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\begin{document}

\title{Long-term N addition reduced the diversity of Arbuscular mycorrhizal
fungi and understory herb of the Korean pine plantation in northern
China}



\author[1]{Wei Wang}%
\author[1]{Yuhan Feng}%
\author[1]{Ruotong Wu}%
\author[1]{Xiaohui Wang}%
\author[1]{Xue He}%
\author[2]{Mengmeng Zhang}%
\author[2]{Kun Li}%
\author[3]{Guangze Jin}%
\author[1]{Fuqiang Song}%
\affil[1]{Heilongjiang University}%
\affil[2]{heilongjiang daxue}%
\affil[3]{Center for Ecological Research}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
With the development of agriculture and industry, the increase in
nitrogen (N) deposition has caused widespread concern among scientists.
Although emission reduction policies have slowed N releases in Europe
and North America, the threat to biodiversity cannot be ignored.
Arbuscular mycorrhizal (AM) fungi play an important role in the
establishment and maintenance of plant communities in forest ecosystems,
both their distribution and diversity have vital ecological functions.
Therefore, we analyzed the effects of long-term N addition on AM fungi
and understory herbaceous plants in Korean pine plantation in northern
China, measured the soil properties, AM fungal and herbaceous community
structure and diversity with different concentrations of NH4NO3
(0,20,40,80 kg N ha-1 year-1) applied for 7 consecutive years. The
results showed that long-term nitrogen fertilization affected soil
properties, decreased soil pH, increased soil ammonium content, and
caused significant fluctuations in P elements. Nitrogen application
improved the stability of soil aggregates by increasing the content of
GRSP (glomus-related soil protein); N addition changed the AM fungal
community composition, and Glomus genus was more adaptable to the acidic
environment treated with the highest nitrogen concentration; with the
increase of N treatment, The species of AM fungi and herbaceous plants
and the biomass of fine roots decreased. In summary, we concluded that
long-term understory application of different concentrations of N
altered soil pH, changed the distribution of N, P elements, and the soil
aggregate fractions, reduced AM fungal and herb diversity. The
importance of AM fungi in maintaining forest ecosystem diversity under
the pressure of global change was verified.%
\end{abstract}%



\sloppy


\textbf{1. Introduction}

The dramatic increase in atmospheric emissions of reactive nitrogen (N)
from human activities (industrial and agricultural activities and fossil
fuel combustion) since the Industrial Revolution has also led to the
rapid increase in N deposition to terrestrial and aquatic ecosystems
((Ackerman, Millet , \& Chen, 2019). N deposition provides a new source
of fertilizer for plants, but excessive N input can also affect
biogeochemical cycles and alter ecosystem structure and function (Yu et
al., 2019). Forest ecosystem species diversity is an important indicator
reflecting the relationship between plants and the environment,
determining the structure and ecological functions of forest
communities, which are mutually constrained and synergistic with
environmental factors. Moderate N input will alleviate forest N
limitation to a certain extent and promote vegetation growth. On the
other hand, excessive N deposition induces ecological problems such as
soil acidification, reduction of biodiversity, and degradation of forest
functions(Gao et al., 2019; Hong, Gan , \& Chen, 2019; Vuorenmaa et al.,
2018). The results of a worldwide network of long-term monitoring of N
deposition since the twentieth century show that China faces more severe
N deposition than the United States, European countries, and other
countries in East Asia(Zhang et al., 2021). Nitrogen deposition remains
a threat to the biodiversity and stability of forest ecosystems (Weldon,
Merder, Ferretti , \& Grandin, 2022).

Herbaceous plants have the highest species diversity in forest
ecosystems and contribute significantly to forest ecosystem structure
and function, but are more sensitive to atmospheric changes and N
deposition(GILLIAM, 2007; McDonnell, Clark, Reinds, Sullivan , \& Knees,
2022; Thrippleton, Bugmann, Kramer-Priewasser , \& Snell, 2016). It has
been demonstrated that excessive N input can have complex effects on
herbaceous community structure and plant biological characteristics,
such as reducing herbaceous diversity and altering herbaceous root
chemistry and biomass(Li et al., 2015). However, most studies have
focused on tropical forests and temperate grasslands, and not enough
studies have been conducted on herbaceous plants in temperate forests.

