoding the target protein of the herbicide, which leads to changes in amino acids or conformational changes in

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protein folding, often resulting in high levels of resistance (Fernández-Moreno et al.,

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2017). NTSR mechanism can decrease herbicide absorption and translocation in the plant, or enhance the degradation of herbicide molecules by plant metabolism, thus

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preventing their deleterious action on their target protein (Délye et al., 2013; Scarabel et al., 2015). NTSR involves a wide diversity of pathways that are inherited in

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complex manners (Babineau et al., 2017). In some cases, both TSR and NTSR may coexist in individuals of various weed populations (Délye et al., 2013).

rate,

survival,

early vigor,

biomass production,

reproductive

and

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germination

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Fitness cost of resistant weeds includes changes in plant performance such as seed

competitive ability, that reduce the frequency of resistant plants in populations (Cousens and Fournier-Level, 2018; Vila-Auib et.al., 2019). Fitness of resistant weeds has been shown to depend on several factors, such as plant species, resistance mechanism involved, specific mutant resistance allele, dominance of the resistance cost, pleiotropic effects on the kinetics of herbicide target proteins, genetic background, and the abiotic and biotic environmental conditions (Yanniccari et al., 2016). From an ecological evolutionary context, rapid herbicide resistance evolution can occur if the gene mutation provides a significant level of resistance and yet show no or negligible fitness cost (Vila-Aiub et al., 2015a). L. rigidum in Greece is one of the most abundant and harmful weed in winter cereals and its control has relied on the extensive use of ACCase and ALS inhibitors during the last 35 years. However, the high selective pressure of their repeated use had resulted in resistance to ALS-inhibitors in several L. rigidum populations found in

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Greece (Kotoula-Syka et al., 2000; Kaloumenos et al., 2012). During the 2013 growing season, wheat growers from northern Greece complained for unsatisfactory control of this weed not only after the application of mesosulfuron-methyl + iodosulfuron-methyl (ALS inhibitor) but also after pinoxaden (ACCase inhibitor). Based on this information, this research was conducted in order to provide data for possible evolution of L. rigidum multiple resistance to ALS- and ACCase inhibitors. Therefore, the objectives of this study were to: 1) determine whether the reduced control of three putative resistant (R) L. rigidum populations collected from wheat fields in northern Greece was due to evolution of multiple resistance to mesosulfuron-

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methyl + iodosulfuron-methyl and pinoxaden, 2) to elucidate the possible presence of a point mutation in the als or/and accase genes, 2) to investigate their biochemical basis of resistance by determining the in vitro catalytic activity of the ALS enzyme in

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the presence of mesosulfuron-methyl + iodosulfuron-methyl, and 4) to study the

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fitness of R L. rigidum populations grown in competition with wheat in comparison

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with that of a susceptible (S) one.

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2. Materials and methods

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2.1. Seed source and plant material

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Matured seeds of L. rigidum plants surviving application of mesosulfuron-methyl + iodosulfuron-methyl or/and pinoxaden at the field-recommended rate were collected in late May 2013 from three winter wheat monoculture fields located in northern Greece [county of Kilkis (R1), Thessaloniki (R2), and Grevena (R3)]. Seeds from each field were considered as putative R populations, while the seeds collected from a field of the Aristotle University Farm of Thessaloniki, which had never been treated with either herbicide, was considered as S population. A representative seed sample was collected from many individuals in each field [S (40o 32'12.8'' Ν, 22ο 59'13.2'' Ε), R1 (40ο 51'36.6'' Ν, 22ο 50'32.6'' E), R2 (40ο 33'23.3'' Ν, 23ο 22'58.5'' Ε), R3 (39ο 55'21.0'' Ν, 21ο 48'02.1'' Ε)], placed in plastic bags and then transferred to laboratory, air-dried, threshed, placed in paper bags and stored in 3–5 °C for further studies. 2.2 Initial screening for L. rigidum resistance

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The three putative R L. rigidum populations along with the S were initially tested for resistance to mesosulfuron-methyl + iodosulfuron-methyl (Hussar Maxx 3% WG, Bayer Hellas) and to pinoxaden (Axial 60 EC Sygenta). Experiments were carried out during 2013-2014. Seeds (15 per pot) were sown in 10 × 10 × 9 cm (0.9 L) plastic pots filled with soil:sand mixture (5:1 by volume) at a depth of 1-2 cm. All pots were placed outdoors in a net protected area of the Aristotle University Farm of Thessaloniki, where they were fertilized and irrigated as required for vigorous plant growth. Seedlings, at the 2- to 3-leaf stage, were thinned to six uniform seedlings in

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each pot. At the 3- to 4-leaf stage, seedlings were treated with mesosulfuron-methyl +

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iodosulfuron-methyl at the recommended rate (1x = 7.5+7.5 g ai ha-1 ) and four times the recommended rate (4x = 30 + 30 g ha-1 ), while pinoxaden was also applied at the

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recommended rate (1x = 45 g ha-1 ) and four times the recommended rate (4x = 180 g

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ha-1 ). The application of herbicides was performed with a portable field plot sprayer (AZO-SPRAYERS, P.O. Box 350–6710 BJ EDE, The Netherlands) using flat-fan

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nozzles (Teejet Spray System Co., P.O. Box 7900, Wheaton, IL 60188) and calibrated to deliver 300 L ha-1 of water at 280 kPa pressure. Control of L. rigidum populations

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was assessed by determining the above ground fresh weight and tiller number of all surviving plants in each pot at 4 weeks after treatment (WAT). This experiment was

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for each treatment.

