Abstract
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.
Highlights
The rhizosphere is a diverse and heterogeneous bio-chemical network, where roots and micro-organisms cohabit in a complex inter active community
Principal component analysis (PCA) was applied to standardised GC-MS data followed by PERMANOVA with species and collection method as main fac tors, as well as their interactions to assess their impact on root traits and root exudate profiles, quantified by GC-MS
Collection method mildly affected above-ground biomass (ANOVA for main effect of collection method, F4, 59 = 3.2, p = 0.018; Fig. 1a), which was higher in H7 than in the other collection methods in both H. lanatus and T. repens, but not R. acetosa
Summary
The rhizosphere is a diverse and heterogeneous bio-chemical network, where roots and micro-organisms cohabit in a complex inter active community. Exudates collected with a hybrid method have been reapplied to soil to quantify their impact on soil-microbe respiration (de Vries et al, 2019), which un derscores the method’s potential for unravelling the mechanistic role that certain root-exuded compounds play in the rhizosphere. Despite these advantages, hybrid methods introduce bias as root washing causes unavoidable stress and root damage (Oburger and Jones, 2018) - the extent to which this impacts exudate composition and rate is not clear. We test these hypotheses in a controlled factorial experiment with three common grassland species
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