DOI: 10.5061/dryad.jwstqjqc0
title: More soil organic carbon is sequestered through the mycelium-pathway than through the root-pathway under nitrogen enrichment in an alpine forest

description: Plant roots and associated mycorrhizae exert a large influence on soil
 carbon (C) cycling. Yet, little was known whether and how roots and
 ectomycorrhizal extraradical mycelia differentially contribute to soil
 organic C (SOC) accumulation in alpine forests under increasing nitrogen
 (N) deposition. Using ingrowth cores, the relative contributions of the
 root-pathway (RP) (i.e., roots and rhizosphere processes) and
 mycelium-pathway (MP) (i.e., extraradical mycelia and hyphosphere
 processes) to SOC accumulation were distinguished and quantified in an
 ectomycorrhizal-dominated forest receiving chronic N addition (25 kg N
 ha-1 yr-1). Under the non-N addition, the RP facilitated SOC accumulation,
 while the MP reduced SOC accumulation. Nitrogen addition enhanced the
 positive effect of RP on SOC accumulation from +18.02 mg C g-1 to +20.55
 mg C g-1 but counteracted the negative effect of MP on SOC accumulation
 from -5.62 mg C g-1 to -0.57 mg C g-1, as compared to the non-N addition.
 Compared to the non-N addition, the N-induced SOC accumulation was
 1.62~2.21 mg C g-1 and 3.23~4.74 mg C g-1, in the RP and the MP,
 respectively. The greater contribution of MP to SOC accumulation was
 mainly attributed to the higher microbial C pump (MCP) efficacy (the
 proportion of increased microbial residual C to the increased SOC under N
 addition) in the MP (72.5%) relative to the RP (57%). The higher MCP
 efficacy in the MP was mainly associated with the higher fungal metabolic
 activity (i.e., the greater fungal biomass and N-acetyl glucosidase
 activity) and greater binding efficiency of fungal residual C to mineral
 surfaces than those of RP. Collectively, our findings highlight the
 indispensable role of mycelia and hyphosphere processes in the formation
 and accumulation of stable SOC in the context of increasing N deposition.
description: <strong>Isolation of
 roots and mycelia using ingrowth cores</strong>
 To isolate roots and
 mycelia, we adopted an ingrowth-core technique modified from Zhang et al.
 (2018) and Keller et al. (2021). Ingrowth cores (6 cm inner diameter and
 15 cm depth) were wrapped with a mesh with different pore sizes: mesh size
 of 2000 µm allowed the ingrowth of fine roots and mycelia (both roots and
 mycelia accessible); 48-µm mesh permitted the growth of mycelia but not of
 fine roots (only mycelia accessible), and 1-µm mesh excluded the growth of
 both roots and mycelia (only the soil) (Fig. 2). The C source in the 2-mm
 mesh cores was mainly derived from roots, mycelia and litter leachates,
 that of the 48-µm mesh cores was derived from mycelia and litter
 leachates, while the 1-µm mesh cores received C only from litter
 leachates. The soil was collected from the mineral layer (0-15cm) at each
 plot. After removing the visible roots, the soil from the same plot was
 homogenized and sieved through a 5-mm mesh. The sieved soil was filled
 into ingrowth cores corresponding to the soil bulk density at 0-15 cm
 depth (0.796 g cm<sup>-3</sup>, approximately 337 g per core). Six sets of ingrowth cores with different mesh-size (1-µm, 48-µm and 2000-μm) were installed in each treatment plot. In total, 108 ingrowth cores (2 N levels * 3 replicates * 6 sets * 3 mesh-sizes) were installed in this coniferous forest. Ingrowth cores were randomly placed in the topmost mineral horizon (0-15cm depth) in each plot in July 2017. The bottom of the ingrowth cores was covered with the corresponding size of the mesh to prevent inputs of roots and mycelia, respectively, and the top was covered by multiple layers of the corresponding size of the mesh to block the entry of coniferous litter but to allow gas and water exchange. When the cores were retrieved, we did not detect any external litter in the cores. To block the influx of new C derived from the saprophytic mycelia outside the cores, we spread a 2 mm-thick layer of silica sand around the cores. Silica sand as a growth substrate effectively reduces the disturbance of saprophytic hyphae (Hagenbo et al., 2017). Ingrowth cores were harvested in August 2019 and August 2020, respectively. Two sets of ingrowth cores were collected in each plot at each sampling date. Cores were transported to the laboratory within the icebox. After the removal of roots, soils inside the cores were sieved through a 2-mm mesh and divided into two subsamples: one subsample stored in -4 °C was used for the analyses of enzyme activities and microbial community composition; the second subsample was air-dried to perform soil aggregate fractionation, SOC determination, and soil biomarkers analysis. <strong>Root and mycelium biomass</strong> Roots inside the 2000-µm mesh cores were manually picked out, washed thoroughly, oven-dried at 60°C for 48 hours and then weighed to determine the total root biomass. The ectomycorrhizal mycelium biomass was estimated using mesh bags (2 cm inner diameter, 15 cm depth; mesh size: 48 µm) filled with different particle sizes of HCl-washed silica sand (60 g, 0.36-2 mm) (Wallander et al., 2001). The mesh bags were randomly buried into the 0-15 cm soil depth in each plot in July 2017, and recovered at the same time as the ingrowth cores. The concentration of ergosterols was measured to characterize the biomass of ectomycorrhizal mycelia in the mesh bags (see details in the Supplementary Methods) (Parrent & Vilgalys, 2007). <strong>Soil aggregate fractionation and SOC concentration</strong> To understand the