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

Herbicides that act by inhibiting protoporphyrinogen oxidase (PPO) are widely used to control weeds in a variety of crops. The first weed to evolve resistance to PPO-inhibiting herbicides was Amaranthus tuberculatus, a problematic weed in the midwestern United States that previously had evolved multiple resistances to herbicides inhibiting two other target sites. Evaluation of a PPOinhibitor-resistant A. tuberculatus biotype revealed that resistance was a (incompletely) dominant trait conferred by a single, nuclear gene. Three genes predicted to encode PPO were identified in A. tuberculatus. One gene from the resistant biotype, designated PPX2L, contained a codon deletion that was shown to confer resistance by complementation of a hemG mutant strain of Escherichia coli grown in the presence and absence of the PPO inhibitor lactofen. PPX2L is predicted to encode both plastid-and mitochondria-targeted PPO isoforms, allowing a mutation in a single gene to confer resistance to two herbicide target sites. Unique aspects of the resistance mechanism include an amino acid deletion, rather than a substitution, and the dual-targeting nature of the gene, which may explain why resistance to PPO inhibitors has been rare.Amaranthus ͉ evolution ͉ waterhemp ͉ weed resistance ͉ herbicide resistance

A population of waterhemp was identified in Adams County, Illinois, that survived treatment of several acetolactate synthase (ALS) inhibitors and a postemergence (POST) application of lactofen, a protoporphyrinogen oxidase (PPO)–inhibiting herbicide. Greenhouse studies were conducted to quantify the responses of this waterhemp population, designated ACR, to multiple PPO inhibitors and various other herbicides with different sites of action. Resistance ratios were obtained by comparing herbicide dose–response curves between the ACR population and a herbicide-susceptible waterhemp population. The ACR population was resistant to lactofen (23-fold) and to five other PPO-inhibiting herbicides (ranging from 2.2- to 6.2-fold). Furthermore, the ACR waterhemp population was 17,000-fold and 18,000-fold resistant to imazamox and thifensulfuron, respectively, two ALS-inhibiting herbicides. In response to atrazine, a Photosystem II inhibitor, the ACR population was 38-fold resistant. Plants within the ACR waterhemp population survived treatment of a herbicide mixture containing lactofen at 175 g ai ha−1, imazamox at 44 g ae ha−1, and atrazine at 1,000 g ai ha−1. Thus, individual plants—not just the population as a whole—displayed multiple herbicide resistance. The ACR population was not resistant to glyphosate or paraquat. This is the first reported weed population from the United States with resistances to herbicides inhibiting three unique sites of action. Furthermore, this research identifies a significant reduction in the number of POST herbicide options available for waterhemp control in soybean production.

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In a survey of herbicide responses among Illinois waterhemp half-sib populations, several were observed with differential responses to imazethapyr and thifensulfuron, two acetolactate synthase (ALS)–inhibiting herbicides. Plants from two waterhemp populations were verified resistant to imazethapyr, but susceptible to chlorimuron, using a nondestructive leaf-disc assay. Sequencing of the ALS gene revealed that imazethapyr-resistant waterhemp plants from both populations had inferred amino acid substitutions at position 653 of ALS. Depending on the population, the serine at position 653 of ALS was substituted with either asparagine (S653N) or threonine (S653T). Waterhemp lines were derived from each population to create uniformly imidazolinone-resistant (IR) waterhemp biotypes, designated IR-62 and IR-101. ALS-inhibitor responses of each IR biotype were compared with a previously identified ALS inhibitor–resistant biotype with a tryptophan to leucine substitution at position 574 (W574L) and an herbicide-susceptible control. Whole-plant dose–response experiments with waterhemp biotypes containing W574L, S653N, or S653T mutations indicated that each biotype was resistant to imazethapyr, but only the biotype with a W574L mutation was resistant to thifensulfuron. In vitro ALS-activity assays revealed unique patterns of cross-resistance among protein extracts derived from each biotype in response to imazethapyr, thifensulfuron, cloransulam, and pyrithiobac. In conclusion, three different forms of target-site–based resistance to ALS inhibitors have been identified in waterhemp.

