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\begin{document}

\title{Roots selectively decompose litter to acquire nitrogen and build new
soil carbon}



\author[1]{Joanna Ridgeway}%
\author[1]{Jennifer Kane}%
\author[1]{Ember Morrissey}%
\author[1]{Hayden Starcher}%
\author[1]{Edward Brzostek}%
\affil[1]{West Virginia University}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Plant-microbe interactions in the rhizosphere shape carbon and nitrogen
cycling in soil organic matter (SOM). However, there is conflicting
evidence on whether these interactions lead to a net loss or increase of
SOM. In part, this conflict is driven by uncertainty in how living roots
and microbes alter SOM formation or loss in the field. To address these
uncertainties, we traced the fate of isotopically labeled litter into
SOM using root and fungal ingrowth cores incubated in a Miscanthus x
giganteus field . Roots stimulated litter decomposition, but balanced
this loss by transferring carbon into more persistent, aggregate
associated SOM. Further, roots selectively mobilized nitrogen from
litter without additional carbon release. Overall, our fundings suggest
that roots can efficiently mine nitrogen and build persistent soil
carbon.%
\end{abstract}%



\sloppy


\textbf{Introduction:}

Managing soils in agricultural systems to sequester carbon (C) in soil
organic matter (SOM) may be a powerful approach to offset anthropogenic
C emissions (Lal, 2004). Soils are the largest terrestrial C pool, and
experimental manipulations like changing vegetation type, increasing
organic inputs, or altering management practices demonstrate the
potential for significant and rapid SOM accumulation (Minasny et al.,
2017; Paustian et al., 2016). However, there is a high degree of
uncertainty in understanding, predicting, and optimizing soil C
accumulation (Sulman et al., 2018). Much of this uncertainty arises
because plant roots and soil microbes, the active drivers of soil
biogeochemical cycling, both build and deplete SOM through
simultaneously occurring processes. As such, our ability to optimize
soil C sequestration relies on improving our understanding of how roots
and microbes drive the transfer of new litter C inputs into SOM.

As per the current understanding of SOM formation, litter inputs are
decomposed into simpler compounds that can be physically protected from
microbial decomposers by occlusion in soil aggregates or sorption to
mineral surfaces (Lehmann \& Kleber, 2015). As such, SOM is often
delineated into three main pools (Fig. 1a): undecomposed or
partially-decomposed particulate organic matter (here, light POM),
aggregate-occluded SOM (here, heavy POM), and mineral associated organic
matter (MAOM) (Lavallee et al., 2020). Light POM accumulation depends
upon the balance between litter inputs to soil and litter decomposition,
and can accumulate with no apparent upper limit but is also vulnerable
to factors like warming that enhance decomposition rates (Benbi et al.,
2014; Cotrufo et al., 2019). Heavy POM is operationally separated from
light POM by density fractionation and is linked with stable soil
aggregates (Lavallee et al., 2020). Accumulation in this pool may
saturate and is vulnerable to factors like soil disturbance and land use
change (Bronick \& Lal, 2005). MAOM is generally considered to be the
most persistent or protected form of SOM (Cotrufo et al., 2013; Liang et
al., 2017). However, optimizing MAOM accumulation may only be practical
in soils like those in degraded agricultural ecosystems that have lost
nearly 50\% of their C since ploughing the prairie (Stockmann et al.,
2015) as MAOM accumulation appears to saturate (Cotrufo et al., 2019;
but see Georgiou et al., 2022). To manage ecosystems for soil C
sequestration, it is critical to understand what drives the transfer of
new litter inputs between these SOM pools to enhance our predictive
understanding of how much soil C can accumulate and how persistent this
soil C may be in a changing climate.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1a:} Litter inputs join SOM as light POM, which is
largely composed of undecomposed litter fragments. As decomposition
progresses, litter-derived SOM can more easily become incorporated into
aggregates in heavy POM or microbial decomposition products and
necromass can preferentially sorb to soil mineral surfaces as
MAOM.\textbf{1b:} Roots and root-associated fungal symbionts can enhance
both retention or loss of litter in light POM (top), heavy POM (middle),
and MAOM (bottom) pools.