The functions of soil microorganisms in forest ecosystems should not be
underestimated, among which AM fungi can establish symbiotic
relationships with most herbaceous plants and play an important role in
the establishment and maintenance of plant communities((Mariotte et al.,
2013; Smith \& Read, 2008; Wang et al., 2019). Arbuscular mycorrhizal
(AM) fungi can promote plant growth((Hoeksema et al., 2010; Xie et al.,
2022)and improve plant resilience(Chen, Arato, Borghi, Nouri , \&
Reinhardt, 2018; Ruiz-Lozano et al., 2016), and mycelium secretion-GRSP
can also promote soil aggregation(Gao, Wang , \& Wu, 2019), affecting
host plants in a range of direct and indirect ways. Most mycorrhizal
fungi depend on the host plant for existence and reproduction and AM
fungi are thought to be susceptible to environmental conditions, such as
climate((Compant, van der Heijden , \& Sessitsch, 2010), plant
species((Kivlin, Hawkes , \& Treseder, 2011), and soil properties, etc.
Excessive N input indirectly affects the structure and diversity of AM
fungi through changes by soil factors (Boeraeve et al., 2022; Ma et al.,
2021), and thus affects the ecological function of AM fungi. The results
of the studies are not the same around the world, which is due to the
influence of the experimental conditions. A global meta-analysis showed
that the negative effect of N addition on AM fungi was mainly a
reduction in total AM fungal abundance and did not significantly affect
the diversity and structure of AM fungi(Han, Feng, Han , \& Zhu, 2020).
However, in a tropical simulated N deposition experiment, it was found
that N addition mainly reduced the diversity and abundance of AM
fungi(Camenzind et al., 2014). AM fungi have been shown to contribute to
the maintenance of forest herbaceous diversity under N application
conditions(Smith \& Stephan, 2021), But studies in northern forest lands
are scarce.

In this experiment, AM fungi, herbaceous plants, and soil properties
were investigated in a northeastern temperate Korean pine plantation
ecosystem that was subjected to a 7-year N addition experiment. We
hypothesized that (1) Long-term nitrogen fertilization will change the
soil pH value, increase soil GRSP content and thus improve the stability
of soil aggregates. (2) Long-term N addition will change the structure
and diversity of AM fungal communities, thus affecting the ecological
functions of AM fungi. (3) Long-term N addition would directly change
AMF diversity by reduce pH, and indirectly by change herbaceous plant
composition.

\textbf{2. Materials and methods}

\textbf{2.1 Study site}

The experiment sample site is located in the Korean pine plantation in
Liangshui National Nature Reserve, Heilongjiang Province, China
(128\selectlanguage{ngerman}°53\selectlanguage{english}'20\selectlanguage{english}"E, 47deg10\selectlanguage{english}'50\selectlanguage{english}"N), Natural N deposition is 12.93 kg N
ha\textsuperscript{-1} year\textsuperscript{-1}. The area has a distinct
temperate continental monsoon climate with an average annual temperature
of -0.3degC, of which the average annual precipitation of 676 mm, a
frost-free period of 100-120 d, and an annual snowpack period of 130-150
d. The Korean pine plantation in this test site was established in 1954
after a clear-cutting of the mixed broadleaved-Korean pine forest. The
soil is dark-brown forest soil (using the Chinese classification), The
dominated species is Korean pine, and undergrowth associated herbs
were\emph{Oxalis corniculate} , \emph{Mitella nuda} , etc.

\textbf{2.2 Experimental design}

The simulated N deposition experiment commenced in 2014, the sample
plots were treated with N application from June to mid-September. The
required NH\textsubscript{4}NO\textsubscript{3} for each treatment was
dissolved in 20 L of water, and CK only adds 20 L of water. Each sample
square 5 m x 30 m, bounded by a 10 m wide buffer strip, with a total of
four gradients of CK (0 kg N
ha\selectlanguage{english}\textsuperscript{-1}year\selectlanguage{english}\textsuperscript{-1}), LN (20 kg N
ha\selectlanguage{english}\textsuperscript{-1} year\selectlanguage{english}\textsuperscript{-1}), MN (40 kg N
ha\selectlanguage{english}\textsuperscript{-1} year\selectlanguage{english}\textsuperscript{-1}) and HN (80 kg N
ha\selectlanguage{english}\textsuperscript{-1} year\selectlanguage{english}\textsuperscript{-1}) ((Song, Zhang, Muller ,
\& Jin, 2019). There were three replicates in each treatment with
completely randomized design.

\textbf{2.3 Soil sample collection and pre-treatment}

Soil samples were collected in mid-July 2020. Soil samples were
collected using soil samplers (0-15 cm deep, 5 cm inside diameter), and
five random samples were collected from each plot and mixed to a
composite sample. Soil samples were sieved and divided into three parts:
One part was stored at -80degC for soil microbial DNA extraction, another
part was air-dried and used for soil chemical property determination,
the rest part was used to determine the composition of soil aggregates
and measures the content of GRSP.