The majority of soybeans planted in the United States are resistant to glyphosate due to introduction of a gene encoding for a glyphosate-insensitive 5-enolypyruvylshikimate-3-phosphate synthase. Gene expression profiling was conducted using cDNA microarrays to address questions related to potential secondary effects of glyphosate. When glyphosate-sensitive plants were treated with glyphosate, 3, 170, and 311 genes were identified as having different transcript levels at 1, 4, and 24 h post-treatment (hpt), respectively. Differentially expressed genes were classified into functional categories, and their possible roles in response to glyphosate are briefly discussed. Gene expression profiling of glyphosate-resistant plants treated with glyphosate indicated that the plants were marginally affected at 1 hpt and then quickly adjusted to glyphosate treatment. Ten, four, and four genes were identified as differentially expressed at 1, 4, and 24 hpt. When gene expression profiles of cotyledons from developing seed were compared between the near-isogenic resistant and sensitive lines, two genes were identified as significantly differentially expressed out of 27000, which was less than the empirical false-discovery rate determined from a control experiment. Quantitative real-time reverse-transcribed Polymerase Chain Reaction was conducted on selected genes and yielded results consistent with those from the microarrays. Collectively, these data indicate that there are no major transcriptomic changes associated with currently used glyphosate-resistant soybean.

While surveying Illinois Amaranthus tuberculatus (Moq) Sauer (tall waterhemp) half-sib populations for herbicide response variability, several were observed to segregate for resistance to atrazine. Studies were conducted on greenhouse-grown A tuberculatus plants to compare atrazine responses among populations that were segregating for resistance (SegR), uniformly sensitive (UniS) or uniformly resistant (UniR). In chlorophyll fluorescence assays, leaves of plants from the SegR and UniS populations displayed changes in fluorescence after treatment with atrazine, indicating that atrazine was inhibiting electron transport of photosystem II in chloroplasts. Sequencing of a fragment of psbA, which encodes the D1 protein, revealed that the SegR population did not contain the amino acid substitution that is typically found in triazine-resistant plants. Whole-plant herbicide dose-response experiments revealed that, relative to the UniS population, atrazine resistances in the UniR and SegR populations were > 770-fold and 16-fold, respectively. The SegR population was also resistant to cyanazine (59-fold), but not to metribuzin, linuron or pyridate. Triazine resistance in the SegR population was shown to be a nuclear inherited trait, unlike maternal inheritance of site-of-action mediated triazine resistance found in the UniR population. Taken collectively, these findings confirm the existence of two distinct triazine resistance mechanisms in A tuberculatus.

A population of common ragweed not controlled by an acetolactate synthase (ALS)-inhibiting herbicide, cloransulam-methyl, was sampled near Dunkirk, IN, the first year of the herbicide's commercialization in 1998. Resistance in the Dunkirk population was confirmed by treating greenhouse-grown seedlings with cloransulam-methyl. ALS activity assays and DNA sequencing were used to identify the resistance mechanism. ALS isolated from plants of the Dunkirk population exhibited an R/S ratio for cloransulam-methyl of >5,000 when compared to ALS activity of populations from Claire City, SD, and V & J Seed Farms. R/S ratios of 4,100 and 110 were observed for two other ALS-inhibiting herbicides, chlorimuron and imazaquin, respectively. DNA sequencing revealed that an inferred leucine for tryptophan substitution at amino acid position 574 in ALS was responsible for the observed herbicide resistance. Additionally, DNA sequencing revealed significant variability among common ragweed ALS alleles. Two fragments of ALS were sequenced from three plants each of the Claire City and Dunkirk populations, totaling 688 nucleotide base pairs, of which 72 were polymorphic.