10.5061/DRYAD.JWSTQJQC0
Zhu, Xiaomin
0000-0001-8213-1992
Chinese Academy of Sciences
Zhang, Ziliang
University of Illinois Urbana-Champaign
Wang, Qitong
Chinese Academy of Sciences
Peñuelas, Josep
0000-0002-7215-0150
Spanish National Research Council
Sardans, Jordi
Spanish National Research Council
Li, Na
Chinese Academy of Sciences
Liu, Qing
Chinese Academy of Sciences
Yin, Huajun
Chinese Academy of Sciences
Liu, Zhanfeng
Chinese Academy of Sciences
Lambers, Hans
0000-0002-4118-2272
University of Western Australia
More soil organic carbon is sequestered through the mycelium-pathway than
through the root-pathway under nitrogen enrichment in an alpine forest
Dryad
dataset
2022
roots
ectomycorrhizal mycelia
microbial C pump
SOC sequestration
N deposition
alpine forests
FOS: Natural sciences
National Natural Science Foundation of China
https://ror.org/01h0zpd94
32171757
The Chinese Academy of Sciences (CAS) Interdisciplinary Innovation Team*
xbzg-zysys-202112
The Second Tibetan Plateau Scientific Expedition and Research*
2019QZKK0301
European Research Council Synergy project*
SyG-2013-610028 IMBALANCE-P
The Spanish Government, grant*
PID2019-110521GB-I00
National Natural Science Foundation of China
https://ror.org/01h0zpd94
31901131
National Natural Science Foundation of China
https://ror.org/01h0zpd94
42177289
The Spanish Government, grant*
PID2020-115770RB-I00
2022-06-21T00:00:00Z
2022-06-21T00:00:00Z
en
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https://doi.org/10.1111/gcb.16263
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CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.
Isolation of roots and mycelia using ingrowth cores 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-3, 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. Root and mycelium biomass
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). Soil aggregate fractionation and SOC concentration 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 (<
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 (Feo + Alo) and dithionite-extractable Fe/Al (Fed + Ald) were
measured by using the extraction method proposed by Gentsch et al (2018).
The Fed + Ald indicates the amount of pedogenic Fe and Al within oxides,
silicates and organic complexes, whereas Feo + Alo 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). SOC chemical composition 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. Microbial community
composition 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+) bacteria. The PLFAs 16:1ω9c, 16:1ω7c, 18:1ω7c, cy17:0, cy19:0 were
used to indicate the relative biomass of Gram-negative (G-) 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).
Extracellular enzyme activity 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. Data calculation and statistical analysis 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 MRCN, SOCN, MRCCK, and SOCCK 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.