Materials and Reagents

1 mm mesh size for soil sieving (Fisherbrand tamis, France)
Plastic pots for plant growing (7 x 7 x 6.4, black pots, France)
5 x 8 mm “Ultra Clean” tin capsules (Elemental Microanalysis, Okehampton, UK, catalog number: D1034)
In the case of calcareous soils: 5 x 8 mm silver capsules (Elemental Microanalysis, Okehampton, UK, catalog number: D2009)
Soil and seeds for plant growing
The soil used to set up this protocol was luvisol with no added nitrogen source collected at La Côte Saint-André (Isère, France) and which was continuously harvested with maize. It was a loamy soil composed of 16.2% clay, 45.4% loam and 28.4% sand, with a total nitrogen content of 1.9 g·kg^-1
Reference materials for ¹³C measurements: IAEA-CH3 (cellulose), IAEA-CH6 (sucrose) (Coplen et al., 2006) and partially ¹³C-labelled material [e.g., D-Glucose (3-13C, 99%)] (Eurisotop, Saint-Aubin, France, catalog number: CLM-1393-0.25)
Calibration material for carbon (C) concentration measurements: aspartic acid (Elemental Microanalysis, Okehampton, UK, catalog number: B2042)
Distilled water
Liquid N₂
Pure ¹³CO₂ (> 99% atom ¹³C; purchased from Cortec Net, Paris, France)
In the case of calcareous soils: hydrochloric acid (HCl) 37% (CAS number: 7647-01-0, e.g., Sigma-Aldrich, Saint-Louis, MO, USA, catalog number: 30721-M)

Equipment

50 ml beaker
Spatula and forceps
Desiccator (Freeze-dryer) (Alpha 1-4 LSC, Martin Christ^®, Osterode am Harz, Germany)
Mixer mill grinder (Mixer Mill MM 200, Retsch^®, Haan, Germany)
Laboratory balance with a resolution of 0.01 mg or better (XP6 microbalance, Mettler Toledo, Greifensee, Switzerland)
Isotope ratio mass spectrometer (Isoprime 100, Elementar UK Ltd., Cheadle, UK), coupled in continuous flow with an elemental analyzer (vario PyroCube in CN mode, Elementar Analysensysteme, Lagenselbold, Germany, or FlashEA 1112, ThermoElectron, Waltham, MA, USA)
Growth chamber equipped for automatic control of light, temperature, moisture, evapotranspiration, irrigation and CO₂ concentration

Software

Isoprime 100 isotope ratio mass spectrometer operating software: Ionvantage^® for Isoprime, build 1.6.1.0 (Elementar Analysensysteme, Lagenselbold, Germany)
Elemental analyzer operating software (pyrocube Software v4.0.1 was used in this protocol, Elementar Analysensysteme, Lagenselbold, Germany)

Procedure

Sieve the soil (1 mm mesh size) and adjust it to 0.17 g of water per gram of dry weight. Afterward, place 170 g of dry weight into pots. Place one seed per pot and do six replicates per plant species and six replicates for unplanted-soil (bulk soil).
Place the pots (six replicates per plant and six replicates for bulk soil) in a growth chamber and set up the day-night period, light intensity and daily temperature according to the studied plant (Guyonnet et al., 2018b). For Brasica napus, Arabidopsis thaliana, Triticum aestivum, Medicago truncatula and Poaceae plants, for example, the day-night period is set at 8 h/16 h, respectively; light intensity is 13.5 klux; maximum daily temperatures ranges from 20 to 22 °C. Control and adjust soil moisture by weighing pots using a laboratory balance and adding distilled water every 2 days (Figure 1).
Three weeks after sowing seeds, inject pure ¹³CO₂ to three replicates per plant and bulk soil during active photosynthesis, according to Haichar et al. (2008 and 2012), in order to maintain a ¹³CO₂ level at 350 μl·L^-1 for one week. Control ¹²CO₂ (soil respiration) and ¹³CO₂ concentrations in the growth chamber. The isotope excess of ¹³C atoms in the chamber must be maintained at > 80% during the first 10 days and > 90% thereafter.
Cultivate the rest of the plants (three replicates per plant) and bulk soil (three replicates for unplanted soil) without ¹³C-labeling in order to determine ¹³C natural abundance in soil and plants.
At the end of labeling, separate the root system from the root-adhering soil (RAS) by shaking each plant and collect the root-adhering soil. Carefully separate the remaining fine roots from the RAS and freeze the RAS immediately in liquid N₂ before storing at -80 °C (Figure 1).
Lyophilise the RAS during 48 h and grind to a fine powder (~100 µm particle size; visual verification is sufficient) using a mixer mill to homogenize before measuring ¹³C enrichment (Figure 2A). The duration of the grinding step should be adapted to each type of soil (approximately 1 min at 30 Hz; repeat if needed until a fine powder is obtained).

