Characterization and inheritance of dicamba resistance in a multiple-resistant waterhemp (Amaranthus tuberculatus) population from Illinois

Waterhemp (Amaranthus tuberculatus (Moq.) Sauer) is one of the most troublesome agronomic weeds in the midwestern US. The rapid evolution and selection of herbicide-resistance traits in A. tuberculatus is a major challenge in managing this species. An A. tuberculatus population, designated CHR, was identified in 2012 in Champaign County, IL, and previously characterized as resistant to herbicides from six site-of-action groups: 2,4-D (Group 4), acetolactate synthase inhibitors (Group 2), protoporphyrinogen oxidase inhibitors (Group 14), 4-hydroxyphenylpyruvate dioxygenase inhibitors (Group 27), photosystem II inhibitors (Group 5), and very long chain fatty acid synthesis inhibitors (Group 15). Recently, ineffective control of CHR was observed in the field after dicamba application. Therefore, this research was initiated to confirm dicamba resistance, quantify the resistance level and investigate its inheritance in CHR. Multiple field trials were conducted at the CHR location to confirm poor control with dicamba and compare dicamba treatments with other herbicides. Greenhouse trials were conducted to quantify the resistance level in CHR and confirm genetic inheritance of the resistance. In field trials, dicamba did not provide more than 65% control, while glyphosate and glufosinate provided at least 90% control. Multiple accessions were generated from controlled crosses and evaluated in greenhouse trials. Greenhouse dicamba dose-response experiments indicated a resistance level of 5 to 10-fold relative to sensitive parental line. Dose-response experiments using F1 lines indicated that dicamba resistance was an incompletely dominant trait. Segregation analysis with F2 and backcross populations indicated that dicamba resistance had moderate heritability and was potentially a multigenic trait. Although dicamba resistance was predominantly inherited as a nuclear treat, minor maternal inheritance was not completely ruled out. To our knowledge, CHR is one of the first cases of dicamba resistance in A. tuberculatus. Further studies will focus on elucidating the genes involved in dicamba resistance.

Metabolic Pathways for S-Metolachlor Detoxification Differ Between Tolerant Corn and Multiple-Resistant Waterhemp

Herbicide resistance in weeds can be conferred by target-site and/or non-target-site mechanisms, such as rapid metabolic detoxification. Resistance to the very-long-chain fatty acid (VLCFA)-inhibiting herbicide, S-metolachlor, in multiple-herbicide resistant populations (CHR and SIR) of waterhemp (Amaranthus tuberculatus) is conferred by rapid metabolism compared with sensitive populations. However, enzymatic pathways for S-metolachlor metabolism in waterhemp are unknown. Enzyme assays using S-metolachlor were developed to determine specific activities of glutathione S-transferases (GSTs) and cytochrome P450 monooxygenases (P450s) from CHR and SIR seedlings to compare with tolerant corn and sensitive waterhemp (WUS). GST activities were greater (~2-fold) in CHR and SIR compared to WUS, but much less than corn. In contrast, P450s in microsomal extracts from CHR and SIR formed O-demethylated S-metolachlor, and their NADPH-dependent specific activities were greater (>20-fold) than corn or WUS. Metabolite profiles of S-metolachlor generated via untargeted and targeted liquid chromatography-mass spectrometry from CHR and SIR differed from WUS, with greater relative abundances of O-demethylated S-metolachlor and O-demethylated S-metolachlor-glutathione conjugates formed by CHR and SIR. In summary, our results demonstrate S-metolachlor metabolism in resistant waterhemp involves Phase I and Phase II metabolic activities acting in concert, but the initial O-demethylation reaction confers resistance.

Biologically-Effective-Dose of Metribuzin, Applied Preemergence and Postemergence, for the Control of Waterhemp (Amaranthus tuberculatus) with Different Mechanisms of Resistance to Photosystem II-inhibiting herbicides

Photosystem II (PS II)-inhibitor herbicide resistance in Ontario waterhemp [Amaranthus tuberculatus (Moq.) Sauer] population is conferred via target-site resistance (TSR) and non-target-site resistance (NTSR) mechanisms. Metribuzin-resistant (MR) A. tuberculatus is due to TSR,. Conversely, in other populations of PS II-resistant A. tuberculatus, plants are resistant to atrazine but metribuzin-sensitive (MS). The objective of this study was to determine the biologically-effective-dose of metribuzin applied PRE and POST for the control of MS and MR A. tuberculatus. Ten field experiments were conducted in 2019 and 2020 to determine the effective doses of metribuzin for 50, 80, and 95% control of MS and MR A. tuberculatus. Metribuzin applied PRE at the calculated doses of 133, 350, and 1070 g ai ha-1 controlled MS A. tuberculatus 50, 80, and 95%, respectively, whereas the calculated doses of 7868 and 17533 g ai ha-1 controlled MR A. tuberculatus 50 and 80%, respectively at 12 WAA. Metribuzin applied POST at the calculated doses of 245 and 1480 g ai ha-1 controlled MS A. tuberculatus 50 and 80%, respectively; the calculated dose for 50% MR A. tuberculatus control was greater than the highest dose (17920 g ai ha-1) included in this study. Metribuzin at 560 and 1120 g ha-1 and pyroxasulfone/flumioxazin (240 g ai ha-1) applied PRE controlled MS A. tuberculatus 88, 95, and 98%, respectively at 12 WAA. The aforementioned treatments controlled MR A. tuberculatus 0, 4, and 93%, respectively at 12 WAA. Metribuzin at 560 and 1120 g ha-1 and fomesafen (240 g ai ha-1) applied POST controlled MS A. tuberculatus 65, 70, and 78%, and MR A. tuberculatus 0, 1, and 49%, respectively at 12 WAA. Based on these results, NTSR PS II-resistant A. tuberculatus (enhanced metabolism) is controlled with metribuzin applied PRE and POST, in contrast TSR PS II-resistant A. tuberculatus (glycine264serine altered target site) is not controlled with metribuzin.

