Sridevi Nkk*, Mithil Jugulm,Dlls Peterson,Mohmmd Asif

aHeartland Plant Innovations, Manhattan,KS 66506,USA

bDepartment of Agronomy,Kansas State University,2004 Throckmorton Plant Sciences Center,Manhattan, KS 66506,USA

Keywords:Clearfield Coaxium Cytochrome P450s GSTs Herbicide resistance Wheat production systems

ABSTRACT Herbicide resistance in crops has extended the scope of herbicide applications to control weeds. The introduction of herbicide resistant crops resulted in a major shift in the way that herbicides are used in many crops, but not necessarily increased the prevalence of herbicide use, especially in wheat. Wheat is one of the most widely grown crops in the world and currently only two major herbicide-resistant wheat groups have been commercialized to manage weeds in a cost-effective manner. However, sustainable wheat production is threatened by the expanding occurrence of herbicide-resistant weed populations with limited efforts to discover new herbicide molecules. Selective control of certain problematic weeds in wheat was impossible until development and introduction of the technologies, Clearfield and CoAXium Production Systems. However, the current limitations of reliance on specific herbicides and evolution of resistant weeds mandate precautions and considerations when using these systems to prevent the loss of existing herbicide resources and continue sustainable wheat production.The focus of this review is to provide an overview of natural pre-existing herbicide resistance and development of herbicide-resistant technologies in wheat. The mechanisms of resistance to herbicides in wheat as well as the weed populations in wheat cropping systems, and implications for weed management are discussed.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751

2. Herbicide resistance in wheat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751

2.1. Herbicide detoxification via GST activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752

2.2. Herbicide detoxification via cytochrome P450 monooxygenases . . . . . . . . . . . . . . . . . . . . . . . . . . 752

3. Wheat production systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752

3.1. Clearfield production system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753

3.2. CoAXium production system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753

3.3. Roundup Ready wheat. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753

3.4. Stewardship of herbicide-tolerant wheat production systems . . . . . . . . . . . . . . . . . . . . . . . . . . 753

4. Herbicide selection and evolution of resistance in weed species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754

5. Herbicide resistant weeds in wheat cropping systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754

5.1. Herbicide resistance in grass weeds in wheat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755

5.2. Herbicide resistance in broadleaf weeds in wheat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756

6. Management of herbicide resistant weeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756

7. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 757

Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 757

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 757

1. Introduction

Wheat (Triticum aestivum L.) contributes significantly to the world's agricultural economy by occupying about 218.5 Mha of arable land, more than any other cultivated food crop, with grain production of 771.7 Mt in 2017 [1]. Sustainable wheat production is critical to meet global food security as the arable land area for cropping is decreasing. Weed infestation and interference during various stages of wheat development is a major impediment and threat to wheat production across the globe.Use of herbicides for weed management in both no-till and non-cropland areas has been highly effective in developed countries [2,3]. A consequence of extensive and intensive use of herbicides has been the evolution and/or spread of herbicide-resistant weeds in many crop production systems,including wheat. According to The International Survey of Herbicide Resistant Weeds, wheat ranks first in reported number of herbicide resistant weed species, a total of 77 species with 140 unique herbicide resistance cases globally[4].

The most common and economically troublesome monocot and dicot weeds in wheat cropping systems include Lolium spp.(annual ryegrass), Alopecurus spp. (black-grass), Phalaris spp.(canary grass),Avena spp.(wild oats),Bromus spp.(brome grass),Raphanus raphanistrum L. (wild radish), Chenopodium album L.(lambsquarters), and Kochia scoparia L. (kochia) [5-10]. When weeds are left uncontrolled yield losses in wheat range from 10%to 50% depending on the weed density and duration of interference [11-13]. Application of herbicides to control weeds has been very effective and efficient in terms of production costs and benefits. However, the adoption of no-till agricultural production [14]and repeated dependence on herbicides for weed management led to selection pressure and thus, the evolution of herbicide-resistant weeds.

Herbicides have physical and chemical properties that allow them to be absorbed after initial contact with plant tissues；they are then translocated to target-sites,followed by injury and death in the case of a susceptible weed or crop plant species.Control of a weed species depends on the mode of action of the herbicide.The mode of action is defined as the sequence of biochemical and physiological events occurring in the plant from the time the plant comes in contact with the herbicide until its complete death. However, when a plant is resistant to a herbicide, the herbicide is ineffective primarily due to two mechanisms:a)alterations in the herbicide targetsite； or b) detoxification (metabolism) or sequestration of herbicide during absorption and translocation before reaching the target site[15-18].Thus,herbicide resistance mechanisms are broadly classified into two types: target-site resistance and non-target-site resistance.