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carried out twice using a randomized complete block design with three replications

2.3 Amplification and sequencing of als and accase gene fragments

The plant material used for the amplification of als and accase genes was taken from plants grown in 18 pots (seven plants per pot) per S and R population as described above. At the 3-to 4-leaf stage, six pots from each of the S and R populations were treated with the four times the recommended rate of mesosulfuronmethyl + iodosulfuron-methyl (30 + 30 g ha-1 ). At the same time, six pots from each of the S and R populations were treated with the four times the recommended rate of pinoxaden (300 g ha-1 ), whereas six pots from each of the S and R populations were kept untreated. Leaf samples were taken from 6 surviving individuals from each herbicide application for each R population. In addition, leaf samples were taken from 12 untreated individuals of the S population. All collected samples were stored at -28

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°C for DNA extraction. Genomic DNA was extracted from 200 mg leaf tissue (from each individual) using the Cetyl trimethylammonium bromide (CTAB) method according to protocol of Doyle and Doyle (1987). The amplification of the als gene fragment containing the Pro-197 codon (400 bp) from the genomic DNA extracted from R and S plants was achieved using the forward

5ʹ-GCCACCAACCTCGTCTCC-3ʹ

and

the

reverse

5ʹ-

CCACCGCCAACATARAGAAT-3ʹ primers. The polymerase chain reaction (PCR) consisted of 0.5 µM of each forward and reverse primer, 0.2 mM deoxyribonucleotide triphosphate (dNTPs), 2 µL of the supplied 10x KAPA Taq thermophylic buffer, 1 µL

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genomic DNA and 0.5 units of standard KAPA Taq polymerase in 20 µL mixture.

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Amplifications were carried out in Applied Biosystems VeritiT M Thermal Cycler using the following cycles: DNA denaturation for 3 min at 95 o C and 35 cycles of 30 s

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denaturation at 95 o C, 30 s annealing at 61 o C and 1 min elongation at 72 o C. A final

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elongation step was performed at 72 o C for 5 min.

Amplification of the accase gene fragment containing the codons Ile-2041, Asp-

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2078, Cys-2088 and Gly-2096 (516 bp) was achieved using the forward primer 5΄AGGACAGCCTGATTCCCATGAG-3΄

and

the

reverse

5΄-

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GCTTCTATGCTCTTCTGAATGG-3. The PCR consisted 0.5µM of each forward and reverse primer, 7.5 μl of One Taq 2x Master Mix with Standard Buffer (M0482L)

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and 1.5 μl genomic DNA in 10 μl mixture. Amplifications were conducted as

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described above except 20 s annealing at 51o C and 40 s elongation at 72 o C. A final elongation step was performed at 72

o

C for 1 min. Amplification of the accase

fragment containing the codons Ile-1781 and Trp-2027 (1000 bp) was achieved using the forward primer 5΄- GCTCAATGACATTGGTATGGTAGCCTGG-3΄ and the reverse primer 5΄TCCAGTTAGCAAGGATGAACAGAGG-3΄. The PCR consisted of 0.5 µM of each forward and reverse primer, 0.2 mM dNTPs, 2 µL of the supplied 10x KAPA Taq thermophylic buffer, 1µL genomic DNA and 0.5 units of standard KAPA Taq polymerase in 20 µL mixture. Amplifications were described above except 20 s annealing at 61 o C and 1 min elongation at 72 o C. A final elongation step was performed at 72 o C for 1 min. Τhe PCR products were separated in 1.5% agarose gel and purified according to the protocol outlined in the NucleoSpin® Extract II kit (MACHEREY NAGEL GmbH & Co. KG. Postfach 10 13 52. D-52313 Düren, Germany). The purified product was sent immediately for sequencing to the International Hellenic University,

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Department of Agriculture, Lab of Agrobiotechnology and Inspection of Agricultural Products in Thessaloniki. Each PCR product was sequenced once, with the forward primer. The sequences were aligned with the CLUSTAL W software using the Bioedit.

2.4 ALS extraction and in vitro activity assay

The plant material used for the ALS activity assay was taken from the above plants used for the amplification of the als and accase genes. At 3-to 4-leaf stage, 3 g

injury

symptoms)

the

application

of four

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of the above ground leaf material from individual R plants that survived (without times the recommended

rate of

mesosulfuron-methyl + iodosulfuron-methyl and untreated S plants were harvested

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and stored at -80 o C. The ALS in vitro assay was conducted according to the method

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of Osuna et al. (2002) and modified by Ntoanidou et al. (2019). Herbicide concentrations used for this in vitro activity assay were 1, 6, 40, 250, 1000 nM

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mesosulfuron-methyl + iodosulfuron-methyl for the resistant populations, while for the susceptible population the respective herbicide concentrations were 0.1, 0.5, 1, 10,

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50, 100 1000 nM. Acetoin was detected by measuring absorbance at 520 nm. The absorbance values were expressed as percentage of control tubes in which the reaction

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took place in the absence of mesosulfuron-methyl + iodosulfuron-methyl. Two

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identical experiments, each with a separate leaf tissue extract from different plants, were conducted per population and each sample per herbicide concentration was assayed in triplicate.

2.5 Fitness of L. rigidum populations grown in competition with wheat

The S and two R (R1, R2) populations of L. rigidum were evaluated for their fitness grown in competition with wheat. Two identical experiments were conducted at the Aristotle University Farm of Thessaloniki during October 2018 to May 2019, using 20 × 25 × 30 cm plastic pots filled with the soil used in the previously described experiments. In each pot, wheat was seeded in two rows spaced 10 cm, whereas L. rigidum was seeded in five densities (0-weed-free wheat control, 2, 4, 6, and 8 plants per pot) as shown in Fig. 1. For each of the two experiments, the randomized complete block design was used with three replications. All pots were placed outdoors

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in a net protected area and were properly irrigated and fertilized to accomplish vigorous growth of the plants during the growing season. All other weeds except L. rigidum were carefully removed manually during the growing season to assure absence of other weed competition. Above ground fresh weight and ear number of wheat as well as fresh weight and tiller number of L. rigidum were determined in early May.