Living roots and their associated fungi alter SOM formation by sending
C-rich exudates to the rhizosphere to enhance decomposition and acquire
N (Bais et al., 2006; Grayston et al., 1997). However, a high degree of
uncertainty remains in whether this increases or decreases soil C
accumulation. In Figure 1b, we diagram potential hypotheses for how
roots could alter litter loss from light POM and the accumulation of new
heavy POM and MAOM through distinct mechanisms. First, root stimulation
of microbial decomposition to mineralize soil N can increase the loss of
unprotected light POM through the rhizosphere priming effect (Cheng et
al., 2014). However, there is also evidence that roots and symbiotic
fungi can outcompete saprotrophic microbes for resources like water and
nutrients leading to the suppression of decomposition (Fernandez \&
Kennedy, 2016). Second, as litter inputs are transferred into more
protected heavy POM, root ingrowth has the potential to both invade
aggregates and increase the formation rate of new aggregates (Six et
al., 2000). Finally, roots can enhance new MAOM formation by increasing
the efficiency of microbial litter decomposition, resulting in greater
microbial biomass production and the formation of microbial necromass
(Liang et al., 2017). This necromass can associate with mineral surfaces
and is the main precursor to MAOM in grassland ecosystems (Angst et al.,
2021). However, roots may also deplete new, litter-derived MAOM as
recent evidence suggests that roots can actively mine MAOM for nutrients
(Jilling et al., 2021) and that root exudate compounds can displace MAOM
from soil minerals (Keiluweit et al., 2015). As such, predicting whether
roots will drive a net gain or loss of soil C is hindered by uncertainty
in how roots impact SOM formation in these different pools.

The extent to which roots and mycorrhizal fungi facilitate SOM formation
or loss in agricultural ecosystems may be modulated by fertilization.
For example, some N-limited plants can dynamically shift C allocation
belowground to root exudation and mycorrhizal symbionts to stimulate
microbial decomposition in the rhizosphere and increase N acquisition
(Brzostek et al., 2014; Kane et al., 2022). When N limitation is
alleviated by fertilization, plants can also reduce belowground C
allocation, suppressing SOM decomposition (Eastman et al., 2021; Frey et
al., 2014). The degree to which fertilization alters SOM cycling also
depends upon the activity of saprotrophic soil microbial decomposers. In
contrast to plants, soil microbes are primarily understood to be energy,
or carbon, limited (Soong et al., 2020). As such, organic fertilizer
that contains C and N can prime microbial activity and decomposition
relative to inorganic N fertilizer (Cui et al., 2022; Ndung'u et al.,
2021). However, uncertainty remains in the extent to which the priming
of microbial activity leads to net soil C losses by enhancing
decomposition or net C gains by promoting the production of microbial
necromass that can form MAOM. Collectively, the effect of fertilization
on SOM formation depends upon the strength of plant-microbe interactions
and the form of fertilizer applied, but the magnitude of this effect is
uncertain.

Given the uncertainty above, our objectives were to: \textbf{1)
determine how living roots and symbiotic fungi influence litter
decomposition and SOM formation in distinct SOM pools and 2) assess how
microbially-driven SOM formation is altered by fertilization.} For the
first objective, we assayed the net effect of the opposing hypotheses
illustrated in Figure 1. For the second objective, we tested two
hypotheses: (1) the effect of living roots on SOM formation would be
strongest in unfertilized soil and (2) organic fertilizer would
accelerate microbial decomposition and SOM cycling to a greater extent
than inorganic fertilizer (SI Figure \textbf{1} ). To meet our
objectives, we measured the effects of living roots and fungi on new SOM
formation from isotopically enriched litter over one growing season. We
incubated litter inputs in soil cores that were open to roots and fungal
ingrowth (root), that excluded roots but were open to fungal ingrowth
(fungal), or that excluded both roots and fungi (none) to quantify the
effect of living roots and fungi on new SOM formation (SI Fig.\textbf{2}
). We installed ingrowth cores in \emph{Miscanthus x giganteus} (herein
miscanthus) plots with different nutrient treatments to investigate the
effect of soil N and C availability on how roots, mycorrhizal fungi, and
saprotrophic microbes drive the transfer of litter C and N into light
POM, heavy POM, and MAOM. We used the bioenergy feedstock crop
miscanthus as a study system because it produces extensive root systems
to overcome nutrient limitation (Dohleman \& Long, 2009; Heaton et al.,
2008) and because miscanthus agriculture typically increases SOM levels
(Harris et al., 2015). Further, because bioenergy offers the potential
to become a C neutral or C negative alternative to fossil fuels, it is
particularly critical to investigate what drives SOM accumulation in
these ecosystems (Hanssen et al., 2020).