2. Herbicide resistance in wheat

Resistance to herbicides in crop plants, including wheat, is primarily achieved via non-target-site mechanisms through detoxification of the herbicides via activity of enzymes such as cytochrome P450 monoxygenases (CYP450s) and glutathione S-transferases (GSTs) [19-24]. Examples of herbicides metabolized by these enzymes are listed in Table 1 and include:chlorotoluron in wheat,corn and cotton[25],fenoxaprop-p-ethyl in wheat and barley [26], bentazoan in soybean [27], atrazine in corn and sorghum [28], clomazone in cotton [29], pyrazosulfuron ethyl inrice[30],and clodinafop in wheat,barley,and corn[22].However,wheat is more sensitive to many herbicides than other crops such as corn,sorghum,rice and soybean.Only a few herbicides can be used for pre-plant,and pre-or post-emergence control of weeds in wheat. Also, the herbicides that are registered have different specific guidelines for use on winter wheat or spring wheat.Synthetic auxins,acetyl CoA carboxylase(ACCase)-,acetolactate synthase (ALS)- microtubule-, photosystem II (PS-II), and longchain fatty acid synthesis-inhibitors(Table 1)play a critical role in weed management in wheat.

Table 1-Herbicides metabolized by cytochrome P450 monooxygenases and glutathione-S-transferases.

Natural resistance in wheat to different herbicides varies depending on cultivar, time of application (pre-planting, preor post-emergence), soil and environmental conditions, and use of protective compounds known as safeners. Therefore,wheat growers should closely follow labeling recommendations to avoid crop injury. Typical herbicide injury symptoms in wheat vary from yellowing of leaf tips to reduction in tiller numbers and more severe symptoms depending on the herbicide mode of action. Herbicide safeners are agrochemicals that enhance herbicide resistance in grass crops and protect them from injury caused by some herbicides [31-34].Additions of safeners, such as mefenpyr diethyl and fenchlorazole ethyl to herbicides, enhance the expression of GSTs[34-36]and cytochrome P450s[37]and play an important role in the detoxification process in crops including wheat.

2.1. Herbicide detoxification via GST activity

GSTs are multifunctional enzymes involved in plant growth and development, cellular metabolism and detoxification of xenobiotic compounds such as pesticides[38].GSTs catalyze the transfer of a tripeptide, γ-glutamyl-cysteinyl-glycine known as glutathione(GSH)to an electrophilic reaction center of many substrates of endogenous and exogenous origin [39]. In plants, GSTs are encoded by a large diverse gene family classified into three types(I,II,and III)based on the amino acid sequence and intron:exon distribution[40].The significance of GSTs in herbicide detoxification was documented in 1970 when atrazine was conjugated with GSH by a maize GST activity,protecting the crop from herbicide injury [24]. Since then, many GSTs induced by herbicides were isolated and characterized for their role in herbicide tolerance,selectivity, and resistance in many crops and weed species[38,41]. GSTs are known to metabolize and detoxify atrazine,alachlor, metolachlor, fluorodifen, chloroacetanilide, and thiocarbamates. Corn contains high levels of GSTs that provide natural tolerance to triazines,alachlor,metolachlor,and s-ethyl dipropyl thiocarbamate(EPTC)[23,24,42,43].However,seed treatment with safener is required in sorghum if alachlor and metolachlor herbicides are used for pre-emergence weed control.Many susceptible crops like wheat, barley, peas, and several broadleaf weeds do not have increased GST activity [44].Nevertheless,these crops can tolerate the herbicides when used with safeners. In wheat, quinolinoxycarboxylic cloquintocet mexyl, mefenpyr diethyl and fenchlorazole ethyl are widely used as post-emergence safeners. These compounds can help reduce injury in wheat from aryloxyphenoxypropionate (AOPP/FOP) herbicides that inhibit the essential enzyme acetyl CoA carboxylase (ACCase). This is because the tau class of GSTs(GSTU) is induced in wheat when fenchlorazole-ethyl is used with dimethenamid and fenoxaprop, thereby increasing the cleavage of the herbicides by glutathionylation to confer resistance [36]. Cloquintocet mexyl is known to increase the hydroxylation rate, cleavage of ether, and glucosylation of the herbicide clodinafop propargyl,another ACCase-inhibitor[22].In addition to safening fenoxaprop-ethyl herbicide in wheat,mefenpyr diethyl enhances the detoxification and selectivity of the unrelated sulfonylurea herbicides such as mesosulfuronmethyl and iodosulfuron methyl-sodium [45]. Various chemical classes of safeners have been developed for each major cereal to protect it from herbicide injury by means of increased GST activity.

