Discussion:
Collectively, this work identifies critical mechanisms of root and
microbial control over SOM stabilization and destabilization in
miscanthus systems that can promote soil C sequestration and support
plant productivity. Root ingrowth did not lead to a net litter C loss
(Fig. 2 ) despite increased litter decomposition due to enhanced
transfer of C into more stable SOM (Fig. 3 ). Notably, we
document the potential for roots to mobilize litter-derived N from POM
without priming litter C loss (Fig. 2 , Fig. 4 ). We
also identified that microbial nutrient or carbon limitation may alter
how microbes grow and decompose litter-derived SOM, with more litter
decomposition and less MAOM stabilization in organically fertilized
soils (Fig. 5 ). Across all treatments, litter-derived OM was
rapidly incorporated into MAOM and appeared to reflect greater microbial
processing despite declining with increasing microbial biomass (Fig.6 ), supporting recent evidence that MAOM may be more dynamic
than previously thought.
It appears that miscanthus roots can mine N without priming soil C
losses (Fig. 2 ) and increase the C:N of litter-derived light
and heavy POM (Fig. 4 ). This raises the question of how
miscanthus accesses N from decomposing litter without priming C losses
that are commonly observed in other ecosystems (Cheng et al., 2014; Zhu
et al., 2014). One plausible mechanism may be that miscanthus roots
engineer their rhizosphere microbiome composition or function to
preferentially decompose N-rich litter compounds like proteins,
potentially by stimulating proteolytic enzyme production (Brzostek &
Finzi, 2011). While the specific mechanism remains uncertain,
preferential N mining from litter has important implications for
miscanthus sustainability (e.g., the propensity of miscanthus to be high
yielding and build soil C). The resulting increase in remaining litter
C:N may make new litter-derived SOM even more resistant to further
decomposition. In addition, there has been a long-standing question of
how miscanthus can maintain relatively high yields with limited N inputs
(Cadoux et al., 2012). Previous research has posited that high nutrient
use efficiency (Beale & Long, 1997) or the promotion of N-fixing
symbionts (Davis et al., 2010) sustains N nutrition by miscanthus. Here,
we show that highly effective N mining may drive the high productivity,
low fertilization demand, and soil C sequestration of miscanthus
cultivation (Dohleman & Long, 2009; Heaton et al., 2008; Smith et al.,
2013).
Our research suggests that roots can actively support the transfer of
litter derived C into more protected forms. We observed that the priming
of litter decomposition from light POM was balanced by stabilization in
heavy POM (Fig. 3 ). The composition of heavy POM is not as
well-characterized as light POM or MAOM, but this pool is commonly
assumed to be composed of stable soil macro- or micro-aggregates
(Lavallee et al., 2020). Aggregate occluded SOM is largely formed
through root and mycorrhizal symbiont activity (Rillig & Mummey, 2006)
and often consists of partially decomposed plant and microbial organic
matter fragments. This pool has a higher activation energy for
decomposition than low C:N compounds like those in MAOM (Williams et
al., 2018) and is more protected from decomposers than free light POM
(Keiluweit et al., 2017; Kögel-Knabner et al., 2008). As such, there is
an opportunity for stabilizing carbon in high C:N, heavy POM rather than
lower C:N MAOM. The N requirements of low C:N SOM retention have often
been cited as a criticism to efforts to use soil C management to
mitigate global change (Schlesinger & Amundson, 2019). Future research
efforts that investigate how roots can build new, stable, and high C:N
SOM could help realize the potential of soil C sequestration to combat
climate change.
Organic fertilization may promote saprotrophic soil microbes that more
effectively decompose litter substrates and less efficiently form more
stable heavy POM and MAOM. We found that the organic fertilizer
treatments had the greatest microbial biomass and light POM
decomposition, in support of our second fertilization hypothesis, but
less litter C and N were incorporated into MAOM (Fig. 5 , SI
Figs. 3, 4 ). On one hand, differences between fertilization
treatments could arise from a shift in the microbial community structure
or function with organic fertilization (Pan et al., 2014). However,
other research at the site has found no significant effects of nutrient
treatment on microbial diversity or mycorrhizal abundance between
treatments (Kane et al. 2023, in review ). On the other hand, C
vs. N limitation over microbial decomposition can regulate the rate and
efficiency of SOM cycling (Averill & Waring, 2018; Schimel &
Weintraub, 2003). As organic fertilization deposits both C and N, our
observations could be explained by the alleviation of C limitation and
induction of N limitation. In support, we observed a reduction in
nitrification rates with organic fertilization relative to unfertilized
plots (SI Fig. 7 ) and other research found that organic
fertilization increases plot-scale microbial respiration (Kane et al.,
2023, in review ). Here, microbial decomposers could increase
decomposition and growth while respiring excess C and immobilizing N in
living biomass rather than forming more microbially-derived MAOM
(Schimel & Weintraub, 2003).