We show that miscanthus roots increased litter decomposition but did not
lead to a net C loss because roots enhanced the incorporation of litter
C into heavy POM. Roots also selectively mobilized litter N from both
POM pools. As such, roots can transfer C into a more persistent SOM pool
while still enhancing N mining. These root effects did not depend on
fertilization. However, organic fertilization enhanced microbial litter
decomposition without increasing litter incorporation in MAOM.



\textbf{Materials and methods:}

\emph{Site description and location selection}

This experiment was performed at the West Virginia University (WVU)
Animal Sciences farm in Morgantown, West Virginia (39\selectlanguage{ngerman}°40'10.2''N,
79°55'53.6''W). This site is located next to the former Baker's Ridge
Mine Site (National Mine Repository 304559) and is managed as a
cool-season grass pasture (detailed site description available in Kane
et al. 2023, \emph{in review} ). Miscanthus plots were established in
2019 using a fully randomized block design with 4 fertilization
treatments replicated 8 times for a total of 32 plots (Kane et al.
2023,\emph{in review} ). Each plot is 5 m\textsuperscript{2} and was
established by planting 25 miscanthus rhizomes using 1
m\textsuperscript{2} grid spacing (site map, SI Fig. \textbf{3a} ).
Plots are fertilized yearly with treatments that include no
fertilization, low-level inorganic N additions (28.5 kgN/ha), high-level
inorganic N additions (57 kgN/ha), and organic fertilization (local
manure, \textasciitilde{}57 kgN/ha). Due to logistical constraints for
sample size, we utilized the control, high-level inorganic, and organic
fertilization treatments for this experiment.

\emph{Experimental design}

We incubated isotopically enriched litter in soil ingrowth cores and
traced the fate of litter C and N into SOM over one growing season. Our
experimental design included 3 levels of root/hyphal ingrowth: root and
fungal ingrowth (root), root exclusion and fungal ingrowth (fungal), and
root and fungal exclusion (none) and 3 fertilization treatments: no
fertilization (control), high-level inorganic fertilization (high N), or
organic fertilization (organic). We randomly selected 5 plots from each
fertilization treatment from those which had successful rhizome
establishment during initial plot development. Within each plot, we
replicated each ingrowth core treatment twice, where we installed
ingrowth cores by 2 of the plot's 25 plants (SI Fig. \textbf{3b} ). This
resulted in a total of 90 experimental ingrowth cores (3 cores x 3
fertilization treatments x 5 plots x 2 locations/plot).

\emph{Ingrowth core construction and installation}

Ingrowth core treatments included root and fungal ingrowth (root), root
exclusion and fungal ingrowth (fungal), and root and fungal exclusion
(none) (SI Fig. \textbf{2} ). Each ingrowth core was constructed with 10
cm long, 4.5 cm diameter rigid plastic 5 mm mesh tubing. The top 2.5 cm
of each core was inserted into 5cm long PVC collars and attached with
elastic sealant. Mesh bases were sewn onto each core with 12 lb. nylon
fishing line and each core was wrapped with mesh that was glued on with
100\% silicon adhesive. Root and fungal ingrowth (root) cores were
constructed with 1.5 mm polyacrylic mesh that allowed fine root
ingrowth. Root exclusion (fungal and none) cores were constructed with
50 um nylon mesh that was too fine for root ingrowth but allowed hyphal
ingrowth (Phillips et al., 2012). Root and fungal exclusion (none) cores
were constructed with the same root exclusion mesh and were also twisted
once or twice a week to break off hyphae and prevent significant fungal
ingrowth and establishment (SI Fig. \textbf{2} ).

