Ingrowth cores
In each plot, we installed five rigid plastic mesh root ingrowth cores (inner diameter 2.27”; Industrial Netting product #RN4465), hereafter referred to as “ingrowth cores”, as well as two PVC cores that were impermeable to root and fungal ingrowth, i.e. “control cores”. Cores were randomly places throughout the plot. The top and bottom of each ingrowth core was covered with window-screen mesh, while the control cores were covered with 1-micron mesh to prevent root and fungal ingrowth. Cores were left in the field for two full growing seasons (Spring 2017 — Fall 2018).
To quantify plant-derived belowground C inputs to the soil, we used a dual ingrowth-core isotopic technique similar to Panzacchi et al. (2016). Each ingrowth core was filled with a C4soil/sand mixture (50:50 by volume) to reduce soil compaction, increase detectability of root-derived C inputs, and enhance root recovery from ingrowth core soils following harvest. The soil was obtained from an agricultural field at the University of Illinois Energy Farm (40º 03’ 046” N, 88º 11’ 046” W) where soils are silt-loam Arguidolls. The field had been under a corn-soy rotation for >100 years, with corn planted most years including the year prior to soil collection. Given that corn is a C4 plant, the soil carried a δ13C signature of -16.0 ± 0.15 (mean ± SE, n = 6), which was significantly enriched in 13C compared to the C3 root material recovered from the ingrowth cores (-28.7 ± 0.15 across all sites, n = 54). Surface soils from the farm field were collected and transported to the laboratory at Indiana University for ingrowth core preparation. There, soils were sieved to 4mm and organic debris was removed. Soils were then mixed with carbonate-free sand in a 50:50 ratio by volume and refrigerated until deployment in the field.
At the beginning of the growing season at each site, ingrowth and control cores were installed in each of the 54 plots. At each core location, a soil core of equal diameter to the ingrowth core was taken, soil was removed and replaced by an ingrowth (or control) core filled with the C4-soil/sand mixture. Care was taken to minimize disturbance of the surrounding soil to prevent significant air gaps between the installed core and forest soil. After two full growing seasons, cores were carefully extracted and transported back to the laboratory for processing.
Roots were removed from each soil core, washed thoroughly, dried at 60 °C for 48 hours and then weighed to 0.0001g. The C4 soil from each core was air-dried. Subsequently, all C4 soil core samples and a subset of root tissue samples were analyzed for total C, N, and δ13C. Specifically, one AM and one ECM end-member plot was selected at each site and root samples from each core within these plots were analyzed, assuming root δ13C is conserved across plots of similar mycorrhizal dominance within a given site. Root tissue and C4 soil subsamples were ground to a powder using a 2010 GenoGrinder (SPEX® SamplePrep) and analyzed for total C and δ13C using an elemental analyzer coupled to a gas-isotope ratio mass spectrometer. Root and C4 soil samples were analyzed at two facilities (the Purdue Stable Isotope Facility with a PDZ Europa Elemental Analyzer coupled to a Sercon 20-22 IRMS, Cheshire, UK, and the BayCEER Laboratory of Isotope Biogeochemistry with a Carlo Erba 1108 Elemental Analyzer coupled to a delta S Finnigan MAT, Bremen, Germany). A subset of samples was analyzed at both facilities, confirming the two facilities reported comparable results (R2 = 0.96). Isotope ratio values were expressed with the delta notation (δ):
δ13C ‰= [(Rsample/Rstandard – 1) × 1000]
where Rsample and Rstandard are the13C : 12C sample and standard ratios, respectively, and Rstandard is referenced to the Vienna Pee Dee Belemnite (VPDB).