2.6. Statistical analysis

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A combined over two experiments analysis of variance (ANOVA) for the initial L. rigidum screening data (fresh weight % of untreated control) was performed using a 2 × 4 × 4 × 3 (2 experiments by 4 populations by 4 herbicide rates by 3 replications)

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split plot approach (the populations were the main plots and the four herbicide rates

significance using the LSD test.

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the sub-plots). Differences among treatment means were compared at the 5% level of

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A combined over two experiments ANOVA was performed for the ALS activity data (absorbance measurements) obtained from the three R population assays using a

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2 × 5 × 3 × 3 (2 experiments by 5 herbicide concentrations by 3 L. rigidum populations by 3 replications) factorial approach. A combined over two experiments

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ANOVA for the ALS activity data (absorbance measurements) obtained from the S

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population assay was also carried out using a 2 × 7 × 3 (2 experiments by 7 herbicide concentrations by 3 replications) factorial approach. As the combined ANOVAs revealed no differences between the two experiments, the data obtained from the two ALS activity assays were pooled and subjected to nonlinear regression analysis using the following log-logistic equation (Seefeldt et.al., 1995):

where C = the lower limit, D = the upper limit, b = the slope at the I50 , and I50 = the mesosulfuron + iodosulfuron concentration (nM) required for 50% inhibition of ALS catalytic activity. The levels of resistance for all populations (R and S) were determined by the resistance ratio (R/S), which was calculated as the respective I50 of the R populations divided by the respective I50 value of the S population.

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For comparison of fitness among L. rigidum populations grown in competition with wheat, a combined over two experiments ANOVA was performed for the fresh weight and tiller number of L. rigidum using a 2 × 3 × 4 × 3 (2 experiments by 3 populations by 4 densities by 3 replications) split-plot approach (populations as main plots and densities as sub-plots). A combined over two experiments ANOVA was also performed for the wheat fresh weight and ear number using the 2 × 3 × 5 × 3 (2 experiments by 3 populations by 5 densities by 3 replications) split-plot approach (populations as main plots and densities as sub-plots). As the ANOVAs indicated no significant differences between the experiments, the data of both wheat and L.

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rigidum parameters determined were pooled and regressed against weed density. In these regression equations, fresh weight and ear or stem number of wheat or weed, respectively, was the dependent variables (y) and density was the independent

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variable (x).

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3. Results

3.1 Initial screening for L. rigidum resistance

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The recommended rate of mesosulfuron-methyl + iodosulfuron-methyl reduced fresh weight of the R1, R2 and R3 by 17, 23 and 16%, while the 4x rate reduced fresh

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weight by 28, 35 and 32%, respectively (Fig. 2). Fresh weight of R1, R2 and R3, after

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application of the pinoxaden recommended rate, was reduced by 24, 68 and 72%, but its reduction due to 4x rate was 54, 89 and 87%, respectively. Tiller number of R1 plants, after application of both herbicides, was higher than R2 and R3 populations, whereas pinoxaden caused higher tiller number reduction in all R populations than mesosulfuron-methyl + iodosulfuron-methyl. All herbicide treatments reduced fresh weight and tiller number of the S population by 100%.

3.2 Amplification and sequencing of als and accase gene fragments

The aligned chromatographs of the als gene fragment (410bp) with Arabidopsis thaliana als gene (Accession number: X51514) did not reveal any point mutations in codon CCT (Pro-197) of the six untreated S individuals (Table 1). However, Glu197/Ala or Pro-197/Gln, Ala-197/Ala, and Gln-197/Gln mutations were detected in one, two, and three R1 individuals, respectively, whereas Pro-197/Ser, Leu-197/Leu,

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and Gln-197/Gln were found in one, three, and two R2 individuals, respectively. Regarding R3 population, three individuals had Thr-197/Thr and three Ser-197/Ser mutations. In general, 16 out of the 18 individuals had a Pro-197 substitution in both alleles, while in 1 R1 and 1 R2 a Pro-197 mutation was found in only one allele. The aligned chromatographs of the accase gene fragments (516 and 1000bp) with A. myosuroides accase (Accession number: AJ310767.1) did not reveal any point mutation in the codons Ile-1781, Trp-2027, Ile-2041, Asp-2078, Cys-2088, Gly-2096 of the same six untreated S individuals (Table 1). However, in two R1 individuals surviving mesosulfuron-methyl + iodosulfuron-methyl application, a Thr-2041/Thr

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substitution was found and in two others an Ile-2041/Asn substitution. The remaining two individuals had wild type Ile-2041. In R2 population, one individual was Ile2041/Asn and one Thr-2041/Thr, with the other 4 being wild type Ile-2041. In R3,

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two individuals were Ile-2041/Asn and the remaining 4 individuals were wild type

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Ile-2041. In 3 out of 8 mutated individuals, the Ile-2041 substitution was detected in both alleles, whereas an Ile-2041substitution was not found in 10 out of 18 individuals

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surviving mesosulfuron-methyl + iodosulfuron- methyl treatment. For plants surviving pinoxaden, three R1 individuals had Asn-2041/Asn mutation

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and the other three were Ile-2041/Asn (Table 2). Also, two R1 individuals, one each with Ile-2041/Asn and Asn-2041/Asn mutation, had a second mutation of Trp-

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2027/Cys. In R2 population, three individuals had Ile-2041/Asn, two had Asn-

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2041/Asn and one had Ile-2041/Thr mutations. For R3, three individuals were Ile2041/Asn and three Asn-2041/Asn mutations. Out of 18 mutated individuals, 8 had the Ile-2041 substitution in both alleles. Several individuals surviving pinoxaden treatment also had mutations in als. In the R1, two individuals had Ala-197/Ala, and one had Gln-197/Gln mutations with the other three being wild type Pro 197. In R2, one individual contained Ser-197/Ser, one Leu-197/Leu, and one Pro-197/Leu mutations with the remaining three being wild type Pro-197. In R3, two individuals were Thr-197/Thr and one individual Ser197/Ser, with the remaining 3 being wild type Pro-197.