\textbf{2.4 Soil physicochemical properties}

The air-dried soil samples were sieved through soil sieves with 2 mm, 1
mm and 0.25 mm aperture, and four groups of soil samples were obtained
(\textgreater{}2 mm, 2-1 mm, 1-0.25 mm, \textless{}0.25 mm)
(Muruganandam, Israel , \& Robarge, 2009), use an electronic balance to
weigh the weight of each particle size. The soil structural stability
was evaluated by the mean weight diameter (MWD) of water-stable
aggregate (Zhao, Chen, Hu , \& Li, 2017). The content of easily
extractable glomalin-related soil protein (EE-GRSP) and total
glomalin-related soil protein (T-GRSP) was determined by improving the
assay method according to Janos et al. (2008). The EE-GRSP was extracted
from 0.5 g soil with 4 ml of 20 mmol L\textsuperscript{-1} of sodium
citrate (pH=7.0) and autoclaved at 121\selectlanguage{english} for 30 min. The T-GRSP was
extracted from 0.5 g soil with 4 ml of 50 mmol L\textsuperscript{-1} of
sodium citrate (pH=8.0) and autoclaved at 121\selectlanguage{english} for 60 min.

The supernatant was collected by centrifugation at 10000 rpm for 10 min
at 4\selectlanguage{english}. The T-GRSP extraction was performed 6 times until the solution
was straw-colored ((Gao, Zhou, Ling, Hu , \& Chen, 2017). An
spectrophotometer (595 nm) was used to determine the GRSP content using
bovine serum protein as the standard product.

The rest air-dried soil samples were used to determine soil pH by pH
meter (FiveEasy FE20) on a mixture of water to soil ratio of 2.5:1. TC
(total carbon), TOC (total organic carbon), and total N (TN) were
determined by the analyzer (multi N/C 2100 TOC/TN Analyze, Germany,
Analytikjene). Soil ammonium N
(NH\textsubscript{4}\textsuperscript{+}-N) and nitrate N
(NO\textsubscript{3}\textsuperscript{-}-N) were determined by continuous
flow analyzer (BRAN+LUEBBEAA3, Germany), and the extract solution was 2
mol*L\textsuperscript{-1} KCl, and soil total phosphorus (TP) and
available phosphorus (AP) were determined by the molybdenum antimony
anti-colorimetric method.

\textbf{2.5 Herbaceous plant diversity, fine root biomass, and AM fungal
colonization rate survey}

We explored herbaceous plant diversity in mid-July 2020. Three small
randomly sample squares (1 mx1 m) were set in each sample square, the
species of herbaceous plants, the number of individuals of the species
(counted as a single plant in the above-ground part), height, and cover
were investigated. The soil was collected at a depth of 0-15 cm using a
soil auger, and the fine roots (\textless{}2 mm in diameter) of
herbaceous plants were selected and their fine root biomass was
measured(Li et al., 2021). We determined the AM fungal colonization rate
of fine roots from herbs by a modified aniline blue staining method
((Zubek, B\selectlanguage{polish}ł\selectlanguage{english}aszkowski , \& Mleczko, 2011).

\textbf{2.6 AM fungal community structure and diversity}

\textbf{2.6.1 Isolation and identification of AM fungal spores}

The wet sieving method was used to obtain spores. The spores were placed
under a stereomicroscope for count and observation. Then transferred
spores to polyvinyl alcohol-lactic acid-glycerol (PVLG) with a pipette,
and morphological characteristics were examined microscopically. It
included spore color, shape, size, sporiferous saccule, spore wall
thickness and type, subtending hyphae width and shape. The spores were
identified according to the photos and feature and feature descriptions
from \emph{VA mycorrhizal fungi identification manual} (Schenck \&
Perez, 1990), INVAM (https://invam.ku.edu/), International AM fungal
classification system (http://www.amf-phylogeny.com/).

AM fungal spore density, species richness, relative abundance (RA),
frequency, importance value (I), and species alpha diversity index were
calculated per 10 g of air-dried soil sample. The degree of AM fungal
dominance was assigned into four categories according to their important
values (I), I \textgreater{} 50\% means dominant species, 30\%
\textless{} I [?] 50\% means subdominant species, 10\% \textless{} I [?]
30\% means accompanying species and I [?] 10\% means rare species.

