In 2006 and 2007, farmers from two counties in Illinois reported failure to control waterhemp with glyphosate. Subsequent onsite field experiments revealed that the populations might be resistant to multiple herbicides. Greenhouse experiments therefore were conducted to confirm glyphosate resistance, and to test for multiple resistance to other herbicides, including atrazine, acifluorfen, lactofen, and imazamox. In glyphosate dose-response experiments, both populations responded similarly to a previously characterized glyphosate-resistant population (MO1). Both Illinois populations also demonstrated high frequencies of resistance to the acetolactate synthase (ALS) inhibitor, imazamox. Additionally, one of the populations demonstrated high frequencies of resistance to both atrazine and the protoporphyrinogen oxidase (PPO) inhibitor, lactofen. Furthermore, using combinations of sequential and tank-mix herbicide applications, individual plants resistant to herbicides spanning all four site-of-action groups were identified from one population. Molecular experiments were performed to provide an initial characterization of the resistance mechanisms and to provide confirmation of the presence of multiple resistance traits within the two populations. Both populations contained the W574L ALS mutation and the ΔG210 PPO mutation, previously shown to confer resistance to ALS and PPO inhibitors, respectively. Atrazine resistance in both populations is suspected to be metabolism-based, because a triazine target-site mutation was not identified. A P106S EPSPS mutation, previously reported to confer glyphosate resistance, was identified in one population. This mutation was identified in both resistant and sensitive plants from the population; however, and so more research is needed to determine the glyphosate-resistance mechanism(s). This is the first known case of a weed population in the United States possessing multiple resistance to herbicides from four site-of-action groups.

Resistance in waterhemp to herbicides that inhibit protoporphyrinogen oxidase (PPO) previously was shown to result from the deletion of a glycine codon at position 210 (ΔG210) in the PPO-encoding gene, PPX2. Research was conducted to determine if this same mechanism accounted for resistance in geographically separated populations—from Illinois, Kansas, and Missouri—and, if so, to determine if the mutation conferring resistance was independently selected. A dose–response study with lactofen indicated that the resistant populations had different levels of resistance. These differences, however, could be accounted for by different frequencies of resistant individuals within populations and, therefore, the dose–response data were consistent with the hypothesis that the populations contained the same resistance mechanism. Direct evidence in support of this hypothesis was provided by DNA sequencing, which showed that nearly all resistant plants evaluated contained the ΔG210 mutation. A variable region of the PPX2 gene was sequenced and resulting sequences were aligned and organized into a phylogenetic tree. The phylogenetic tree did not reveal clear clustering by either geography or phenotype (resistant vs. sensitive). Possibly recombination within the PPX2 gene has masked its evolutionary history.

Recent advances in sequencing technologies (next-generation sequencing) offer dramatically increased sequencing throughput at a lower cost than traditional Sanger sequencing. This technology is changing genomics research by allowing large scale sequencing experiments in nonmodel systems. Waterhemp is an important weed in the midwestern United States with characteristics that makes it an interesting ecological model. However, very few genomic resources are available for this species. One half of a 70 by 75 picotiter plate of 454-pyrosequencing was performed on total DNA isolated from waterhemp, generating 158,015 reads of an average length of 271 bp, or a total of nearly 43 Mbp of sequence. Included in this sequence was a nearly complete sequence of the chloroplast genome, sequences of several important herbicide resistance genes, leads for simple sequence repeat (SSR) markers, and a sampling of the repeated elements (e.g., transposons) present in this species. Here we present the waterhemp genomic data gleaned from this sequencing experiment and illustrate the value of next-generation sequencing technology to weed science research.

Waterhemp is a major weed of field crops in the Midwestern United States. Its prevalence is at least partly due to its rapid evolution of resistance to many groups of herbicides over the last two decades. In light of its dioecy and anemophily, pollen movement in waterhemp is considered to be an important factor in the spread of herbicide resistance genes. Thus, the biology and dispersal profile of waterhemp pollen are critical determinants for understanding and predicting the spatial population dynamics of herbicide resistance in this species. In this study, pollen longevity was investigated with greenhouse experiments, and pollen dispersal and the effect of pollen competition were investigated in field plots. Pollen dispersal was determined by measuring the frequency of seeds produced on receptor plants positioned at various distances from a pollen source, which flowered in synchrony with the receptor plants. Results indicated that waterhemp pollen can remain viable up to 120 h, implying a low limitation of pollen dispersal by its longevity. Effective pollen dispersal declined exponentially with distance, with most pollen fertilizing recipient plants within 50 m of the pollen source. However, long distance pollen dispersal (800 m) was also observed. We also saw evidence for pollen swamping in this species. Under conditions of pollen competition among distinct genotypes, pollination success was inversely related to distance between pollen donors and receptors. However, relative pollen density may also play an important role in determining the rate of long distance gene flow. This study confirmed the potential of waterhemp pollen to effect long distance gene flow and provides supporting data for quantitative spatial modeling of waterhemp resistance dynamics.