3.3. ALS activity assay

The

I50

values

for

mesosulfuron-methyl

+

iodosulfuron-methyl

revealed

significant differences in ALS resistance between S and R populations of L. rigidum.

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The I50 values for mesosulfuron-methyl + iodosulfuron-methyl for the S population was 21 nM, but was 337, 227 and 191 nM for the R1, R2 and R3 populations, respectively. The estimated RF values for the ALS enzyme were 16, 11 and 9 for R1, R2 and R3, respectively (Table 3, Fig. 3).

3.4 Fitness of L. rigidum populations grown in competition with wheat

Fresh weight of wheat, averaged over the three populations, was reduced by 12, 21, 30, and 34% and wheat ear number was reduced by 16, 22, 28, and 26% from the

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competition of 2, 4, 6, and 8 L. rigidum plants per pot, respectively (data not shown). Based on weed population by density interaction data presented in Fig. 4, the linear equation was the best fit for the regression performed between fresh weights or ear

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numbers against density of each population. The decreasing order of the calculated

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negative slopes for wheat fresh weight of S, R2 and R1 was 14.7 > 10.1 > 8, respectively, while the respective order for ear number negative slopes was 1.5 > 1.3

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> 0.8.

Fresh weight of L. rigidum grown in competition with wheat, averaged over the

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three populations, was increased from 39 to 41, 81, 95 g/pot and tiller number increased from 21 to 24, 40, and 50 with increasing density from 2, to 4, 6, and 8

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plants per pot, respectively (data not shown). The linear equation was the best fit for

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the regression performed between fresh weight and tiller numbers against density of each population (Fig. 4). The decreasing order of the calculated slopes for fresh weight of L. rigidum S, R1 and R2 populations was 18.1 > 6.7 > 6.3, respectively, while the order for tiller number was 6.9 > 5.6 > 3.2 for S, R2 and R1.

4. Discussion

The unsatisfactory control of R1, R2 and R3 L. rigidum populations with mesosulfuron-methyl + iodosulfuron-methyl and pinoxaden was due to evolved multiple resistance to these herbicides. Similar results were reported by others (Boutsalis et al., 2012; Matzrafi et al., 2017) who found L. rigidum populations with resistance

to

chlorsulfuron,

diclofop-methyl,

tralkoxydim,

cycloxydim,

and

pinoxaden. The lower control by mesosulfuron-methyl + iodosulfuron-methyl as

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compared with that of pinoxaden could be attributed to different level and type of resistance resulted from the agricultural practices applied by farmers. The amino acid substitution of Pro197 by Ala, Glu, Gln, Thr, Ser or Leu in all individuals (six from each of the R1, R2 and R3 population) surviving mesosulfuronmethyl + iodosulfuron-methyl treatment confirms the results obtained from the initial screening experiments and supports the evidence of resistance to this herbicide mixture. The Pro-197 substitutions by Ala, Gln, Leu, or Ser were also reported by Kaloumenos et. al. (2012) in their R L. rigidum populations originating from Greece. The detection of mutations in both alleles of 16 (five from R1 and R2, and six from

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R3 populations) out of 18 individuals surviving mesosulfuron-methyl + iodosulfuronmethyl application shows clearly that these plants were homozygous (RR), suggesting that resistance is widespread in these populations.

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Ile-2041 substitutions by Thr and Asn occurred in 8 (four R1, two R2 and two

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R3) out of the above 18 individuals surviving mesosulfuron-methyl + iodosulfuronmethyl treatment showing multiple resistance to pinoxaden and mesosulfuron-methyl

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+ iodosulfuron-methyl in these populations. Similar results were reported by others (Guo et al., 2015; Guo et al., 2016) who found either the Asp-2078/Gly and Trp-

aequalis individuals.

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574/Leu mutations or Ile-2041/Asn and Pro-197/Arg ones in the same R Alopecurus

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Ile-2041 substitution by Asn in 17 out of 18 individuals (one individual had Ile-

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2041/Thr) surviving pinoxaden application confirms the results of the initial screening experiments and supports the evidence of resistance to this herbicide. Similar results were reported by others (Delye et al., 2003, 2005; Zhang and Powles, 2006a) who detected Asn-2041 mutation in R L. rigidum and A. myosuroides individuals. The detection of Ile-2041 substitution by Asn mutation in both alleles of 8 (three R1, two R2 and three R3) out of 18 individuals survived pinoxaden treatment indicates that less than 50% of the individuals were homozygous (RR), suggesting that resistance to this ACCase herbicide not as abundant in the populations compared to ALS inhibitors. The presence of Trp-2027/Cys mutation in two R1 individuals that also had Ile2041/Asn or Asn-2041/Asn mutation, supports the coexistence of two mutations in the same accase gene. The Trp-2027/Cys mutation was also detected in R A. myosuroides individuals by Delye et al. 2003 (Delye et al., 2003), whereas other

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researchers (Yu et al., 2007; Zhang and Powles, 2006a, 2006b) also found R L. rigidum individuals carrying simultaneously two different accase point mutations. The 9 (three R1, three R2 and three R3) out of the 18 individuals that survived pinoxaden application also had mutations at Pro 197. This confirms the evidence of multiple resistance evolution to pinoxaden and mesosulfuron-methyl + iodosulfuronmethyl in these populations. Zangeneh et al. (2018) found that 94 and 75% of the 30 evaluated L. rigidum populations revealed resistance to ACCase- and ALS-inhibitors, respectively, whereas approximately 69% of these populations included individuals with multiple resistance to both herbicides. Preston et al. (1996) reported that a L.

