A Comparative Evaluation of Rhizosphere Metabolites Collection Methods: Impacts on Metabolic Diversity, Composition and Pathways.

Rhizosphere metabolites play a pivotal role in plant-soil-microbe interactions, yet the absence of standardised sampling method presents challenges for data comparability and a unified understanding of rhizochemistry dynamics. In this study, the rhizosphere metabolites of spring wheat (Triticum aestivum L.) were systematically evaluated using three commonly used sampling methods - rhizosphere soil extraction, hydroponic collection, and in situ sampling. We assessed the differences in metabolite abundance, diversity, composition, and metabolic pathways among the three methods. The results revealed that rhizosphere soil extraction method is simple and repeatable, but it tends to underestimate the rare metabolic features compared to the other two methods. The hydroponic collection method offered a more comprehensive rhizochemical profile, however, it may not reflect the actual soil environment in which plants grow. The in situ method mostly captured the rhizochemical dynamics under natural conditions but was technically complex and less reproducible. Pathway enrichment of differential metabolic features showed a distinct pattern, with hydroponic emphasising plant-centric primary metabolism, and rhizosphere soil extraction and in situ methods reflecting pathways more associated with environmental interaction and ecological adaptation. Overall, our results highlight that the sampling methods significantly influences the abundance and composition of the detected rhizochemical profile, thus the rhizosphere metabolites sampling methods should be considered according to the experimental objectives of future research.