2.2. Herbicide detoxification via cytochrome P450 monooxygenases

Cytochrome P450s are heme-containing monooxygenases involved in both biosynthetic and detoxification pathways in many organisms including plants[46,47].Cytochrome P450s are mixed or multi-functional oxidoreductases that use NADPH and/or NADH to cleave oxygen molecules to form an organic substrate with a functional group and water. Some cytochrome P450s are tissue- or substrate-specific and regulated by specific genes.However, many different cytochrome P450s can act on a single substrate, and a single cytochrome P450 can have several substrates [48-51]. Chlorotoluron is metabolized by cytochrome P450s through oxidative N-demethylation and hydroxylation of the ring-methyl group in wheat and barley [52]. Similar to their role in increasing GST activity,safeners also increase cytochrome P450 activity. Wheat seedlings treated with safeners naphthalic anhydride and phenobarbital increase the activity of several cytochrome P450s that catalyze hydroxylation of diclofop herbicides and lauric acid [53,54]. To assess whether one or more cytochrome P450s are involved, irreversible inhibitors of lauric acid hydroxylase such as acetylenic compounds (10- and 11-dodecynoic acids) showed that a single cytochrome P450 in wheat was responsible for catalyzing both diclofop and laurate hydroxylation [54]. However, herbicides such as bentazon and chlorotoluron are not affected by these inhibitors.

3. Wheat production systems

In addition to non-target-site herbicide detoxification,target-site resistance in crops can be obtained by altering,amplifying or over expressing the target gene.However,only alteration of the target gene has been successfully employed in the development and commercialization of herbicide resistant crops[55-57].Herbicides that inhibit amino acid biosynthesis are ideal for developing target-site herbicide resistant crops[58].The 3 major amino acid biosynthesis inhibitors include,ALS,5-enol pyruvylshikimate 3-phosphate synthase (EPSPS) and glutamine synthetase (GS).Generally, herbicide resistant crops are commercialized under the trade names Clearfield, Roundup Ready, and LibertyLink,respectively[57].These were developed by selection,mutagenesis or genetic engineering and transformation [59]. In wheat,target-site resistance, particularly, ALS- and ACCase-inhibitor resistant cultivars,has been developed through mutagenesis and commercialized as Clearfieled and Coaxium wheat productions systems,respectively.

3.1. Clearfield production system

Herbicide resistant wheat cultivars were developed to meet farmer demand to control weeds in wheat without crop injury or loss of grain yield. BASF first developed the Clearfield technology in corn hybrids in the early 1990s.The technology was subsequently applied in canola, rice, sunflower, and finally in wheat in the late 1990s and early 2000s. Clearfield wheat cultivars resistant to imidazolinone (ALS-inhibitor)herbicides were commercially released for cultivation in the Great Plains and Pacific Northwest wheat growing regions of the USA in 2002 and 2003. ALS-inhibitors are herbicides that inhibit the enzyme acetolactate synthase and control grass and broadleaf weeds in many cropping systems. The ALS enzyme catalyzes the indispensable first step in biosynthesis of branched-chain amino acids leucine,isoleucine,and valine[60,61].

Clearfield wheat genotypes were developed by ethyl methanesulfonate(EMS)mutagenesis.The mutation occurred in the ALS gene on the long arm of chromosome 6D [62-64].Mutants with single nucleotide polymorphisms in homologous ALS genes in the A and B genomes were also obtained by mutagenesis. Single gene or two-gene imidazolinoneresistant wheat cultivars were developed and released for commercial cultivation following backcrossing and advanced plant breeding procedures [65,66]. Each imidazolinoneresistant wheat line carries a mutation that produces a serine to asparagine substitution at amino acid position 653 in the ALS protein relative to the Arabidopsis thaliana consensus sequence.The Clearfield wheat production system allows the use of imazamox herbicide (Beyond, BASF, EPA Reg. No.241-244) to control many weeds including downy brome(Bromus tectorum L.), feral rye (cereal rye), jointed goatgrass,wild oat, mustards, and volunteer wheat and barley. “Clearfield Plus” wheat is similar to Clearfield wheat, but has two mutant genes conferring resistance to imidazolinone herbicides in wheat varieties.Two-gene cultivars have“Plus”or“+”in the name indicating ALS mutations on both the A and D,or B and D, genomes. More than 20 Clearfield winter and spring wheat cultivars have been developed by BASF in collaboration with private and public breeding programs.