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rigidum population in Australia was found containing multiple resistance not only to ALS (chlorsulfuron) and ACCase (diclofop, tralkoxydim, fluazifop, haloxyfop), but also to PS II (chlorotoluron, simazine) inhibitors. Moreover, Burnet et al. (1994) a

L.

rigidum

population

that exhibited

multiple resistance

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found

to

ALS

e-

(chlorsulfuron, triasulfuron, sulfometuron, imazaquin, imazapyr), ACCase (diclofop, tralkoxydim, sethoxydim), PS II (atrazine, simazine, ametryn, metribuzin), PS I

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(paraquat), and cell division (metolachlor) inhibitors. Multiple resistance occurs often in L. rigidum populations.

al

The higher mesosulfuron-methyl + iodosulfuron-methyl I50 values for ALS in the three L. rigidum R populations as compared with that of the S one shows clearly that

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resistance is due to altered ALS enzyme. In addition, these results confirm the

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findings of the whole-plant response assays and are in agreement with those reported by others (Kaloumenos et al., 2012; Yu et. al., 2010). The higher RF to mesosulfuronmethyl + iodosulfuron-methyl of the R1 population as compared with that of the R2 and R3 populations could be attributed to different amino acid substitutions in the codon Pro-197 (Delye et al., 2013; Papapanagiotou et al., 2015). The S population was more competitive against wheat than R1 and R2 populations and reduced wheat fresh weight and ear number by a greater amount than R1. The S population also had higher fresh weight and tiller number compared to R populations. These findings suggest that a fitness cost is associated with multiple target-site resistance in these populations. Vila-Aiub et al. (2009) identified a fitness cost associated with P450 metabolic resistance in L. rigidum accompanied with an impaired ability to compete for resources. Matzrafi et al. (2017) also found individuals from S L. rigidum population having significantly higher early vigor and grain weight as compared with plants of two R (substitution of isoleucine 1781 to

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leucine or cysteine 2088 to arginine) populations. Vila-Aiub et al. (2015a, 2015b) also found L. rigidum plants homozygous for the Asp-2078-Gly mutation exhibiting a significantly lower ACCase activity and plant growth rate (30%) than plants carrying the Ile-1781-Leu mutation and S wild-type genotypes. On the contrary, Zangeneh et al. (2016) found that competitive ability of one S and two R L. rigidum populations (with lle-1781/Leu or lle-2041/Asn) against wheat was similar, suggesting no apparent fitness penalty associated to ACCase-inhibitor resistance. Kaloumenos et al. (2012) also reported that R L rigidum populations originating from Greece, in the absence of competition, had similar growth pattern to that of S populations, meaning

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that the resistance-endowing ALS mutations did not result in detectable resistance fitness cost. Concerning fitness difference between the R populations studied by Kaloumenos et al. (2012) and those investigated in this study could be attributed to

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different type of resistance (populations with resistance to ALS inhibitors vs

e-

populations with multiple resistance to ALS and ACCase inhibitors), different resistant als and accase ratio (RS, RR), different origin (environment by genotype

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interaction), different experimental approach used (absence of competition for the R populations with ALS resistance vs weed/wheat competition for the R ones with

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multiple resistance), and different environmental conditions prevailing during the experiments.

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This study has shown that the unsatisfactory control of the R L. rigidum

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populations with mesosulfuron-methyl + iodosulfuron-methyl and pinoxaden was due to evolved multiple target-site resistance to these herbicides. The S population had higher competitive ability against wheat than R1 and R2 populations suggesting the occurrence of fitness cost due to multiple target-site resistance.

Acknowledgements We thank the Associate Editor and two anonymous reviewers for their constructive comments and grammatical changes, which helped us to improve the manuscript. References

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Babineau, M., Mathiassen, S.K., Kristensen, M., Kudsk, P., 2017. Fitness of ALSinhibitors herbicide resistant population of loose silky bentgrass (Apera spicaventi). Front. Plant Sci. 8, 1660. Boutsalis, P., Gurjeet S.G., Preston, C., 2012. Incidence of herbicide resistance in rigid ryegrass (Lolium rigidum) across Southeastern Australia. Weed Technol. 26, 391-398. Burnet, M.W.M., Hart, Q., Holtum, J.A.M., Stephen B. Powles, S.B., 1994. Resistance to nine herbicide classes in a population of rigid ryegrass (Lolium rigidum). Weed Sci. 42, 369-377.

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Cousens, R.D., Fournier-Level, A., 2018. Herbicide resistance costs: what are we actually measuring and why? Pest Manag. Sci. 74, 1539-1546. Cruz-Hipolito, H., Osuna, M.D., Domínguez-Valenzuela, J.A., Espinoza, N., De

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Prado, R., 2011. Mechanism of resistance to ACCase-inhibiting herbicides in

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wild oat (Avena fatua) from Latin America. J. Agric. Food Chem. 59, 7261– 7267.

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Délye, C., Zhang, X-Q, Chalopin, C., Michel, S., Powles, S.B., 2003. An isoleucine residue within the carboxyl-transferase domain of multidomain acetyl-CoA

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carboxylase is a major determinant of sensitivity to aryloxyphenoxypropionate but not to cyclohexanedione inhibitors. Plant Physiol. 132, 1716–1723.

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Délye, C., Zhang, X.Q., Michel, S., Matejicek, A., Powles, S.B., 2005. Molecular

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bases for sensitivity to acetyl-coenzyme A carboxylase inhibitors in black grass. Plant Physiol. 137, 794-806. Delye, C., Jasieniuk, M., Le Corre, V., 2013. Deciphering the evolution of herbicide resistance in weeds. Trends Gen. 29, 649-658. Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytoch. Bulletin 19, 11-15. Fernández-Moreno, B.T., Alcántara-de la Cruz, R., Smeda, R.J., and De Prado, R., 2017. Differential Resistance Mechanisms to Glyphosate Result in Fitness Cost for Lolium perenne and L. multiflorum. Front. Plant Sci. 8, 1796. Guo, W., Yuan. G., Liu, W., Bi, Y., Du, L., Zhang, C., Li, Q., Wang, J., 2015. Multiple resistance to ACCase and AHAS-inhibiting herbicides in shortawn foxtail (Alopecurus aequalis Sobol.) from China. Pestic. Biochem. and Physiol. 124, 66-72.