Our research supports recent theory that some component of MAOM may
dynamically cycle (Kleber et al., 2021; Sokol et al., 2022). Soil C
persistence theory has evolved to rely on two fundamental assumptions,
that (1) microbial growth and necromass production are tightly coupled
to the stabilization of microbially-derived MAOM and (2) that MAOM is
physically inaccessible to decomposers, leading to slow turnover rates
and long-term persistence (Cotrufo et al., 2013; Lehmann & Kleber,
2015). While our MAOM appeared to reflect greater microbial processing
due to a lower, constrained C:N (Fig. 4c ), microbial biomass
was weakly negatively, rather than positively, correlated with MAOM
formation (Fig. 6a ). In contrast to the first assumption, this
pattern supports recent findings that microbial growth and efficiency
may not drive MAOM stabilization (Craig et al., 2022; Sokol et al.,
2022). In contrast to the second assumption, the rapid transfer of
litter carbon into MAOM (Fig. 2 ) and the loss of native MAOM
over a single growing season (Fig. 6b ) suggest that some part
of MAOM may turnover rapidly. Further, declining MAOM formation with
microbial and root biomass (Fig. 6a , SI Fig. 5 )
support recent findings that MAOM can be continually processed by
microbes (Fossum et al., 2022) and that roots can destabilize MAOM
(Jilling et al., 2021; Li et al., 2021). However, other studies observe
that microbial growth efficiency promotes MAOM formation (Kallenbach et
al., 2016; Ridgeway et al., 2022) and that MAOM generally persists
longer than POM pools (Heckman et al., 2022). As such, continuing
research should be directed at determining where and under what
conditions MAOM cycles dynamically.
While our experiment identified several important ways living roots and
soil microbes control litter decomposition and SOM formation, some
mechanisms may not have been fully captured. Our experiment was designed
to separate the effects of roots vs. mycorrhizal fungi on litter C and N
transformations, but our data only identifies a root effect despite the
presence of mycorrhizal fungal symbionts (SI Fig. 8 ). However,
the lack of differences between fungal ingrowth and total exclusion
cores could be linked to the greater dependence of AM plants on root
than hyphal foraging for nutrient uptake (Chen et al., 2016). Future
efforts should quantify mycorrhizal fungal ingrowth to better
investigate the contribution of symbiotic fungi to root-driven SOM
transformations. In addition, our observations of no significant
interaction between fertilization and ingrowth treatments (SI Table 1)
do not support our first fertilization hypothesis that roots would have
the greatest effect in unfertilized soils. However, this pattern may
have been driven by the stand age of miscanthus. These plots were in the
third year of growth whereas older, more nutrient limited stands exhibit
greater differences in root C allocation and N acquisition (Kantola et
al., 2022). As such, future efforts to investigate how nutrient
availability alters living root impacts on SOM formation should leverage
ecosystems with longer-term fertilization history. Despite these
limitations, our data has identified several important mechanisms of SOM
stabilization in situ and provides the foundation for future
efforts to study how living roots and fungi alter SOM dynamics with more
sophisticated measurements, under different environmental conditions, or
across different ecosystems and plant-microbe interactions.
This work has expanded our mechanistic understanding of how living roots
shape ecosystem processes in agricultural systems. Our finding that
miscanthus roots can simultaneously prime N release from litter without
an additional C release and build stable soil C has important
implications for the sustainability of bioenergy production as well as
the viability of restorative agricultural to offset carbon emissions.
Our findings also have important implications for our understanding of
the formation and stability of mineral bound carbon and nitrogen. The
lack of a positive correlation between microbial and root biomass with
MAOM formation, the rapid transfer of litter into MAOM, and the loss of
native MAOM suggest that the mechanisms that control MAOM formation and
loss are more dynamic than current theories assert (Heckman et al.,
2022; Kleber et al., 2021; Sokol et al., 2022). Overall, our work
suggests that living roots can mine N while stabilizing soil C. This
knowledge can help improve the predictive understanding of SOM cycling
that is critical to meeting the goals of restorative agriculture.