3.2. CoAXium production system

The Colorado Wheat Research Foundation and Colorado State University initiated and developed the CoAXium Wheat Production System in collaboration with Albaugh LLC and Limagrain Cereal Seeds. AXige is the patented trait that confers the herbicide tolerance and CoAXium is the system.CoAXium/AXigen wheat cultivars are resistant to the ACCaseinhibitor herbicide quizalofop-p-ethyl (Aggressor, Albaugh LLC, EPA Reg. No. 42750-313). CoAXium wheat production system allows the use of quizalofop-p-ethyl (Aggressor) to control many grassy weeds such as feral rye, downy brome,jointed goatgrass, and ALS-resistant grasses. However, other herbicides should be used to control broad leaf weeds.ACCase-inhibitors interfere with lipid biosynthesis by inhibiting the ACCase enzyme that catalyzes the primary reaction of converting acetyl co-enzyme A to malonyl coenzyme A, in de novo synthesis of fatty acids [67,68]. The AXigen trait in wheat was also developed by EMS mutagenesis, specifically by treating winter wheat (cv. Hatcher) with 60 mmol L-1EMS and screening the M2and M2:3populations with quizalofop to identify herbicide-tolerant plants [69].Twenty mutants tolerant to quizalofop were identified, all with the amino acid substitution alanine to valine at position 2004 (A2004V) in the ACCase protein, where the position corresponds to the Alopecurus myosuroides reference sequence.The substitution was found in each of the A,B,and D genomes and each one confers resistance to ACCase herbicides [69].However,A2004V mutation in B genome confers lower level of resistance to quizalofop compared to the mutation in the A or D genomes and specifically, the A2004V change does not confer resistance to the cyclohexanedione class of ACCaseinhibitor herbicides. CoAXium is commercialized as a twogene (A and D genome) trait obtained by crossing the individual mutant lines. Such lines confer high levels of resistance to quizalofop relative to single-gene AXigen genotypes. ‘Incline AX' and ‘LCS Fusion AX' released in 2018 were the first hard red winter wheat (HRWW) with CoAXium technology. Future CoAXium varieties are being developed by both public and private breeding programs to provide diverse germplasm options for growers.

3.3. Roundup Ready wheat

Glyphosate resistant wheat was developed by Monsanto by insertion of a cassette containing the coding sequence of CP4 EPSPS from Agrobacterium strain CP4 into Bobwhite background. The Roundup Ready wheat lines MON 71800 was developed with the idea to benefit farmers and improve weed control in wheat and rotation cropping systems.The benefits include reductions in herbicide injury to wheat, simplified control of herbicide-resistant weed biotypes, elimination of other cereals and off-type wheat within a crop,and increased control or suppression of perennial weeds [70,71]. However,the risks outweigh the advantages of Roundup Ready wheat and include difficulty and increased cost to control volunteer wheat, development of glyphosate resistant weeds in roundup ready dependent cropping systems, gene flow and out crossing into non-GMO wheat, increased seed costs, and loss of farmer saved seed options for wheat.In addition,there is risk of contamination of wheat segregated for sale as non-GMO and increased costs in the market system [70,71].Although farmers were ready to embrace the technology consumer rejection of the end product and fear of loss of potential markets around the world has led to the withdrawal of the Environmental Protection Agency (EPA) application by Monsanto and it should be noted that Roundup Ready wheat is not currently grown.

3.4. Stewardship of herbicide-tolerant wheat production systems

The CoAXium wheat production system provides winter wheat farmers with an option to use a new herbicide mode of action in addition to BASF's Clearfield cultivars,which have been in the market for nearly 20 years.Both technologies were generated by mutagenesis and are thus marketed as non-GMO wheat with wider global acceptance. However, to use these technologies more effectively and to reduce the risk of developing herbicide resistant weed populations, certain guidelines should be followed when producing CoAXium and/or Clearfield cultivars. The herbicide Aggressor controls only grassy weeds, and to control broadleaf weeds in CoAXium/AXigen cultivars, a broadleaf herbicide must also be used. Adjuvants recommended when spraying Aggressor include methylated seed oil(MSO),crop oil concentrate(COC)and non-ionic surfactants(NIS). The herbicide can be applied with nitrogen-based foliar fertilizers and also tank-mixed with a wide array of broadleaf herbicides for broadleaf weed control.However, it is critical not to tank-mix Aggressor with MCPA or 2,4-D amine formulations due to antagonistic interactions (http://www.coaxiumwps.com/stewardship/).

