A simple, cost-effective and optimized protocol for collecting root exudates from soil grown plants

Root exudates play a pivotal role in belowground interactions in both ecological and agricultural contexts. The metabolic composition of exudates profoundly influences the dynamics of these interactions, thereby shaping the intricate relationships between plants, microbes, and soil environments. Recent advances in mass-spectrometry have facilitated the analysis of root exudate metabolic composition to a greater depth. Previously used methods primarily analyze root exudates in hydroponic systems, or employ hybrid methodologies, which cultivate plants in soil and transitioning them briefly to hydroponic systems for exudate collection. Modern day ecological studies demand that exudates are collected in their natural habitats, because this will provide a more ecologically meaningful exudate metabolic profile. However, collecting exudates from soil grown plants poses several challenges with regard to the collection procedures, amongst others, the need for recovery after excavation of the roots, the collection period, and the solution in which to collect. Here, we present an optimized, cost-effective protocol for root exudate collection from potted plants, which is readily adaptable to field-grown specimens. Using tomato plants grown in pots, we examined and optimized various parameters: the collection medium (water versus nutrient solution), the use of wetted glass beads versus roots submerged in water, the recovery phase post-substrate removal, and the duration of exudation. Employing liquid chromatography-mass spectrometry (LC-MS), we assessed total amount of exudate, the number of features and background noise. Subsequent to data processing and statistical analyses, we assessed the chemical classes within exudates and variations in key metabolites among the different methods. Our results showed that each of the tested parameters can influence the outcome in different ways. Omitting the recovery phase increased the numbers of features and exudate amounts, likely due to adding metabolites from damaged roots, whereas the exudation medium and the duration of exudation had fewer effects. Based on our results, we propose to collect exudates in beakers containing ultrapure water, and to collect exudates for 4 h after a 24 h recovery phase. This is a straightforward and economical approach for collecting root exudates from soil-grown plants which is suitable for LC-MS analysis. •A simple method to collect root exudates from soil-grown plants is in beakers with pure