Feb 05, 2026
Determining Root and Fungal Derived Soil Organic Carbon Accumulate Rates
- 1Dartmouth College
- Tumber-Dávila LabTech. support email: [email protected]
Protocol Citation: Thomas Muratore 2026. Determining Root and Fungal Derived Soil Organic Carbon Accumulate Rates. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge1exyv47/v1
Manuscript citation:
Muratore, Thomas J., Nikhil R.Chari, Richard P.Phillips, Benton N.Taylor, Melissa A.Knorr, and Serita D.Frey. 2026. “Increased Root-Derived Carbon Buffers Soil Carbon Loss under Simultaneous Warming and Nitrogen Addition.” Ecology107(3): e70351. https://doi.org/10.1002/ecy.70351
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 23, 2026
Last Modified: February 05, 2026
Protocol Integer ID: 240478
Keywords: fungal derived soil organic carbon accumulate rate, c4 soil, organic matter formation from belowground source, derived soil fill, soil substrate, net incorporation of new belowground carbon, rates of soil, dissolved organic carbon, defined soil substrate, soil, soil fill, organic carbon, c3 vegetation input, c3 vegetation, organic matter formation, new belowground carbon, hyphae inputs from hyphae, δ¹³c contrast between c3 vegetation input, hyphae outer core, hyphae input, isotopic tracer, belowground source, combined root, determining root
Abstract
This protocol quantifies net incorporation of new belowground carbon (C) into a defined soil substrate using the δ¹³C contrast between C3 vegetation inputs and a C4-derived soil fill. The design partitions root + hyphae inputs from hyphae-only inputs using a nested mesh insert, and uses paired no-ingrowth controls to correct for plot-specific background shifts and water-mediated (dissolved organic carbon) inputs. This method can be used to estimate rates of soil organic matter formation from belowground sources; however, it does not reflect long-term stability and persistence of SOM.
Scope and principle
- Isotopic tracer: C4 soil (relatively enriched δ¹³C) packed into cores placed under C3 vegetation (relatively depleted δ¹³C).
- Biological partitioning: A hyphae-only insert (37 µm mesh) nested within a root + hyphae outer core (3 mm mesh) separates hyphal-only from combined root + hyphal inputs.
- Control correction: A paired no-ingrowth control (1 µm mesh wrap) corrects for non-ingrowth isotopic shifts (e.g., plot specific background changes, water movement, DOC inputs).
Guidelines
Experimental design
Core types used in this protocol
- Root + hyphae ingrowth core (outer mesh cylinder) with a hyphae-only insert inside
- Paired no-ingrowth control (1 µm mesh wrapped)
- Solid PVC “no input” cores installed in a nearby reference area to exclude both roots and DOC entry. Reference control (recommended if dissolved inputs are a concern)
Replication guidance (site-adaptable)
- Deploy cores across replicated plots appropriate for your study design (e.g., treatment × replicate plots).
Minimum per plot:
- 1 root + hyphae ingrowth core containing a hyphae-only insert
- 1 paired no-ingrowth control core
Record at minimum: plot ID, core ID, core type, install date, retrieval date, microsite notes, and deviations (lost cores/plots, damage, etc.).
Materials
Materials and equipment
Core materials
1. Root + hyphae ingrowth core (outer cylinder)
- Rigid plastic mesh cylinder (e.g., Industrial Netting #RN4465 or equivalent)
- Dimensions: 5.77 cm diameter × 10 cm height
2. Hyphae-only insert (inner cylinder)
- Mesh fabric: 37 µm (permits hyphae; excludes roots) folded into a cylinder shape
- Dimensions: 2 cm diameter × 7 cm height
3. No-ingrowth control core
- Packed identically to ingrowth cores, then wrapped in 1 µm mesh (excludes roots and hyphae; permits water movement)
4. Caps / litter exclusion
- Window screen (or similar) and inert fasteners (zip ties/bands)
Fill substrate preparation
- Surface soil from a long-established C4-dominated field (0–5 cm)
- Sand (to create a sand:soil mixture)
- 4 mm sieve
- Containers for mixing and incubation
Field and lab supplies
- Labels (waterproof), permanent marker
- Trowels/soil corers/shovels for installation
- Plastic bags for transport and storage
- Cooler / refrigerator (4 °C)
- 2 mm sieve for processing
- Drying ovens (105 °C for soils; 60 °C for roots)
- Grinder for soil homogenization
- EA-IRMS access for %C and δ¹³C
Prepare isotopically distinct fill substrate (C4 soil + sand)
Collect 0–5 cm soil from a long-established C4 site. Remove
coarse debris (roots, stones, litter).
Sieve soil to <4 mm.
Allow soil to incubate in the lab at room temperature for at least 2 months. This allows all labile C4 plant material to be mineralized and allows the C4 signature to equilibrate.
Mix the sieved C4 soil with sand to create a sand:soil mixture.
The mix you decide should be aimed at matching your sites textures or increasing "clean" carbon free sand to improve the detectability of carbon inputs.
Record the mass ratio and apply it consistently across all
cores.
I recommend mixing the exact amount needed for each core before filling.
Core construction and packing (critical steps for reproducibility)
Label all components and assign unique IDs:
- Outer (root + hyphae) core ID
- Hyphae-only insert ID
- Paired no-ingrowth control core ID
Pack the hyphae-only insert first (fungal sleeve) with the C4:sand mixture.
- Standardize to a target bulk density across all cores.
- Add the sand:C4 soil mixture in small increments.
- Gently tamp to remove large voids; avoid over-compaction that could restrict hyphal entry.
- Pack to a target bulk density (mass-based target per insert volume recommended).