The herbicide Beyond controls both grass and broadleaf weeds in the Clearfield system. Application times of the herbicide and adjuvants used differ between one-gene and two-gene Clearfield cultivars. Beyond with MSO can be applied post-tiller initiation until the jointing stages of wheat in 2-gene Clearfield cultivars, but MSO cannot be used on 1-gene cultivars at any time due to crop injury.NIS can be applied to 2-gene cultivars from the 2-leaf to second-joint stages, but only after tiller initiation until jointing on 1-gene cultivars. In addition, a nitrogen-based fertilizer such as ammonium sulphate (AMS) or 28% urea ammonium nitrate(UAN) should be added to the spray solution for both Clearfield cultivars.

It is important to be aware that the AXigen trait is not compatible with the Clearfield system, i.e., Aggressor will kill Clearfield wheat and Beyond will kill CoAXium wheat.Both herbicides will kill wheat without the herbicideresistant traits. Farmers must sign a stewardship agreement in order to purchase and plant seed of either technology, indicating that they will only plant certified seed and cannot save the harvested seed for future planting.It is recommended that growers should rotate crops,herbicide modes of action, not use these production systems more than two years in a row, use a fallow period,and use glyphosate in fallows to limit weed seed production.The stewardship guidelines and recommendations are intended to help,preserve and maximize the efficacy of the technologies and to prevent the selection and spread of herbicide-resistant weeds as a result of repeated use of specific herbicides. The summary of these two wheat production systems are given below in a table(Table 2).

4.Herbicide selection and evolution of resistance in weed species

Widespread and recurrent application of herbicides to control weeds exerts a strong selection pressure and ultimately results in the evolution and spread of herbicide-resistant weeds. Weed populations naturally contain individual plants that are resistant to herbicides, regardless of the actual herbicide [72,73], but the frequencies are likely to be extremely low and it is usually impossible to distinguish susceptible individuals in the population.Repeated applications of herbicides with the same mode of action select the herbicide-resistant individuals that survive andreproduce. Continued use of herbicides with the same mode of action results in rapid spread of resistant individuals that eventually become the dominant component of the population.

Table 2-Summary of wheat production systems in use.

Besides the selection pressure from herbicide use other factors such as the biological characteristics of individual weed species, genetic factors, characteristics of herbicides,and agronomic practices also play important roles in the evolution and spread of herbicide resistance in weed species[74].Characteristics that facilitate rate of increase in herbicide resistant populations include plant fecundity, germination rates, extended germination periods, seed dispersal mechanisms, seed dormancy, and initial frequency of resistant individuals.The genetic factors influencing rates of herbicide resistance include mutation frequency, and fitness costs of resistance genes in the presence and absence of herbicide use[72,75].When the initial frequency of resistant plants is high,there will be a rapid development of resistance. On the other hand,if resistance genes are rare in the weed population,the increase in frequency of resistant plants could take many years or not develop at all. Herbicide characteristics such as structural properties, site/mode of action, residual activity,and agronomic practices such as applying lower than recommended rates can influence development of herbicide resistance. Time of application or weed size, climatic conditions,and crop rotation practices are important factors. Recent advances in agronomic practices involving no-till and reduced-till are dependent on herbicides, and hence increase the selection pressure imposed upon weed populations.

5. Herbicide resistant weeds in wheat cropping systems

Many weed species in wheat cropping systems have become resistant due to increased reliance on herbicide use and the selection pressure that is imposed. To date, about 330 individual reports of resistance have occurred across wheat growing areas of the world [4]. Many weeds have evolved resistance to different chemical classes and mode-of-action herbicides. In addition, there are reports of cross resistance and multiple resistance to herbicides due to either target-site alterations or non-target-site-based mechanisms in several agronomically problematic weed species[4].