\textbf{2.6.2 High throughput analysis of AM fungi}

Total DNA was extracted from mixed soil (0.5 g) using Fast DNA\selectlanguage{ngerman}® Spin Kit
for Soil. And then AM fungus-specific primers AML1 and AML2 were
selected as the first round of primers ((Lee, Lee , \& Young, 2008),
AMV4.5NF (AAGCTCGTAGTTGAATTTCG) and AMDGR (CCCAACTATCCCTAT-TAATCAT) were
selected as the second round of primers for amplification with the
nested PCR amplification method ((Sato, Suyama, Saito , \& Sugawara,
2005). The PCR amplification products were detected and recovered by 2\%
agarose gel electrophoresis, and Illumina's MiseqPE300 platform was used
for sequencing. Raw sequences were uploaded to the NCBI Sequence Read
Archive (SRP426700).

\textbf{2.7 Statistical analysis}

One-way ANOVA and Duncan's multiple tests (\emph{P} \textless{} 0.05)
were performed using IBM SPSS Statistics 26 (IBM Corporation, New York)
software to compare the means of plant, AM fungi, and soil
physicochemical property data. \textbackslash{}soutTwo-way ANOVA was
used to analysis the interaction effect between N addition and soil
agglomerate particle size on GRSP. The correlation between soil
physicochemical properties and herbaceous plants diversity was
calculated by Pearson's test (two-tailed) at two significant levels,
\emph{P} \textless{} 0.05 and\emph{P} \textless{} 0.01. RDA analysis was
used to reveal the effect of soil properties on the community
composition of AM fungi. Structural equation modeling (SEM) was used to
predict causal pathways of N addition, key soil physicochemical
indicators, and AM fungi on herbaceous plant diversity. Build prior
models and evaluate model fit using chi-square, RMSEA, and GFI.

\textbf{3. Result}

\textbf{3.1 Soil physical and chemical properties}

\textbf{3.1.1 Soil chemical properties}

The soil pH gradually decreased with the increase of N addition
concentration, and the pH value in HN treatment significantly decreased
to 5.24 (\emph{P}\textless{}0.05) (Table 1). TP content was
significantly increased by 16.7\% in the MN treatment. The AP content
was significantly reduced after N addition. There were significant
changes in NH\textsubscript{4}\textsuperscript{+}-N and
NO\textsubscript{3}\textsuperscript{-}-N content. The
NH\textsubscript{4}\textsuperscript{+}-N content increased with the
concentration of N addition increasingly, and there was a significant
increase in MN and HN treatments. The
NO\textsubscript{3}\textsuperscript{-}-N content in the LN treatment was
significantly increased and was significantly decreased in the HN
treatment.

\textbf{3.1.2 Soil aggregate and glomalin-related soil protein}

The weight percentage of soil aggregate varied with its size fractions
(Fig.1). In all N addition treatments, MWD values ranged from 1.48-1.74
mm. The content of large aggregates (2-5 mm) and MWD value was
significantly higher in the HN treatment compared to the CK.

N addition exerted significant effect on the EE-GRSP and T-GRSP content
of soil aggregate. The contents of EE-GRSP ranged from 1.36-4.29, and
T-GRSP ranged from 3.73-13.50 mg·g\textsuperscript{-1} (Fig.2).
\textbackslash{}soutThough two-way ANOVA analyze, there were interaction
effect between N addition and aggregate fractions on GRSP
(\emph{P}\textless{}0.01). Among all treatments, the GRSP content of
soil aggregate \textless{}0.25 mm was the highest; GRSP content varied
according to the amount of N addition. N addition increased the content
of EE-GRSP and T-GRSP in soil aggregates, and reached the maximum in the
MN treatment. N addition increased the EE-GRSP content of soil
aggregates by 60.6\% to 118.7\%, 12.0\% to 77.2\%, 5.0\% to 60.5\%, and
16.3\% to 56.2\% for each particle size (\textless{}0.25 mm, 0.25-1 mm,
1-2 mm, and 2-5 mm), respectively. N addition increased the T-GRSP
content of soil aggregates by 45.5\% to 87.6\%, 25.5\% to 88.4\%, 44.5\%
to 105.2\% and 51.1\% to 86.4\% for each particle size, respectively.

\textbf{3.1.3 Relationship between the stability of soil aggregates and
the content of GRSP content under N addition conditions}

In the present study, Korean pine plantation soils were dominated by
medium aggregates (1-2 mm) with MWD values ranging from 1.48 to 1.74 mm,
which have been identified as an indicator of soil stability, and
long-term (7 years) HN treatment (80 kg N
ha\textsuperscript{-1}year\textsuperscript{-1}) increased the soil
macro-aggregates content and thus improved soil stability. Pearson
correlation analysis of undisturbed soil GRSP content and MWD showed
that both EE-GRSP and T-GRSP were significantly positively correlated
with MWD values (\emph{P} \textless{}0.01) (Fig. 3).