- Place the packed hyphae-only insert into the main (outer) core.
- NOTE: I used a plastic bag sealer to close the ends of the 37 um mesh on both sides.
Pack the root + hyphae core
- Standardize to a target bulk density across all outer cores.
- Example implementation: BD = 1.0 g cm⁻³ for the packed sand:C4 soil mixture.
- Have the C4:sand mixture weighted and ready to go.
- attach a piece of 1 um mesh on the bottom of the core to prevent soil from spilling out. I used super strength super glue with no issues over 2 growing seasons in a temperate forest with freeze thaw.
- Position the insert vertically and centered within the outer mesh cylinder.
- Fill and pack the outer compartment while holding the insert still.
- Physically hold the insert stationary (hand or clamp/stand) to prevent tilting or shifting.
- Add the sand:C4 soil mixture around the insert in small lifts (e.g., 1–2 cm).
- Lightly tamp after each lift until the outer core is filled to the intended height.
- Inspect for bypass gaps.
- Confirm the insert remains centered.
- Confirm there are no obvious channels between insert and outer compartment.
- Re-pack and re-center if needed.
Prepare the no-ingrowth control cores (Control cores)
- Wrap a piece of rigid 3 mm rigid mesh in 1 um mesh.
- Attach a piece of 1 um mesh on the bottom of the core to prevent soil from spilling out. I used super strength super glue with no issues over 2 growing seasons in a temperate forest with freeze thaw.
- Pack the control core identically as the root + hyphae core above.
Field installation
- At each plot, carefully remove the surface litter layer without mixing it into mineral soil.
- Create a hole matching the outer core dimensions to 10 cm depth.
- Insert the core so the top is flush with the soil surface (or at a consistent reference depth across all plots).
- Fill any air gaps along the outside of the core with the same sand:C4 soil mixture to minimize preferential flow and root bypass.
- Record installation metadata: Date, plot ID, core IDs, core type, microsite notes.
- Deployment duration guidance: install during the growing season; harvest after the desired interval (e.g., after 1–2 growing seasons).
Harvest and storage
- Carefully excavate cores to avoid losing soil from within the core.
- Remove external root fragments from the outside surface of the core (do not disturb internal soil).
- Seal cores in plastic bags
- Store at 4 °C for ≤3 days before processing.
Core processing (soil and roots)
- Separate the hyphae-only insert from the outer core.
Process compartments separately:
- Hyphae-only insert soil
- Outer (root + hyphae) compartment soil
- Sieve soils to <2 mm.
- Remove fine roots (<2 mm) from the outer compartment for:
- Biomass (fine-root production)
- Isotopic endmember characterization (δ¹³C and %C), as applicable
- Dry soils at 105 °C, then finely grind to homogenize.
- Analyze dried, ground soils for total C and δ¹³C by EA-IRMS (or equivalent).
You may wish to perform additional analyses on the soil. For instance you could fractionate the soil into POM and MAOM to see which pool of SOM the new C accumulated in. See my protocol, Mineral Associated Organic Matter (MAOM) and Particulate Organic Matter (POM) Size Fractionation Protocol
Data Analysis
Two-endmember mixing model: fraction of soil C derived from new belowground inputs
For each ingrowth compartment (outer or insert), compute: f = (δ¹³C_ingrowth − δ¹³C_control) / (δ¹³C_input δ¹³C_control)
Where:
δ¹³C_ingrowth = δ¹³C of recovered C4 fill soil from the ingrowth compartment after deployment
δ¹³C_control = δ¹³C of recovered soil from the paired 1 µm no-ingrowth control in the same plot after deployment
δ¹³C_input = δ¹³C of the belowground input endmember. For root + hyphae compartment: weighted average δ¹³C of recovered root tissues contributing to that core (site-specific; may include multiple species)
For hyphae-only inserts: δ¹³C of hyphal inputs
Estimating fungal δ¹³C when hyphae cannot be sampled
If hyphal δ¹³C cannot be directly measured, estimate fungal
δ¹³C as:
Fungal δ¹³C = Root δ¹³C + 2‰
This applies a discrimination offset consistent with published estimates of enrichment during C transfer from host plants to mycorrhizal fungi (Ekblad et al., 2016).
Convert fraction to areal accumulation of new belowground-derived SOC
Compute new belowground-derived SOC accumulation (g C m⁻²):
C_new = C_conc × f × BD × (d × 10,000)
Where:
C_conc = soil C concentration after deployment (convert %C to g g⁻¹ as needed)
f = fraction from mixing model
BD = bulk density of packed sand:C4 soil mixture (empirically measured/confirmed from packing records)
BD = bulk density of packed sand:C4 soil mixture (empirically measured/confirmed from packing records)
d = core depth (cm; 10 cm in this protocol)
10,000 = conversion from cm² to m²
Annualization
If deployed across multiple growing seasons, compute annual
net accumulation:
C_new,annual = C_new / (number of growing seasons)
Quantifying fine-root production from ingrowth cores
After sieving (<2 mm), separate live fine roots (<2 mm diameter; typically 1st–3rd order) using forceps.
If needed, sort by genus/species using morphology (color, branching, texture) and a stereomicroscope; document criteria.
Exclude dead roots using discoloration, brittleness, and cortex separation (Persson & Stadenberg, 2009).
Dry cleaned roots at 60 °C for 48 h and weigh.
Determine root %C via elemental analysis (or apply a justified %C estimate).
Compute annual fine-root production (g C m⁻² yr⁻¹ or per core scaled appropriately): Fine-root production = (M_live_roots × (%C/100)) / (years deployed)
(For a two-year deployment, divide by 2.)