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Guo, W., Lingling L.V., Zhang, L., Li, Q., Wu, C., Lu, X., Liu, W., Wang, J., 2016. Herbicides cross resistance of a multiple resistant short-awn foxtail (Alopecurus aequalis Sobol.) population in wheat field. Chilean J. Agric. Res. 72, 163-169. Heap, I., 2019. International Survey of Herbicide Resistant Weeds.[Online].Available: http://www.weedscience.org (10.24.2019) Kaloumenos, N., Adamouli, V.N., Dordas, C.A., Eleftherohorinos, I.G., 2011. Corn poppy (Papaver rhoeas) cross-resistance to ALS inhibiting herbicides. Pest Manag. Sci. 67, 574-585. Kaloumenos, N.S., Tsioni, V.C., Daliani, E.G., Papavassileiou, S.E., Vassileiou, A.G.,

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Lautidou, P.N., Eleftherohorinos, I.G., 2012. Multiple Pro-197 substitutions in the acetolactate synthase of rigid ryegrass (Lolium rigidum) and their impact on chlorsulfuron activity and plant growth. Crop Prot. 38, 35-43.

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Kotoula-Syka, E., Tal, A., Rubin, B., 2000. Diclofop-resistant Lolium rigidum from

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northern Greece with cross-resistance to ACCase inhibitors and multiple resistance to chlorsulfuron. Pest Manag. Sci. 56, 1054-1058.

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Manalil, S., 2014. Evolution of herbicide resistance in Lolium rigidum under low herbicide rates: An Australian experience. Crop Sci. 54, 461-474.

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Matzrafi, M., Gerson, O., Rubin, B., and Peleg, Z., 2017. Different mutations endowing resistance to Acetyl-CoA carboxylase inhibitors results in changes in

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ecological fitness of Lolium rigidum populations. Front. Plant Sci. 8, 1078.

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Ntoanidou, S., Madesis, P., Diamantidis, G., Eleftherohorinos, I., 2017. Trp574 substitution in the acetolactate synthase of Sinapis arvensis confers crossresistance to tribenuron and imazamox. Pestic. Biochem. Physiol. 142, 9-14. Ntoanidou, S., Madesis, P., Eleftherohorinos, I., 2019. Resistance of Rapistrum rugosum to tribenuron and imazamox due to Trp574 or Pro197 substitution in the acetolactate synthase. Pestic. Biochem. Physiol, 154, 1-6. Osuna, M.D., Vidotto, F., Fischer, A.J., Bayer, D.E., De Prado, R., Ferrero, A., 2002. Cross resistance to bispyribac-sodium and bensulfuron-methyl in Echinochloa phyllopogon and Cyperus difformis. Pestic. Biochem. Physiol. 73, 9–17. Papapanagiotou, A.P., Kaloumenos, N.S., Eleftherohorinos, I.G., 2012. Sterile oat (Avena sterilis L.) cross-resistance profile to ACCase- inhibiting herbicides in Greece. Crop Prot. 35, 118-126.

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Papapanagiotou A.P., Paresidou, M.I., Kaloumenos, N.S., Eleftherohorinos, I.G., 2015. ACCase mutations in Avena sterilis populations and their impact on plant fitness. Pestic. Biochem. Physiol. 123, 40-48. Petit, C., Bay, G., Pernin, F., Délye, C., 2010. Prevalence of cross and multiple resistance

to

the

acetylcoenzyme

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clodinafop, and pinoxaden in black grass (Alopecurus myosuroides Huds.) in France. Pest Manag. Sci. 67, 168-177. Preston, C., Tardif, F.J., Christopher, J.T., Powles, S.B., 1996. Multiple resistance to dissimilar herbicide chemistries in a biotype of Lolium rigidum due to enhanced

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activity of several herbicide degrading enzymes. Pestic. Biochem. Physiol. 54, 123–134.

Scarabel, L., Panozzo, S., Varotto, S., Sattin. M., 2011. Allelic variation of the

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ACCase gene and response to ACCase-inhibiting herbicides in pinoxaden-

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resistant Lolium spp. Pest Manag. Sci. 67, 932-941. Scarabel, L., Pernin, F.,

and Délye. C., 2015. Occurrence, genetic control and

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evolution of non-target-sit based resistance to herbicides inhibiting acetolactate synthase (ALS) in the dicot weed Papaver rhoeas. Plant Sci. 238, 158-169.

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Seefeldt, S.S., Jensen, H., Fuerst, E.P., 1995. Log-logistic analysis of herbicide doseresponse relationships, Weed Technol. 9, 218-227.

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Spoor, W., McCraw, J.M., 1984. Self-incompatibility in Lolium - A reply. Heredity

Vila-Aiub, M.M., Neve, P., Powles, S.B., 2009. Evidence for an ecological cost of enhanced herbicide metabolism in Lolium rigidum. Journal of Ecology 97, 772– 780.

Vila-Aiub, M.M., Gundel, P.E., Preston, C., 2015a. Experimental methods for estimation of plant fitness costs associated with herbicide-resistance genes. Weed Sci. Special Issue, 203-216. Vila-Aiub, M.M., Yu, Q., Han, H., Powles, S.B., 2015b. Effect of herbicide resistance endowing Ile-1781-Leu and Asp-2078-Gly ACCase gene mutations on ACCase kinetics and growth traits in Lolium rigidum. J. Exper. Bot. 66, 4711-4718. Vila-Aiub, M.M., Yu, Q., Powles, S.B., 2019. Do plants pay a fitness cost to be resistant to glyphosate? New Phytol. 223, 532-547.