Target-site herbicide resistance in weeds can be conferred by point mutations resulting in amino acid substitutions, over expression,and/or amplification of target genes or through gene regulatory changes (in gene promoters) in target-sites. Alterations caused by point mutations in the target-site prevent or reduce the binding of herbicide to the target. Amplification or increased expression of the herbicide target gene can produce more target protein without affecting the normal functioning of the plant [76,77]. Target-site based herbicide resistance mechanisms are more common to some mode-of-action herbicides than others,such as ACCase-[78],ALS-[79,80]and PS II-inhibitors[81]due to the flexibility of the protein structures to function normally along with inhibition of herbicide binding.

Non-target-site herbicide resistance involves one or a combination of several mechanisms that limit the quantity of herbicide reaching the target-site. These mechanisms include reduced absorption, decreased translocation, and enhanced metabolism of the herbicides. The cytochrome P450 and GST families that are involved in rapid detoxification of herbicides in crop plants such as wheat can also provide resistance to herbicides in weed species.

The most commonly reported mechanism of resistance to ACCase- and ALS-inhibitors in weed species involves alteration(point mutation)leading to a single amino acid substitution in the target protein. In plants there are two isoforms of the ACCase gene, plastidic ACCase and cytosolic ACCase essential for biosynthesis of primary and long-chain fatty acids, respectively[82]. Cytosolic ACCase in all plant species is homomeric and function similarly but structural differences in plastidic ACCase make plants sensitive to ACCase-inhibitors.In dicots,ACCase in chloroplast is heteromeric and insentive to herbicides,whereas it is homomeric in all grass species and sensitive to ACCase herbicides[83].Thus,ACCase inhibiting herbicides are popularly known as graminicides due to their selective control of grass weeds in many cropping systems.Many grass weeds are resistant to ACCase-inhibitors in different cropping systems. So far, 11 mutations affecting seven amino acid residues in the ACCase gene and herbicide sensitivity have been identified[4,74,78,84].

Similarly,ALS mutations involve 26 different substitutions at eight amino acid positions [83]with changes at the proline197 amino acid position being the most common,with eleven substitutions conferring resistance to the sulfonylurea class of herbicides [85]. Aspartate376glutamate and trptophan574leucine substitutions confer high-level, broad spectrum resistance to five chemical classes of ALS-inhibitors[86-88]. However, the examples of herbicide resistance and their mechanisms discussed here are weeds and herbicides widely used in wheat cropping systems.

5.1. Herbicide resistance in grass weeds in wheat

Downy brome, commonly called cheat-grass, is a winter annual grass weed with wide adaptability to different soil and climate conditions. It is a troublesome weed of winter wheat and infestation can cause wheat yield losses up to 92%[89,90]. The ALS-inhibitors, e.g., sulfosulfuron, propoxycarbazone,and pyroxsulam,are commonly used to selectively control downy brome in winter wheat. Imazamox can also provide control of downy brome in Clearfield wheat.However,ALS-resistant downy brome identified in Clearfield wheat in Montana had cross-resistance to other chemical classes of ALS-inhibitors. The ALS resistant downy brome had mutations in the target-site,leading to Ser653Asn substitution[91].Feral rye,also a damaging winter annual grass weed in wheat,reduces grain quality and yield by approximately 69% at 50-200 rye plants per square meter [92,93]. Imazamox provides inconsistent control of feral rye but improved control can be achieved when it is tank-mixed with MCPA-ester to increase uptake of the herbicide [94]. Jointed goatgrass is an invasive grass species introduced from Europe and is highly problematic in western United States. Non-selective herbicides such as glyphosate, paraquat or glufosinate can be applied during fallow or prior to planting to control jointed goatgrass.Selective herbicides can also be used depending on the production system. Imazamox provides selective control of feral rye, downy brome, jointed goatgrass and other grass weeds in Clearfield wheat systems. However, there is differential response to imazamox between the three species due to differences in absorption, translocation and metabolism of the herbicide.Absorption of14C imazamox in feral rye,downy brome and jointed goatgrass varies depending on the adjuvant used.Translocation and metabolism of the herbicide was higher in feral rye than in jointed goatgrass [95], which also has potential to become resistant to imazamox through gene flow between wheat and jointed goatgrass due to cross compatibility, and genetic similarities and similar growth habits [96]. Wheat (genomes AABBDD) and jointed goatgrass(CCDD)have the D genome in common,and hybridization and natural backcrossing in the field could lead to transfer of herbicide resistance genes from wheat to jointed goatgrass through normal chromosome recombination[97].

