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Abstract Canada thistle is a pervasive perennial weed, causing challenges to agricultural and natural ecosystems globally. Although research has focused on the phenology, genetics, and control of Canada thistle, little is known about the rhizosphere microbiome and the role plant–microbe interactions play in invasion success. This study investigated the rhizosphere microbiome of Canada thistle across diverse climates, soils, and crops in the U.S. northern Great Plains. Soil and rhizosphere samples were collected and bacterial 16S and fungal ITS2 sequencing were performed to characterize the core microbiome and identify potential factors contributing to invasion success. Amplicon sequencing revealed a stable core microbiome that was detected in the Canada thistle rhizosphere across all locations. The core microbiome was dominated by the bacterial phyla Actinobacteriota and Proteobacteria and fungal phyla Ascomycota and Basidiomycota. Differential abundance analysis showed rhizosphere fungal communities were enriched in pathogen-containing genera with a 1.7-fold greater abundance of Fusaria and a 2.6-fold greater abundance of Gibberella compared to bulk soil. Predictive functional profiling showed rhizosphere communities were enriched (p

Abstract The widespread evolution of glyphosate-resistant (GR) Bassia scoparia in the U.S. Great Plains poses a serious threat to the long-term sustainability of GR sugar beet. Glyphosate resistance in B. scoparia is due to an increase in the EPSPS (5-enolpyruvyl-shikimate-3-phosphate) gene copy number. The variation in EPSPS gene copies among individuals from within a single GR B. scoparia population indicated a differential response to glyphosate selection. With the continued use of glyphosate in GR sugar beet, the effect of increasing glyphosate rates (applied as single or sequential applications) on the fitness of GR B. scoparia individuals with variable EPSPS gene copies was tested under field conditions. The variation in EPSPS gene copy number and total glyphosate rate (single or sequential applications) did not influence any of the reproductive traits of GR B. scoparia, except seed production. Sequential applications of glyphosate with a total rate of 2214 g ae ha−1 or higher prevented seed production in B. scoparia plants with 2–4 (low levels of resistance) and 5–6 (moderate levels of resistance) EPSPS gene copies. Timely sequential applications of glyphosate (full recommended rates) can potentially slow down the evolution of GR B. scoparia with low to moderate levels of resistance (2–6 EPSPS gene copies), but any survivors (highly-resistant individuals with ≥ 8 EPSPS gene copies) need to be mechanically removed before flowering from GR sugar beet fields. This research warrants the need to adopt ecologically based, multi-tactic strategies to reduce exposure of B. scoparia to glyphosate in GR sugar beet.