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Yanniccari, M., Vila-Aiub, M., Istilart, C., Acciaresi, H., and Castro, A.M. 2016. Glyphosate resistance in perennial ryegrass (Lolium perenne L.) is associated with a fitness penalty. Weed Sci. 64, 71–79. Yu, Q., Collavo, A., Zheng, M.Q., Owen, M., Satin, M., Powles, S.B., 2007. Diversity of acetyl-coenzyme A carboxylase mutations in resistant Lolium rigidum populations: evaluation using clethodim. Plant Physiol. 145, 547-558. Yu Q., Han, H., Vila-Aiub, M.M., Powles. S.B., 2010. AHAS herbicide resistance endowing mutations: effect on AHAS functionality and plant growth. J. Exper. Bot. 61, 3925–3934.

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Mutations of the ALS gene endowing

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Yu, Q., Heping, H., Powles, S.B., 2008.

resistance to ALS-inhibiting herbicides in Lolium rigidum populations. Pest Manag. Sci. 64, 1229-1236.

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Zhang, X.Q., Powles, S.B., 2006a. Six amino acid substitutions in the carboxyl-

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transferase domain of the plastidic acetyl-CoA carboxylase gene are linked with resistance to herbicides in a Lolium rigidum population. New Phytol. 172, 636-

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645.

Zhang, X.Q., Powles, S.B., 2006b. The molecular bases for resistance to acetyl co-

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enzyme A carboxylase (ACCase) inhibiting herbicides in two target-based resistant biotypes of annual ryegrass (Lolium rigidum) Planta 223, 550-557.

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Zangeneh, H.S., Chamanabad, H.R.M., Zand, E., Asghari, A., Alamisaeid, Kh.,

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Travlos, I.S., Alebrahim, M.T., 2016. Study of fitness cost in three rigid ryegrass populations susceptible and resistant to acetyl-CoA carboxylase inhibiting herbicides. Front. Ecol. Evol. 4, 142. Zangeneh, H. S., Chamanabad, H.R.M., Zand, E., Asghari, A., Alamisaeid, Kh., Travlos, I.S., Alebrahim M.T., 2018. Cross-and multiple herbicide resistant Lolium rigidum Guad. (rigid ryegrass) biotypes in Iran. J. Agric. Sci. Technol. 20, 1187-1200.

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10:2

20

10:6

10:4

10:8

Fig.1. Schematic presentation of wheat/L. rigidum density pattern (10:0 wheat pure stands, 10:2, 10:4, 10:6, 10:8) to assess wheat/weed responses (wheat = open circles vs. L. rigidum =

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Pr

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blue triangles).

100

a 80

S R1

a ab

R2

ab

ab

b

Tiller number (% of untreated)

Fresh weight (% of untreated)

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R3

60 c 40

d

d

20 0

e e

7.5+7.5

30+30

45

Mesosulfuron-methyl+iodosulfuron-methyl

100

180

21

a

b 80

R1

b

R2

c

R3

d

d d 60 40

e f

20 0

g g 7.5+7.5

30+30

45

Mesosulfuron-methyl+iodosulfuron-methyl

Pinoxaden

S

a

180 Pinoxaden

Herbicide rate (g ai/ha)

Herbicide rate (g ai/ha)

Fig. 2. Fresh weight and tiller number of one S and three R (R1, R2, R3) L. rigidum populations after the application of mesosulfuron-methyl + iodosulfuron-methyl and

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pinoxaden at field-recommended and four times the field-recommended rate. Columns

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Pr

e-

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within each graph with different letter indicate significant difference at P < 0.05.

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ALS activity (% of untreated)

100

22

80 60 R1

40

S

20 0

0 0.5 1 1.5 2 2.5 3 3.5 Mesosulfuron-methyl + iodosulfuron-methyl [Log(x+1)]

80 60

R2

40

S

20

0

0 0.5 1 1.5 2 2.5 3 3.5 Mesosulfuron-methyl + iodosulfuron-methyl [Log(x+1)]

f

80

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60 40

R3 S

20

pr

ALS activity (% of untreated)

100

e-

0 0 0.5 1 1.5 2 2.5 3 3.5 Mesosulfuron-methyl + iodosulfuron-methyl [Log(x+1)]

Fig. 3. In vitro ALS enzyme activity for one S and three R (R1, R2, R3) L. rigidum populations

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in the presence of mesosulfuron-methyl + iodosulfuron-methyl (nM).

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f 20

y = 37.6 - 1.5x (R² = 0.74) R1 y = 41 - 0.8x (R² = 0.94) R2 y = 36 - 1.3x (R² = 0.50) S

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10 0

2 4 6 8 Lolium rigidum density (plants/pot)

0

2 4 6 8 Lolium rigidum density (plants/pot)

75

Pr

120

80

al

40

S y = -13.5 + 18.1x (R² = 0.90) R1 y = 30 + 6.7x (R² = 0.88) R2 y = 20.5 + 6.3x (R² = 0.83)

0

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0

30

e-

0

40

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y = 263.2 - 14.7x (R² = 0.93) (R² = 0.93) R1 y = 254.2 - 8x (R² = 0.80) R2 y = 233 - 10.1x

S

100

140 L. rigidum fresh weight (g/pot)

Wheat ear number/pot

200

L. rigidum tiller number/pot

Wheat fresh weight (g/pot)

300

0

23

2 4 6 8 Lolium rigidum density (plants/pot)

60 45 30

S y = 2.5 + 6.9x (R² = 0.92) R1 y = 17.5 + 3.2x (R² = 0.98) R2 y = 3.5 + 5.6x (R² = 0.90)

15 0

0

2 4 6 8 Lolium rigidum density (plants/pot)

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Fig. 4. Linear equation and coefficient of determination of wheat fresh weight and ear number against L. rigidum density, as well as linear equation and coefficient of determination of L. rigidum fresh weight and tiller number grown with wheat.