Rigid ryegrass(Lolium rigidum Gaudin),a native to Mediterranean countries,the Arabian Gulf and Indian sub-continent,is an invasive grass species introduced to North and South America,South Africa and Australia[98].It was introduced to Australia during the 19th century as a forage grass, but subsequently became a major weed in wheat crops in Southern and Western Australia. ALS- and ACCaseinhibiting herbicides were commonly used for control [99],but many populations have developed high levels of resistance to chlorsulfuron, sulfometuron, diclofop-methyl, and clethodim [100,101]. It has developed cross resistance and multiple resistance to other mode-of-action herbicides, including microtubule- and PS II-inhibitors, as well as ALS and ACCase-inhibitors[102-104].Rigid ryegrass has evolved resistance to seven mode-of-action herbicides in Australian wheat cropping systems [4,105]. Rigid ryegrass populations in Western Australia are resistant to ALS herbicides due to target-site or non-target-site resistance mechanisms. Resistance to sulfometuron-methyl was associated with alterations at the proline197 position in the ALS protein. Four amino acid substitutions (P197S, P197T, P197Q, and P197R)were detected in one population,whereas another population had P197S and W574L mutations that conferred resistance to both imazapyr and sulfometuron-methyl [106]. Non-target site resistance to chlorsulfuron due to metabolism of the herbicide was found in the same study.

Low and sub-optimal doses of herbicides have been shown to trigger polygenic metabolic resistance in many weeds,including rigid ryegrass [107,108]. In a study to assess the potential for evolution of herbicide resistance and phenotypic variation in the level of resistance to suboptimal rates of diclofop-methlyl, a susceptible rigid ryegrass biotype (VLR1)was selectively sprayed with various lower than recommended rates (37.5 g ha-1). The low rates of diclofop-methyl selected genetic variants and produced resistant phenotypes through genetic recombination and selection over three generations [109]. The genes involved in metabolic herbicide resistance in rigid ryegrass were investigated by transcriptomic RNA-Seq analysis. Two cytochrome P450s(CytP450), glucosyltransferase (GT), and nitronate monooxygenase (NMO) showed constitutively increased expression in resistance compared to susceptible individuals in a segregating F2population,along with expression differences in metabolism and signal transduction-related genes [110].Species closely related to rigid ryegrass, such as perennial ryegrass(L.perenne L.)and Italian ryegrass(L.multiflorum Lam.)have evolved resistance to glyphosate which is applied during fallow periods in wheat rotations in Australia and other countries.

Wild oat (Avena fatua) is another economically troublesome weed in wheat production systems across the world.Wild oat resistant to ACCase-inhibitors has been reported in 17 countries, with most populations having multiple resistance[4].In Western Australia,individuals from four wild oat populations resistant to ACCase-inhibitors were sequenced and identified with mutations in the ACCase gene causing one, two or three amino acid substitutions [111]. In some resistant individuals target-site mutations were not found；these plants had non-target-site resistance mediated by enhanced metabolism of diclofop-methyl[112],thus showing that an individual plant can have two independent resistance mechanisms for the same herbicide.

5.2. Herbicide resistance in broadleaf weeds in wheat

Many broadleaf weeds economically impact wheat production. Some of the most common summer weeds are field bindweed(Convolvulus arvensis L.),wild buckwheat(Polygonum convolvulus L.), and kochia (Basia scoparia (L.) Scott) whereas winter broadleaf weeds include wild mustards (Brassica spp.),common chickweed (Stellaria media (L.) Vill), prickly lettuce(Lactuca serriola L.), Russian thistle (Salsola tragus L.), field pennycress(Thlaspi arvense L.),and shepherd's-purse(Capsella bursa-pastoris (L.) Medik). Along with control of summer annual broadleaf weeds, spring herbicide applications are also effective for winter weed control. However, weather conditions and timing (weed and crop growth stage) are critical to achieve good control. ALS and synthetic auxin herbicides are widely used to control these weeds. Sulfonylurea herbicides, metsulfuron, prosulfuron, and triasulfuron are effective on most winter annual broadleaf weeds but control of mustards is difficult with sulfonyl ureas alone as these plants bolt early. Tank mixing with 2,4-D improves the control of mustards. Dicamba and 2,4-D are often combined and applied to achieve broad-spectrum broadleaf weed control compared to when used alone. However, care should be taken to avoid injury to wheat when applying these herbicides because of a very narrow window of application time during which both herbicides can be safely applied.

