The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F 2 populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology.5-enolpyruvylshikimate-3-phosphate synthase | herbicide resistance | mobile genetic element | evolution | Palmer amaranth

Imidazolinone herbicides, which include imazapyr, imazapic, imazethapyr, imazamox, imazamethabenz and imazaquin, control weeds by inhibiting the enzyme acetohydroxyacid synthase (AHAS), also called acetolactate synthase (ALS). AHAS is a critical enzyme for the biosynthesis of branched-chain amino acids in plants. Several variant AHAS genes conferring imidazolinone tolerance were discovered in plants through mutagenesis and selection, and were used to create imidazolinone-tolerant maize (Zea mays L), wheat (Triticum aestivum L), rice (Oryza sativa L), oilseed rape (Brassica napus L) and sunflower (Helianthus annuus L). These crops were developed using conventional breeding methods and commercialized as Clearfield* crops from 1992 to the present. Imidazolinone herbicides control a broad spectrum of grass and broadleaf weeds in imidazolinone-tolerant crops, including weeds that are closely related to the crop itself and some key parasitic weeds. Imidazolinone-tolerant crops may also prevent rotational crop injury and injury caused by interaction between AHAS-inhibiting herbicides and insecticides. A single target-site mutation in the AHAS gene may confer tolerance to AHAS-inhibiting herbicides, so that it is technically possible to develop the imidazolinone-tolerance trait in many crops. Activities are currently directed toward the continued improvement of imidazolinone tolerance and development of new Clearfield* crops. Management of herbicide-resistant weeds and gene flow from crops to weeds are issues that must be considered with the development of any herbicide-resistant crop. Thus extensive stewardship programs have been developed to address these issues for Clearfield* crops.

The selection of herbicide-resistant weed populations began with the introduction of synthetic herbicides in the late 1940s. For the first 20 years after introduction, there were limited reported cases of herbicide-resistant weeds. This changed in 1968 with the discovery of triazine-resistant common groundsel. Over the next 15 yr, the cases of herbicide-resistant weeds increased, primarily to triazine herbicides. Although triazine resistance was widespread, the resistant biotypes were highly unfit and were easily controlled with specific alternative herbicides. Weed scientists presumed that this would be the case for future herbicide-resistant cases and thus there was not much concern, although the companies affected by triazine resistance were somewhat active in trying to detect and manage resistance. It was not until the late 1980s with the discovery of resistance to Acetyl Co-A carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors that herbicide resistance attracted much more attention, particularly from industry. The rapid evolution of resistance to these classes of herbicides affected many companies, who responded by first establishing working groups to address resistance to specific classes of herbicides, and then by formation of the Herbicide Resistance Action Committee (HRAC). The goal of these groups, in cooperation with academia and governmental agencies, was to act as a forum for the exchange of information on herbicide-resistance selection and to develop guidelines for managing resistance. Despite these efforts, herbicide resistance continued to increase. The introduction of glyphosate-resistant crops in the 1995 provided a brief respite from herbicide resistance, and farmers rapidly adopted this relatively simple and reliable weed management system based on glyphosate. There were many warnings from academia and some companies that the glyphosate-resistant crop system was not sustainable, but this advice was not heeded. The selection of glyphosate resistant weeds dramatically changed weed management and renewed emphasis on herbicide resistance management. To date, the lesson learned from our experience with herbicide resistance is that no herbicide is invulnerable to selecting for resistant biotypes, and that over-reliance on a weed management system based solely on herbicides is not sustainable. Hopefully we have learned that a diverse weed management program that combines multiple methods is the only system that will work for the long term.

SummaryResistance occurs when a genetic change allows a population of weeds to survive a herbicide treatment to which the original population was susceptible. Individual plants of weed species that are resistant to a particular herbicide are typically present in untreated populations at very low frequencies. These few resistant individuals survive a herbicide application and reproduce, whereas susceptible individuals are killed and do not reproduce. The percentage of resistant individuals increases over time as the herbicide treatment is repeated. Weed scientists began identifying resistant weed biotypes (genotypes) about 40 years ago, and the number of weeds with resistant biotypes has increased in recent years. Use of a few modes of herbicide action in the major row crops, cotton (Gossypium hirsutum), corn (Zea mays), and soybean (Glycine max), has selected for resistance in certain weeds. Widespread use of the acetolactate synthase (ALS) inhibiting herbicides and glyphosate has led to resistance to one or both of these modes of action in weeds including Palmer amaranth (Amaranthus palmeri), common cocklebur (Xanthium strumarium), and horseweed (Conyza canadensis). Growers should diversify weed management tactics to avoid selecting more resistant weeds. Scout to detect uncontrolled weeds early and prevent movement of possibly resistant weed seed among fields. To reduce the rate of resistance buildup, practice rotation of all management factors where possible, including type of tillage, crops grown, and herbicide modes of action. Crop monoculture and continuous use of the same modes of action will accelerate resistance buildup and increase the difficulty and cost of weed control. What is Herbicide Resistance?Herbicide resistance is the inherited ability of a weed biotype to survive and reproduce despite exposure to a dose of herbicide that previously was effective on an unselected population. Application of a herbicide may reveal individuals within a population that already possess the capacity to survive exposure. Repeated, successive use of one herbicide, or herbicides with the same mode of action, increases the likelihood that resistant individuals will survive and reproduce. How are Weed Populations Selected for Resistance?The rate at which a resistant weed population is selected depends on the number and frequency of herbicide applications the population receives, the size of the population and its genetic diversity, and characteristics of the herbicide target site. Resistance buildup is accelerated when the management of crops does not include diverse tactics that limit herbicide use such as crop rotation and mechanical weed management. For example, there may be more opportunities for resistance buildup in conservation tillage because weeds are not killed by mechanical disturbance and non-selective herbicides such as glyphosate, paraquat, or glufosinate are used for pre-plant burndown. What are Herbicide Modes of Action?Mode of action describes the plant process affected by the herbicide that results in d...

An in vivo shikimate accumulation assay with excised leaf tissue was developed to provide a fast and reliable method for identifying glyphosate-resistant plants. The assay is based on glyphosate-induced accumulation of shikimate. There was a linear accumulation of shikimate in excised leaf discs of soybean and canola treated with 250 μM glyphosate for 48 h. The IC50 for the accumulation of shikimate in soybean and corn leaf discs was 34 and 87 μM, respectively. Leaf discs excised from glyphosate-resistant corn or soybean did not accumulate shikimate when treated with 500 μM glyphosate. Leaf discs taken from a number of field-grown plants accumulated shikimate in a glyphosate dose–dependent manner. The accumulation of shikimate was dependent on light and the age of the leaf from which the disc was taken. The assay worked either in 96-well microtiter plates or in vials, and it clearly differentiated between glyphosate-resistant and -susceptible crops in which the resistance is due to an alteration of the target site for glyphosate. The assay was simple and robust and has the potential to be used as a high throughput assay to detect glyphosate resistance in weeds.