Unveiling belowground allelopathy: 1H-NMR spectroscopy reveals metabolic crosstalk and novel sterols in Cyperus rotundus and Sorghum bicolor co-cultures

Root exudates are crucial for driving belowground ecosystem dynamics and subterranean chemical interactions. <i>Cyperus rotundus</i> (purple nutsedge) is a highly invasive weed, notoriously resistant to conventional control and causing significant crop losses globally. <i>Sorghum bicolor</i> is well-known for producing allelopathic compounds via root exudates. The metabolic shifts occurring during the direct interaction between <i>C. rotundus</i> and allelopathic crop <i>S. bicolor</i> are yet unexplored. In this study, we established an <i>in vitro</i> protocol for the isolation of root exudates from solid growth medium and employed a non-targeted ¹H-NMR spectroscopy approach to fingerprint the hydrophilic and lipophilic root exudates within an <i>in vitro</i> co-culture system. A total of 21 metabolites, including carbohydrates, phenolics, peptides and fatty acids were identified. Notably, three new sterols including (3β)-3-hydroxy-6-methylstigmast-20(22)-en-14-oic acid, 6-methylstigmast-20(22)-en-14-oic acid derivative 1 and 6-methylstigmast-20(22)-en-14-oic acid derivative 2 were univocally assigned. Comparative analysis revealed that co-cultivation induced specific metabolic changes rather than a general increase in exudation. Specifically, the cyanogenic glycoside dhurrin, a primary allelochemical in <i>S. bicolor</i>, significantly decreased in co-cultures, while phytoalexins B, detected in individual <i>C. rotundus</i> exudates, was absent in co-cultures. However, no significant reduction in root biomass was observed for either species over the 12-day experimental period. These findings suggest that while root-root proximity triggers specific secondary metabolites shifts, these changes did not translate into immediate growth inhibition under the tested conditions. This study provides the first non-targeted metabolic profiling of the direct belowground interaction between <i>S. bicolor</i> and <i>C. rotundus</i> and highlights the complexity of modulating allelopathic responses in sterile environments.