Figure 1. Plant growth, labeling and harvesting in order to measure root exudation rate
Weigh 8 mg (adjust the target mass to obtain a sufficient amount of carbon, commonly > 10 μg of C depending on the C concentration of the sample and IRMS sensitivity) of lyophilized RAS samples into 5 x 8 mm “Ultra Clean” tin capsules to the nearest 0.01 mg (Figure 2B).
Note: In calcareous soils, inorganic carbon (carbonates) can distort the ¹³C measurement of organic carbon. In this case, remove inorganic C by acid fumigation. Weigh soil samples into silver capsules instead of tin. Add a small amount (50-150 μl) of distilled water in each capsule to moisten the soil. Place each open capsule in a desiccator with a beaker of fuming (37%) hydrochloric acid (HCl) during 6 to 8 h. Dry at 60 °C. Crimp the capsule and place it in a new tin capsule.
Carefully seal the tin capsules using forceps into a cubical or spherical shape (Figure 2C).
Measure the ¹³C/¹²C ratio and C concentration of each sample by EA-IRMS (Figure 2D) after optimizing the source parameters and calibration according to good practice (e.g., Dunn and Carter, 2018). Intersperse reference materials with the samples to allow for the normalization of the data and quality control.

Figure 2. Scheme of soil sample preparation for the ¹³C/¹²C ratio measure using Elemental Analyser-Isotope Ratio Mass Spectrometer (EA-IRMS)

Data analysis

Apply blank corrections and normalize the C concentrations and ¹³C/¹²C ratio with the measured values of reference and calibration materials (Coplen et al., 2006; Dunn and Carter, 2018).
C isotope composition is measured by IRMS as a ¹³C/¹²C ratio, expressed by the instrument software as δ¹³C in ‰:

δ¹³C = [(¹³C/¹²C)_sample/(¹³C/¹²C)_PDB - 1] x 10³

with (¹³C/¹²C)_sample, the ¹³C/¹²C ratio of the soil sample, (¹³C/¹²C)_PDB is the ¹³C/¹²C ratio of PDB (Pee Dee Belemnite, the international standard used to anchor the δ¹³C scale at 0‰) and equals 0.0112372.
Calculate the ¹³C concentration in RAS using the equation:

[¹³C] = [C] x [(δ¹³C_t - δ¹³C_t-0) - (δ¹³C_control - δ¹³C_control-0)] x (¹³C/¹²C)_PDB/10³

where,
[¹³C] estimates the concentration of exudates in the soil or the assimilation by microorganisms present in the soil in mg of ¹³C by kg of soil (equivalent to dry weight),
[C] is the carbon concentration of the soil in mg·kg^-1,
δ¹³C_t is the C isotope composition of organic carbon in the planted soil with ¹³C-labeling,
δ¹³C_t-0 is the C isotope composition of organic carbon in the planted soil without ¹³C-labeling,
δ¹³C_control is the C isotope composition of the organic carbon in of the bulk soil with ¹³C-labeling,
δ¹³C_control-0 is the C isotope composition of the organic carbon in the bulk soil with ¹³C-labeling.
Note: δ¹³C_t-0 and δ¹³C_control-0 (i.e., without ¹³C-labeling) are determined in order to take into account the ¹³C natural abundance in bulk and planted soils. In the majority of experiments, these values will be identical and can therefore be removed from the equation in step 3.

Example of calculations:
For the soil planted with Bromus erectus with ¹³C-labeling (δ¹³C_t), a δ¹³C of 480.5‰ was measured for the root-adhering soil. The δ¹³C of the bulk soil with ¹³C-labeling (δ¹³C_control) was -5.5‰. Without ¹³C-labeling, the δ¹³C of the root-adhering soil (δ¹³C_t-0) was equal to -22.5 ± 0.3 ‰ and the δ¹³C of the bulk soil (δ¹³C_control-0) was -22.7 ± 0.2 ‰. The C concentration ([C]) of the soil was 1.67 ± 0.2 % (16.7 x 10³ mg·kg^-1).
Using the equation in step 3, a·concentration of exudates in the soil of 91.2 mg ¹³C·kg^-1 (dry weight) was measured.

Acknowledgments

The method to measure root exudate content was developed in the Microbial Ecology laboratory jointly with Dr. L. Simon and published by Guyonnet et al. (2018a) Ecology and Evolution DOI: 10.1002/ece.3.4383. We thank the “Serre et chambres climatiques” platform (Université Lyon1, FR BioEnviS) for growing the plants, as well as the “Ecologie Isotopique” platform (Université Lyon 1, UMR 5023) for elemental and isotopic analysis. This work was supported by the French National Research Agency (ANR-18-CE32-0005, DIORE).

Competing interests

No competing interests to declare.

References

Coplen, T. B., Brand, W. A., Gehre, M., Gröning, M., Meijer, H. A. J., Toman, B. and Verkouteren, R. M. (2006). New guidelines for δ¹³C measurements. Anal Chem 78(7): 2439-2441.
Dunn, P. J. H. and Carter, J. F. (2018). Good practice guide for isotope ratio mass spectrometry. In: Dunn P. J. H. and Carter, J. F. (Eds.). 2nd Edition.
Guyonnet, J. P., Cantarel, A. A. M., Simon, L. and Haichar, F. e. Z. (2018a). Root exudation rate as functional trait involved in plant nutrient-use strategy classification. Ecol Evol 8(16): 8573-8581.
Guyonnet, J. P., Guillemet, M., Dubost, A., Simon, L., Ortet, P., Barakat, M., Heulin, T., Achouak, W. and Haichar, F. E. Z. (2018b). Plant nutrient resource use strategies shape active rhizosphere microbiota through root exudation. Front Plant Sci 9: 1662-1662.
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