Selective ancestral sorting and de novo evolution in the agricultural invasion of Amaranthus tuberculatus

The relative role of hybridization, de novo evolution, and standing variation in weed adaptation to agricultural environments is largely unknown. In Amaranthus tuberculatus, a widespread North American agricultural weed, adaptation is likely influenced by recent secondary contact and admixture of two previously isolated subspecies. We characterized the extent of adaptation and phenotypic differentiation accompanying the spread of A. tuberculatus into agricultural environments and the contribution of subspecies divergence. We generated phenotypic and whole-genome sequence data from a manipulative common garden experiment, using paired samples from natural and agricultural populations. We found strong latitudinal, longitudinal, and sex differentiation in phenotypes, and subtle differences among agricultural and natural environments that were further resolved with ancestry-based inference. The transition into agricultural environments has favoured southwestern var. rudis ancestry that leads to higher biomass and environment-specific phenotypes: increased biomass and earlier flowering under reduced water availability, and reduced plasticity in fitness-related traits. We also detected de novo adaptation to agricultural habitats independent of ancestry effects, including marginally higher biomass and later flowering in agricultural populations, and a time to germination home advantage. Therefore, the invasion of A. tuberculatus into agricultural environments has drawn on adaptive variation across multiple timescales—through both preadaptation via the preferential sorting of var. rudis ancestry and de novo local adaptation.

Fitness Cost Associated With Enhanced EPSPS Gene Copy Number and Glyphosate Resistance in an Amaranthus tuberculatus Population

The evolution of resistance to pesticides in agricultural systems provides an opportunity to study the fitness costs and benefits of novel adaptive traits. Here, we studied a population of Amaranthus tuberculatus (common waterhemp), which has evolved resistance to glyphosate. The growth and fitness of seed families with contrasting levels of glyphosate resistance was assessed in the absence of glyphosate to determine their ability to compete for resources under intra- and interspecific competition. We identified a positive correlation between the level of glyphosate resistance and gene copy number for the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) glyphosate target, thus identifying gene amplification as the mechanism of resistance within the population. Resistant A. tuberculatus plants were found to have a lower competitive response when compared to the susceptible phenotypes with 2.76 glyphosate resistant plants being required to have an equal competitive effect as a single susceptible plant. A growth trade-off was associated with the gene amplification mechanism under intra-phenotypic competition where 20 extra gene copies were associated with a 26.5 % reduction in dry biomass. Interestingly, this growth trade-off was mitigated when assessed under interspecific competition from maize.

Hybridization potential between Amaranthus tuberculatus and Amaranthus albus

The genus Amaranthus is composed of numerous annual herbs, several of which are primary driver weeds within annual production agricultural systems. In particular, Amaranthus tuberculatus, a dioecious species, is noteworthy for rapid growth rates, high fecundity, and an expanding geographic distribution. Interspecific hybridization within and between the subgenera Amaranthus and Acnidia is reported both in controlled environment and field studies, however a gap in knowledge exists with the subgenus Albersia. Interspecific hybridization may contribute to genetic diversity, and may contribute to the current range expansion of A. tuberculatus. Recently, a herbicide resistance survey of A. tuberculatus across five Midwestern states reported alleles of PPX2 similar to sequences of Amaranthus albus, a monoecious species. Here, we seek to generate empirical data for the hybridization potential of A. albus and A. tuberculatus through replicated, controlled crosses in a greenhouse. Of 65,000 progeny screened from A. albus grown with A. tuberculatus males, three were confirmed as hybrids. Hybrids were dioecious, possessed phenotypic traits of both species, and had limited to no fertility. DNA content analysis of backcross progeny suggested a polyploid state may be required for hybrid formation. Screening of 120 progeny of A. tuberculatus females grown with A. albus identified no hybrids, though a skew to female progeny was observed. The female skew may be due to apomixis or auto-pollination, the spontaneous generation of male flowers on otherwise female plants. Our results indicate that introgression between A. albus and A. tuberculatus will occur less frequently than what has often been reported from hybridization studies with different pairs of Amaranthus species.