Ingrowth cores were prepared in the lab using isotopically enriched
litter amendments and soil harvested from the corresponding plot. In
April 2021, soils from the top 10 cm were collected from each future
ingrowth core location and were brought back to the lab where they were
sieved to 2 mm and stored at 5°C when not being processed. Soils were
homogenized within each nutrient treatment (control, high N, or organic)
and were mixed with sand that had been acid washed and separated from
particles less than 53 um diameter in a 9:1 soil:sand ratio to prevent
soil compaction. 250 mg of isotopically enriched corn leaf litter,
generated as described in Ridgeway et al., 2022, was used as the
substrate in each ingrowth core. This addition rate was selected to be
lower than litter production at the site to limit experimental artefacts
from introducing a new decomposition substrate and high enough to ensure
that the \textsuperscript{13}C inputs were traceable into SOM pools.
This litter had a \%C of 41.7\% (±0.17\%), C:N of 18.8 (±0.64),
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C of 7020 (\selectlanguage{ngerman}±49), and \selectlanguage{greek}δ \selectlanguage{english}\textsuperscript{15}N of
34,800 (\selectlanguage{ngerman}±310) and was dried and coarsely ground. Each core was filled
with corresponding soil, and the labeled litter was gently mixed in to
the top 2 cm.

Within 5 days of initial soil collection, the assembled cores were
transported to the field location where they were installed into the top
10 cm of soil in each corresponding treatment plot (SI Fig.\textbf{3a}
). This occurred in April 2021 when miscanthus shoots were beginning to
emerge. Within each plot, ingrowth cores were installed 8'' north of
visibly emerged miscanthus shoots (SI Fig. \textbf{3b} ). After 20
weeks, the ingrowth cores were carefully cut from the soil in September
2021 and were brought back to the lab for processing. Although each
treatment combination began with a planned replicate of n=10, two cores
were removed from analysis due to animal interference. Additionally,
five cores intended for the root exclusion fungal ingrowth treatment
(fungal) were invaded by roots. After determining that these cores did
not significantly vary from the rest of the root ingrowth (root) cores,
these were also analyzed as root ingrowth (root) cores. Given these
adjustments, the total replication ranged from 5-15 for each treatment
(provided in SI table 1).

\emph{Soil fractionation}

Ingrowth cores were destructively harvested in September and litter C
and N inputs were traced into SOM pools (Ridgeway et al., 2022). A 5 g
subsample of dry soil from each core was separated into light POM, heavy
POM, and MAOM by density and size fractionation as described in Lavallee
et al. (2020). In brief, the light POM was separated through density
floatation in 1.85 g/mL sodium polytungstate salt solution. The
remaining soil was separated into heavy POM and MAOM fractions by size
separation where the MAOM fraction passes through a 53 um sieve.

\emph{Tracing litter C and N fate}

To trace the fate of \textsuperscript{13}C and \textsuperscript{15}N
litter amendments, the soil fractions were analyzed for \%C, \%N,
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C, and \selectlanguage{greek}δ \selectlanguage{english}\textsuperscript{15}N using a Thermo
Fisher Delta V+ isotope ratio mass spectrometer interfaced with a Carlo
Erba NC2500 Elemental Analyzer. First, the proportion of litter-derived
C or N in each soil fraction (\emph{f\textsubscript{litter}} ) was
determined with two endmember mixing models (eq. 1) (Derrien \& Amelung,
2011; Poeplau et al., 2018). Here, the C and N isotope signatures were
measured from the enriched litter substrate and each of the 3 SOM
fractions from control, high N, and organic soils.

eq. 1: \emph{f\textsubscript{litter} =}\(\ \frac{sample\ isotope\ signature-SOM\ isotope\ signature}{litter\ isotope\ signature-SOM\ isotope\ signature}\)

Next, the litter C and N recovered in each SOM pool (shown in
Fig.\textbf{2} , Fig. \textbf{5} ) was determined for each ingrowth core
(eq. 2). Here, the mass proportion of each SOM fraction was determined
from lab fractionation and the \%C, \%N, \selectlanguage{greek}δ \selectlanguage{english}\textsuperscript{13}C, and
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{15}N were measured on an elemental analyzer as
described above. The distribution of litter C between the SOM fractions
(shown in Fig. \textbf{3} ) was calculated as the litter mass in each
SOM fraction out of the total litter mass remaining in the ingrowth core
soil.