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24

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Table 1 Nucleotide and deduced amino acid sequence alignment of als gene fragments from 6 individuals taken from the S population and 6 survived individuals from each of the three R L. rigidum populations after the application of mesosulfuron-methyl + iodosulfuron-methyl. These individuals were also used for accase sequencing. Populations

Susceptibilitya

Codon positionc

Genotype

Plants with a specific genotype

6

ALS mutation S

S

CCTb

Pro-197/Pro-197

R1

R R R

SMG GCG CAG

Glu/Ala or Pro/Gln Ala-197/Ala-197 Gln-197/Gln-197

R2

R R R

YCG CTG CAG

R R

ACG TCG

pr

1 2 3

Pro-197/Ser-197 Leu-197/Leu-197 Gln-197/Gln-197

1 3 2

Thr-197/Thr-197 Ser-197/Ser-197

3 3

e-

Pr

R3

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f

a

ACCase mutation

S R R

R2

S R R

S R a S, susceptible; R, resistant. R3

ATTb

Ile-2041/Ile-2041

6

ATT ACT AWT

Ile-2041/Ile-2041 Thr-2041/Thr-2041 Ile-2041/Asn-2041

2 2 2

ATT AWT ACT

Ile-2041/Ile-2041 Ile-2041/Asn-2041 Thr-2041/Thr-2041

4 1 1

ATT AWT

Ile-2041/Ile-2041 Ile-2041/Asn-2041

4 2

al

R1

rn

S

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S

b

IUPAC – IUB nucleotide for ALs codes S= G or C, M= A or C, Y= C or T.

b

IUPAC – IUB nucleotide for ACCase codes, W= A or T.

c

The ALS codon positions refer to the standard with A. thaliana (X51514.1)

c

The ACCase codon positions refer to the standard with A. myosuroides accase (AJ310767.1)

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Table 2 Nucleotide and deduced amino acid sequence alignment of accase gene fragments from 6 individuals taken from the S population and 6 survived individuals from each of the three R

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L. rigidum populations after the application of pinoxaden. These individuals were also used

Susceptibilitya

Codon positionc

Genotype

pr

Populations

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for als sequencing.

Plants with a specific genotype

e-

ACCase mutation

R1

R R R

R2

R R R

R3

R R

Ile-2041/Ile-2041

6

Trp-2027/Trp or Cys Asn-2041/Asn-2041 Ile-2041/Asn-2041

2 3 3

AWT AAT AYT

Ile-2041/Asn-2041 Asn-2041/Asn-2041 Ile-2041/Thr-2041

3 2 1

AWT AAT

Ile-2041/Asn-2041 Asn-2041/Asn-2041

3 3

Pr

S

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ATTb

S

rn

al

TGK AAT AWT

ALS mutation

S

S

CCGb

Pro-197/Pro-197

6

R1

S R R

CCG GCG CAG

Pro-197/Pro-197 Ala-197/Ala-197 Gln-197/Gln-197

3 2 1

R2

S R R R

CCG TCG CTG CYG

Pro-197/Pro-197 Ser-197/Ser-197 Leu-197/Leu-197 Pro-197/Leu-197

3 1 1 1

R3

S R R

CCG ACG TCG

Pro-197/Pro-197 Thr-197/Thr-197 Ser-197/Ser-197

3 2 1

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S, susceptible; R, resistant.

b

IUPAC – IUB nucleotide for ALS codes Y= C or T.

b

IUPAC – IUB nucleotide for ACCase codes, W= A or T, K= G or T, Y = C or T.

c

The ALS codon positions refer to the standard with A. thaliana (X51514.1)

c

The ACCase codon positions refer to the standard with A. myosuroides accase (AJ310767.1)

f

a

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Table 3

factor (RF) values for three R populations.

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Estimated mesosulfuron-methyl + iodosulfuron-methyl I50 ALS activity values and resistance

a

b

I50 (95% CL) (nΜ) 337 (190-485)

R2

227 (135-318)

R3

191 (158-223)

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21 (16-26)

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S

R2

16

67

0.922

0.84 (0.1)

11

67

0.903

1.38 (0.07)

9

40

0.964

0.60 (0.06)

1

60

0.835

1.02 (0.9)

al

R1

Res.MS

b Slope (SE)

Pr

Populations

e-

Μesosulfuron-methyl + iodosulfuron-methyl c

RF

a

S, susceptible; R, resistant (R1, R2, R3)

b

I50 : Μesosulfuron-methyl + iodosulfuron+methyl concentration for 50% reduction of the

L. rigidum ALS activity. c

RF (resistance factor) = I50 (R population) / I50 (S population)

Res.MS, residual mean of squares

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28

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Highlights - Lolium rigidum populations with coexisted multiple resistance to herbicides. - Pro-197 mutation resulted in resistance to ALS inhibiting herbicides. - Ile2041 mutation resulted in resistance to ACCase inhibiting herbicides.

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- Lolium rigidum fitness cost was associated with multiple resistance.

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Graphical abstract Pro197, Ile2041 and Trp2027 substitution in Lolium rigidum populations confers coexisted multiple resistance to ALS and ACCase inhibitors

ACCase sequence of the survived R11-R16 plants after pinoxaden application Trp2027

Ile2041

A. myosuroides AJ310767 Lolium (S)

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pr

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R11 R12 R13 R14 R15 R16

ALS sequence of the survived R11-R16 plants after pinoxaden application

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A. thaliana X51514 Lolium (S) R11 R12 R13 R14 R15 R16

Pr

Pro197