Roots are crucial in plant adaptation through the exudation of various compounds which are
influenced and modified by environmental factors. Buckwheat root exudate and root system
response to neighbouring plants (buckwheat or redroot pigweed) and how these exudates affect
redroot pigweed was investigated. Characterising root exudates in plant–plant interactions presents
challenges, therefore a split-root system which enabled the application of differential treatments to
parts of a single root system and non-destructive sampling was developed. Non-targeted metabolome
profiling revealed that neighbour presence and identity induces systemic changes. Buckwheat and
redroot pigweed neighbour presence upregulated 64 and 46 metabolites, respectively, with an overlap
of only 7 metabolites. Root morphology analysis showed that, while the presence of redroot pigweed
decreased the number of root tips in buckwheat, buckwheat decreased total root length and volume,
surface area, number of root tips, and forks of redroot pigweed. Treatment with exudates (from the
roots of buckwheat and redroot pigweed closely interacting) on redroot pigweed decreased the total
root length and number of forks of redroot pigweed seedlings when compared to controls. These
findings provide understanding of how plants modify their root exudate composition in the presence
of neighbours and how this impacts each other’s root systems.