physico-chemical protection of SOC in the RP and MP under N addition, soils were physically fractionated into three size fractions to examine the allocation of C and biomarkers among macroaggregates (Macro: 250~2000 µm), microaggregates (Micro: 53~250 µm) and slit-clay (&lt; 53 µm) by using the wet-sieving technique (Six et al., 1998). The proportions of SOC and the concentrations of biomarkers in the three fractions were measured to characterize the role of physical protection by aggregates. The SOC and total N (TN) concentrations in bulk soil and size fractions were analyzed using an elemental analyzer (Vario MACRO, Elementar Analysensysteme GmbH, Hanau, Germany). To assess the protection of SOC by minerals, two forms of Fe and Al oxides, oxalate-extractable Fe/Al oxides (Fe<sub>o</sub> + Al<sub>o</sub>) and dithionite-extractable Fe/Al (Fe<sub>d</sub> + Al<sub>d</sub>) were measured by using the extraction method proposed by Gentsch et al (2018). The Fe<sub>d</sub> + Al<sub>d</sub> indicates the amount of pedogenic Fe and Al within oxides, silicates and organic complexes, whereas Fe<sub>o</sub> + Al<sub>o</sub> represents poorly crystalline oxyhydroxides (Gentsch et al., 2018). The concentrations of Fe and Al oxides in extracts were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES, Optima 8300, Perkin Elmer, USA). <strong>SOC chemical composition</strong> A range of major biomarkers, which are widely accepted to trace plant-derived and microbial-derived C, respectively, were selected to reveal the changes of the chemical composition of SOC in two pathways under N addition (Barré et al., 2018; Liang et al., 2019). Air-dried soil (1 g) was sequentially extracted (solvent extraction, base hydrolysis, and CuO oxidation) to isolate solvent-extractable free lipids (long-chain fatty acids), cutin- and suberin-derived compounds and lignin-derived phenols (vanillyls, syringyls and cinnamyls), respectively, according to standard protocols (Otto & Simpson, 2007; Tamura & Tharayil, 2014). Since the direct contribution of microbial living biomass to soil amino sugars is negligible, amino sugars are good indicators of microbial necromass (Liang et al., 2017, Joergensen, 2018). Four types of amino sugars, including glucosamine, galactosamine, manosamine, and muramic acid, were tested in this study. By assessing them in soils, we can investigate microbial necromass dynamics at the community-level (i.e., fungi and bacteria) and evaluate the contributions of necromass to SOC storage under different environmental conditions (Joergensen, 2018; Liang et al., 2019). The detailed chemical extractions and analyses of plant and microbial biomarkers are provided in Supplementary Methods. <strong>Microbial community composition</strong> Soil microbial community composition was characterized using the phospholipid fatty acids (PLFAs) methods (see details in Supplementary Methods) (Bossio & Scow, 1998). The identification of the extracted fatty acid was based on a MIDI peak identification system (Microbial ID Inc., Newark, DE, USA). The PLFAs i15:0, α15:0, i16:0, i17:0, α17:0 were used to indicate the relative biomass of Gram-positive (G<sup>+</sup>) bacteria. The PLFAs 16:1ω9c, 16:1ω7c, 18:1ω7c, cy17:0, cy19:0 were used to indicate the relative biomass of Gram-negative (G<sup>-</sup>) bacteria. The PLFA 18:2ω6c was used as an indicator of saprotrophic fungal biomass. The PLFAs 10Me16:0, 10Me17:0 and 10Me18:0 were used to indicate actinomycete (AC) biomass. Microbial community composition was assessed by the ratio of saprotrophic fungal biomass to bacterial biomass (F/B ratio). <strong>Extracellular enzyme activity </strong>The activities of three extracellular enzymes involved in the decomposition of lignin and fungal residues were measured as described by Saiya-Cork et al. (2002) (see details in Supplementary Methods). The β-N-acetyl-glucosaminidase（NAG）participates in chitin and peptidoglycan degradation, hydrolyzing chitobiose to glucosamine (Sinsabaugh et al., 2009). NAG activity was measured fluorometrically using 4-methylumbelliferyl N-acetyl-β-D-glucosaminide as the substrate. Phenol oxidases (POX) and peroxidases (PER) play an important role in degrading polyphenols, and their activities were measured colorimetrically using L-dihydroxyphenylalanine (DOPA) as the substrate. <strong>Data calculation and statistical analysis</strong> To isolate the effects of root and mycelium on the SOC dynamics and associated microbial characteristics (i.e., SOC, biomarkers concentrations, fungal and bacterial biomass, and enzymes activities), net changes of the observations mediated by the root-pathway and mycelium-pathway were quantified by the difference of corresponding variables between the 2-mm mesh cores and 48-µm mesh cores, or between the 48-µm cores and 1-µm mesh cores, respectively (Fig. 2). The recent concept proposed by Zhu et al (2020) highlighted the contribution of microbial necromass to the SOC pool (i.e., MCP efficacy). Based on this concept, the changes of MCP efficacy (i.e., the contribution of increased microbial residual C to the increased SOC) under N addition were calculated as follow: Changes of MCP efficacy (% SOC) under N addition =  , where MRC<em><sub>N</sub></em>, SOC<em><sub>N</sub></em>, MRC<sub><em>CK</em></sub>, and SOC<em><sub>CK</sub></em> represent the concentration of microbial residual C and SOC in the N-addition plots and the non-N addition plots, respectively. Additionally, the contribution of increased plant-derived C to the increased SOC induced by N addition was calculated using Eq. 1 but replacing microbial residual C with plant-derived C.