Kochia(Kochia scoparia L.Schrad)is a summer annual weed in crops including wheat with emergence in early spring extending to late August to early September. The residual activity of ALS-herbicides applied early in spring controls late emerging kochia in wheat but is ineffective on ALS-resistant kochia.Dicamba,is more effective but must be applied before jointing. ALS-resistant kochia is widespread in the USA and Canada with a few populations also exhibiting cross resistance to PS II-, EPSPS-inhibitors and synthetic auxins [4].Resistance in kochia to ALS-inhibitors is conferred by three different target-site mutations (Pro197, Asp376, or Trp574) as well as two-mutation combinations (Pro197 + Trp574 and Asp376 + Trp574) in highly resistant individuals [113]. The mechanism of glyphosate resistance in kochia in wheat fallow fields is increased copy number of the EPSPS gene [114], and the mechanism of resistance to synthetic auxins is unknown.

Russian thistle (Salsola tragus L.) is another weed that poses a severe management problem in summer fallow,spring wheat and other crops, such as pulses, canola and mustard, in the USA [115,116]. Season-long competition from Russian thistle can reduce wheat yields by as much as 50% [117]. ALS-inhibitor resistant Russian thistle was first reported in wheat fields in Washington and Montana in 1987 and currently more than 75% of the wheat fields are infested [118,119]. Glyphosate is widely sprayed prior to planting wheat and post-harvest as a burndown application. Repeated applications led to increased numbers of glyphosate resistant Russian thistle populations and the mechanism of glyphosate resistance is yet to be investigated [120]. Other broadleaf weeds have also evolved resistance to commonly used ALS-inhibitors used in wheat cropping systems, and appropriate weed management strategies must be implemented to minimize the spread of resistant weeds.

6. Management of herbicide resistant weeds

With increased presence of herbicide-resistant weeds in cropping systems, we must discourage practices that impose high selection pressures and increase gene frequencies and try to prevent the increase and spread of mutant weed biotypes by implementing integrated control measures. Integrated weed management strategies include the use of cultural, mechanical and chemical tools to control weeds and to maximize the longevity of individual herbicides in cropping systems.The most common strategy is to reduce the intensity of selection of resistant individuals by sequential application of different herbicide mixtures or rotation of herbicides with different modes of action over multiple growing seasons. It is very important to follow label recommendations to prevent metabolic resistance due to low rates,which help to reduce the survival and reproduction of resistant individuals [75]. Management strategies that reduce and eliminate the spread of resistance by means of pollen movement, seed production, and propagule dispersal should be implemented. However, without integrated management practices it is not possible to produce weed free crops, and producers should take advantage of cultivars with greater competitiveness [121]. Understanding the physiological, genetic, biochemical, and molecular mechanisms by which weeds evolve herbicide resistance provides an insight on the survival of weeds to herbicide applications. Unraveling the multiple herbicide resistance mechanisms will have significant impact to delay the evolution of resistance and contribute to discovery of alternate new herbicides, wiser use of existing herbicide resources, and more sustainable strategies for weed control.

7. Conclusions

Herbicide resistant wheat production systems offer exceptional and viable weed control strategies in wheat cropping systems.Beyond herbicide can be effectively used to control both grass and broadleaf weeds in Clearfield wheat production and Aggressor shows excellent control of grassy weeds in the CoAXium system. Two-gene Clearfield Plus and CoAXium wheat varieties provide robust resistance to respective herbicides and better weed control options compared to their single gene counterparts. Clearfield wheat production systems have been widely adopted by farmers due to effective management of problematic weeds in wheat over the last 15 years. However, the evolution of resistant weeds has limited the use of Clearfield wheat.The introduction CoAXium wheat varieties provided farmers an option to control ALS resistant weeds. High yields are produced when Beyond and Aggressor are applied to small and actively growing weeds compared to tall and mature weeds. In addition to these target herbicides, the CoAXium system mandates broadleaf herbicides such as 2,4 D,dicamba, metsufuron, thifensulfuron, and huskie, or in combination, to control many broad leaf weeds. Preemergence herbicides such as trifluralin, pyroxasulfone,and sulfosulfuron,or in combination,with other herbicides depending on the wheat variety grown are applied to control weeds in these wheat production systems. The benefits of these systems can be preserved and protected by following label recommendations, crop rotations, seed rate that is competitive against weeds and integrated weed management strategies. Furthermore, the stewardship guidelines and recommendations are intended to help,preserve and maximize the efficacy of wheat production technologies and to prevent the selection and spread of herbicide-resistant weeds as a result of repeated use of specific herbicides.

Acknowledgments

The research was supported by Heartland Plant Innovations,Inc.,Manhattan, Kansas.