eq. 2: litter mass = dry soil mass in each core \(\times\) SOM
fraction mass proportion \(\times\) SOM fraction \%C/100 or
\%N/100 \(\times\)\emph{f\textsubscript{litter}}

\emph{Root biomass, root colonization, and microbial biomass}

All roots that were inside of the cores were separated from soils and
washed in the lab. Dry root biomass was measured, and microbial biomass
C was measured from a subsample of soil from each core using chloroform
slurry fumigations (Witt et al., 2000) followed by persulfate digestion
to CO\textsubscript{2} (Doyle et al., 2004; Kane et al., 2022). In
brief, soils were extracted in potassium sulfate with and without
chloroform for 4 hrs. Filtered supernatant was digested in persulfate
solution where dissolved C was oxidized to CO\textsubscript{2}. Total
CO\textsubscript{2} and \selectlanguage{greek}δ \selectlanguage{english}\textsuperscript{13}CO\textsubscript{2} was
measured on a Picarro G2201 (Picarro Inc). Microbial biomass C was
calculated as the difference between chloroform-fumigated and
non-fumigated samples scaled by 2.64 (Vance et al., 1987) and litter
C-derived microbial biomass was determined using two endmember isotope
mixing models.

A sample of roots were separated for root arbuscular mycorrhizal (AM)
colonization measurements. To remove pigment, root samples were cleared
in 10\% potassium hydroxide followed with 85\% ethanol to leach excess
pigmentation. Roots were acidified in 5\% hydrochloric acid and then
stained for 5 minutes in 0.05\% trypan blue (Comas et al., 2014). AM
colonization was determined by suspending root samples in water on a 1x1
cm gridded petri dish and measuring how often arbuscules or hyphae were
present at each root-gridline intersect (Giovannetti \& Mosse, 1980).

\emph{Net mineralization and nitrification}

Net N mineralization and net nitrification were measured immediately
after ingrowth core harvest. These were expressed as the difference in
pools of ammonium (NH\textsuperscript{4+}) and nitrate
(NO\textsuperscript{3-}) between an initial sample that was extracted
within 24 hours of collection and a sample that was incubated for 2
weeks at room temperature. Inorganic N was extracted from 5 g of soil
from each core in 10 mL of 1M KCl solution, and dissolved inorganic N
was determined through phenol-hypochlorite and azo-dye colorimetric
assays for NH\textsuperscript{4+} and NO\textsuperscript{3-},
respectively (Finzi et al., 1998).

\emph{Statistical analysis}

To determine the extent to which ingrowth core treatments and
fertilization treatments altered the fate of litter C and N amendments,
we performed two-way analyses of variance in R version 3.5.1 (R Core
Team 2021). Model factors were ingrowth core treatment, fertilization
treatment, and their interaction. Post-hoc comparisons between groups
were made using the Tukey's HSD test. Differences were considered
statistically significant at an alpha level of 0.05 (p\textless{}0.05)
and marginally significant at an alpha level of 0.10 (p\textless{}0.10).
Linear regression was used to investigate the effect of living roots or
microbial decomposers on litter C incorporation into MAOM. Data was
checked for normality and heteroscedasticity. Outliers, defined as
samples where decomposer biomass was greater than 2 standard deviations
from the mean, were omitted from linear regression.

\textbf{Results:}

\emph{Root impacts on litter C and N transformations did not depend on
fertilization}

Root ingrowth core treatments and fertilization treatments both altered
the fate of litter C and N in SOM, but the root effect did not depend on
fertilization. All p-values for ingrowth core treatment x fertilization
treatment interactions are above 0.05 (SI Table 2) and root biomass did
not vary across fertilization treatment (SI Fig. 4a). As such,
subsequent data shown for each factor are aggregated over the other
factor.

\emph{Root ingrowth reduces litter N remaining in SOM}

Root ingrowth did not significantly alter litter C in total SOM
(Fig.\textbf{2a} , p\textgreater{}0.10) but reduced the litter N in
total SOM by 20\% relative to both root exclusion treatments (Fig.
\textbf{2b} , p\textless{}0.001). Within the SOM fractions, root
ingrowth reduced both litter C (Fig. \textbf{2a} , light green, p=0.001)
and litter N (Fig.\textbf{2b} , light green, p\textless{}0.001)
remaining in the unprotected light POM fraction.

