1. Plant-microbe interactions are critical for ecosystem functioning and drive rhizosphere processes. Root exudates are an important soil carbon (C) input, as well as a mechanism for communication between plants and rhizosphere microbes, but are notoriously difficult to extract and characterise. Common methods produce either substantial noise from the soil or do not mimic natural systems. Optimising methods for root exudate collection in soil is crucial for advancing our understanding of root-microbe interactions under changing environmental conditions.

2. Hybrid root exudate collection methods, where plants are grown in soil and transferred to hydroponics for exudate collection after root washing, might offer an ecologically relevant alternative to existing approaches. However, this method causes potential root damage as well as osmosis and subsequent leaking of cell contents. Here, we assessed different ‘root recovery’ periods after root washing and before hybrid root exudate collection, by comparing root exudate quantity and quality with both damaged root extracts and with leachates collected from the intact root-soil system. This was done across three common grassland species representing three functional groups.

3. We found that root exudate profiles of the shortest recovery period (0 days) were similar to damaged root extracts and were very high in C. With an increasing period of root recovery, profiles were more similar to leachates collected from the intact root-soil system, and C concentrations decreased. While both hybrid and leachate collection methods separated species by their root exudate profiles, the hybrid method was less variable in terms of the amount of C measured and provided a more diverse and abundant metabolome with better identification of metabolites.

4. Our results show that a recovery period after root washing of at least 3 days is critical to prevent root damage bias in hybrid collection methods, and that our hybrid method yields exudates that discriminate between species. Our data also suggest that exudates collected with this hybrid method are ecologically valid, which is vital for gaining a mechanistic understanding of their role in ecosystem functioning.

1. 植物-微生物相互作用对于生态系统功能和驱动根际过程至关重要。根系分泌物是重要的土壤碳（C）输入，也是植物和根际微生物之间交流的机制，但众所周知难以提取和表征。常见的方法要么会从土壤中产生大量噪音，要么不会模仿自然系统。优化土壤中根系分泌物收集的方法对于加深我们对不断变化的环境条件下根系与微生物相互作用的理解至关重要。

2. 混合根系分泌物收集方法，即植物在土壤中生长，并在洗根后转移至水培法以收集分泌物，可能为现有方法提供生态相关的替代方案。然而，这种方法会导致潜在的根部损伤以及渗透和随后的细胞内容物泄漏。在这里，我们通过比较受损根提取物和从完整根-土壤系统收集的渗滤液的根分泌物数量和质量，评估了根清洗后和混合根分泌物收集前的不同“根恢复”期。这是针对代表三个功能组的三种常见草原物种进行的。

3. 我们发现最短恢复期（0 天）的根系分泌物轮廓与受损根部提取物相似，并且 C 含量非常高。随着根系恢复时间的增加，轮廓与从完整根部收集的渗滤液更加相似 -土壤系统和C浓度下降。虽然混合法和渗滤液收集方法都通过根系分泌物特征来分离物种，但混合法在测量的 C 量方面变化较小，并且提供了更加多样化和丰富的代谢组，可以更好地识别代谢物。

4. 我们的结果表明，洗根后至少 3 天的恢复期对于防止混合收集方法中的根损伤偏差至关重要，并且我们的混合方法产生的渗出物可以区分物种。我们的数据还表明，用这种混合方法收集的渗出物在生态上是有效的，这对于从机械角度理解其在生态系统功能中的作用至关重要。