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/601987/articles/653304-roots-selectively-decompose-litter-to-acquire-nitrogen-and-build-new-soil-carbon}

\emph{}

\emph{Root ingrowth alters the balance of C in SOM pools}

Of the litter C that remained in SOM, root ingrowth altered the balance
of C between SOM pools. Root ingrowth decreased the proportion of litter
C remaining in light POM by 32\% (Fig. \textbf{3a} , p\textless{}0.001)
and increased the proportion of litter C incorporation into protected
heavy POM by 30\% (Fig. \textbf{3b} , p=0.001) relative to both root
exclusion treatments. Roots did not significantly alter the
incorporation of litter C into MAOM (Fig. \textbf{3c} ). There were no
significant differences between fungal only and fungal exclusion
treatments.

\textbf{Hosted file}

\verb`image12.emf`  available at \url{https://authorea.com/users/601987/articles/653304-roots-selectively-decompose-litter-to-acquire-nitrogen-and-build-new-soil-carbon}

\emph{}

\emph{Roots mine light and heavy POM for litter N}

Root ingrowth selectively mined N from organic matter in both POM pools.
Root ingrowth preferentially reduced the litter N remaining in light and
heavy POM fractions (Fig. \textbf{2b}, green light POM N is 55 \% lower
with root ingrowth, p\textless{}0.001; blue heavy POM N is 26\% lower
with root ingrowth, p\textless{}0.01). In turn, root ingrowth increased
the C:N ratio of litter-derived SOM in light POM (Fig. \textbf{4a},
p\textless{}0.001) and heavy POM (Fig. \textbf{4b}, p\textless{}0.001).

\emph{Organic fertilization reduces litter retention in SOM}

Organic fertilization reduced litter C and N remaining in the soil
relative to control treatments, but there were no significant
differences between control and high N fertilization treatments.

Net litter C remaining in SOM was reduced by 14\% under the organic
fertilization treatment (Fig. \textbf{5a} , p\textless{}0.01) relative
to the unfertilized control treatment soils. Within the SOM fractions,
the loss of litter C was driven by an 18\% reduction in litter C
incorporation into MAOM (Fig. \textbf{5a} , brown, p=0.018). Organic
fertilization reduced litter N remaining in total SOM by 12\%
(Fig.\textbf{5b} , p=0.020) relative to unfertilized control treatments.
Within the SOM fractions, the loss of litter N was primarily driven by a
16\% reduction in litter N incorporation into MAOM (Fig. \textbf{5b} ,
brown, p\textless{}0.001).

Organic fertilization treatments had 25\% greater microbial biomass (SI
Fig. \textbf{4b} , p=0.09) relative to unfertilized treatments.
Microbial decomposition in organic fertilization treatments was more
effective with less litter C remaining in each SOM pool per gram
microbial biomass compared to control fertilization (SI Fig. \textbf{5,
a-c} ). However, this decomposition was less effective for litter N than
litter C, with no significant difference in litter N in POM pools per
gram of microbial biomass across nutrient treatments (SI Fig. \textbf{5,
d-e} ). Litter C and N incorporation into MAOM was lower per gram of
microbial biomass with organic fertilization compared to control
fertilization (SI Fig.\textbf{5c, 5f} ).

\textbf{Hosted file}

\verb`image36.emf`  available at \url{https://authorea.com/users/601987/articles/653304-roots-selectively-decompose-litter-to-acquire-nitrogen-and-build-new-soil-carbon}

\emph{}

\textbf{Discussion:}

Collectively, this work identifies how roots and soil microbes drive SOM
loss and formation in miscanthus systems that can promote soil C
sequestration and support plant productivity. Root ingrowth did not
promote a net litter C loss from soil (Fig. \textbf{2} ) despite
increased light POM decomposition due to the enhanced transfer of C into
heavy POM (Fig. \textbf{3} ). Notably, we document the potential for
roots to mobilize litter-derived N from POM without priming litter C
loss (Fig. \textbf{2} , Fig. \textbf{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 formation from litter in organically fertilized soils
(Fig.\textbf{5} ).

It appears that miscanthus roots can mine N from litter without
stimulating corresponding litter C losses (Fig. \textbf{2} ) and can
increase the C:N of litter-derived light and heavy POM (Fig.\textbf{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. Overall, our results
suggest that miscanthus may also meet its N nutrition by effectively
mining N from litter and SOM.

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 litter C
incorporation in heavy POM (Fig. \textbf{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; K\selectlanguage{ngerman}ögel-Knabner et al., 2008). As such,
there is an opportunity to build soil 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, persistent,
and high C:N SOM could help realize the potential of soil C
sequestration to combat climate change.

We found that the organic fertilizer treatments had the greatest
microbial biomass and litter-derived light POM decomposition, in support
of our second fertilization hypothesis, but less litter C and N were
incorporated into MAOM (Fig. \textbf{5} , SI Figs. \textbf{4, 5} ). 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,\emph{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.
\textbf{6} ) and other research found that organic fertilization
increases plot-scale microbial respiration (Kane et al., 2023, \emph{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).

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. \textbf{7} ). 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). As such, our
experiment may not have isolated fungal effects on litter decomposition
and SOM formation. Future efforts should quantify mycorrhizal fungal
ingrowth to better investigate the contribution of symbiotic fungi to
root-driven SOM transformations. In addition, our observations that
fertilization did not impact root biomass (SI Fig \textbf{4a} ) and that
there was no significant interaction between fertilization and ingrowth
treatments (SI Table \textbf{2} ) do not support our first fertilization
hypothesis that roots would have the greatest effect in unfertilized
soils. While miscanthus root systems do not always respond to
fertilization treatments(Amougou et al., 2011), this pattern may have
been driven by the stand age of miscanthus in our experiment. 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 formation \emph{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 transfer C into a more persistent form of
SOM has important implications for the sustainability of bioenergy
production as well as the viability of restorative agricultural to
offset carbon emissions. Overall, our work suggests that living roots
can selectively mine N while sequestering soil C. This knowledge can
help improve the predictive understanding of SOM cycling that is
critical to meeting the goals of restorative agriculture.



\textbf{Acknowledgements:}

We would like to acknowledge the West Virginia University Animal
Sciences Farm for providing access to the field site. We thank Matthew
Craig, Mark Burnham, William Peterjohn, Chris Walter, and Stephanie
Juice for helpful conversations about experimental design, measurements,
and data analysis. We also thank Emel Kangi, Bashar Sadat, and Robin
Paulman for help with lab work, field measurements, and data collection.
This material is based upon work supported by the National Science
Foundation Graduate Research Fellowship for Ridgeway under Grant No.
DGE-1102689. Support for Brzostek and Starcher was provided by the DOE
Center for Advanced Bioenergy and Bioproducts Innovation (U.S.
Department of Energy, Office of Science, Office of Biological and
Environmental Research under Award Number DE-SC0018420). Support for
Kane was provided by the US Department of Agriculture (USDA-NIFA
2019-67019-29307). Support for Morrissey was provided by the US
Department of Agriculture (USDA-AFRI awards 2019-67019-29307,
2022-67019-36499). Additional support for Morrissey was provided by the
USDA NIFA Hatch Program, in association with project number WVA00755.
Any opinions, findings, and conclusions, or recommendations expressed in
this publication are those of the author(s) and do not necessarily
reflect the views of the U.S. Department of Energy.

\textbf{Competing Interests:}

The authors declare that they have no competing interests.



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\textbf{Supplemental Information:}

SI Figure 1: Hypotheses for how fertilization alters root-stimulated
microbial decomposition

SI Figure 2: Ingrowth core experimental design

SI Figure 3: Site map, fertilization treatments, ingrowth core placement
example

SI Table 1: replication for each treatment

SI Table 2: 2-way ANOVA p-values

SI Figure 4: Root and Microbial Biomass by nutrient treatment

SI Figure 5: litter C and N in light POM, Heavy POM, and MAOM per
microbial biomass

SI Figure 6: Net nitrification between fertilization treatments

SI Figure 7: Root mycorrhizal colonization example

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