\textbf{3.2 Understory herbs}

\textbf{3.2.1 Herb plants communities}

There were 33 species of herb plants were investigated in this
experiment (Table 2). N addition significantly altered the dominance of
the first five plants in Table 2 (\emph{P} \textless{}0.05), which were
\emph{Chrysosplenium sinicum, Aegopodium alpestre, Dryopteris
crassirhizoma, Eranthis stellata} and\emph{Phryma leptostachya} .
Compared with CK, 9.1\% of the species were not detected in the LN
treatment, 27.3\% in the MN treatment, and 33.3\% in the HN treatment.

\textbf{3.2.2 Herb diversity, fine root biomass, and AM fungal
colonization rate}

The Shannon index and Simpson index of herb plants decreased gradually
with increasing gradient of N addition concentration, and the MN and HN
treatments were significantly lower than CK (Table 3); The fine root
biomass and AM fungal colonization rate decreased significantly with the
increase of N addition concentration gradient, and the biomass decreased
by 78.6\% in the HN treatment, the AM fungal colonization rate decreased
significantly in the MN and HN treatments, and decreased by 21.95\% in
the HN treatment. These indicated that herb diversity and AM fungal
colonization rate of fine roots were significantly reduced when the
applied N concentration exceeded 40 kg N
ha\textsuperscript{-1}year\textsuperscript{-1} in this experiment. The
fine root biomass of herb plants was significantly reduced when the
applied N concentration exceeded 20 kg N ha\textsuperscript{-1}
year\textsuperscript{-1}.

\textbf{3.3 AM fungal community structure and diversity}

\textbf{3.3.1 Morphological identification of AM fungi}

There were 4461 AM fungal spores isolated from all soil samples, and 9
genera and 20 species were identified morphologically. Among them, 7
species belonging to \emph{Acaulospora} are the most diverse, accounting
for 35\% of the total number of species, followed by the
genus\emph{Glomus} with four species, accounting for 20\% of the total
number of species.

The relative abundance (RA) and importance values (I) of AM fungal
spores under the N application treatment were analyzed (Table 4). The
soil samples from the CK had the most abundant spore species, with 17 AM
fungal species identified, while\emph{Acaulospora spinosa} and
\emph{Claroideoglomus lamellosum} only appeared in CK and not found in
other treatment. Gradual reduction of spore species in soil samples with
increasing N addition concentration, and 14 species of AM fungi were
isolated and identified in the LN treatment. There were also 14 species
in the MN treatment, in which\emph{Sclerocystis sinuosa} was unique and
never found in other treatments. It was found that HN treatment reached
the least AM fungal species of 10. In summary, the increasing of N
addition concentration gradually reduced the spore species.

\textbf{3.3.2 Spore density, species abundance and diversity index of AM
fungi}

The highest AM fungal spore density was found in the MN treatment with
40.13 spores g\textsuperscript{-1} (Table 5), followed by the HN and LN
treatments with 36.9 and 36.43 spores g\textsuperscript{-1}, and the
lowest 35.27 spores g\textsuperscript{-1} in CK. With increasing N
addition concentration, the species abundance of AM fungi gradually
decreased, with a significant decrease in the LN and MN treatment
(\emph{P}\textless{}0.05), and there were only 8 species
g\textsuperscript{-1} in the HN treatment. All AM fungal species
diversity differed significantly in the N addition treatment compared to
the CK, both Simpson's index and Shannon's index were highest in the CK,
the LN treatment was significantly lower compared to the CK and MN
treatments, while the HN treatment had the lowest diversity index.

\textbf{3.3.3 Bioinformatical analyses}

In the present study, we identified 398299 sequences from the total
dataset, optimized to 391960 sequences. The coverage of OTUs in all soil
types was more than 99\%. The CK had the largest OTUs richness of AM
fungi (Table 6). The highest AM fungal diversity was found in the CK by
Shannon index and Simpson index analysis, and gradually decreased with
the increase of N addition concentration, and the lowest AM fungal
diversity was found in the high N treatment.

The results of the community structural composition showed that the
majority of OTUs sequences belonged to \emph{Glomus} (Fig. 4). The
relative abundance of the\emph{Glomus} increased with N addition and was
significantly higher than CK in the HN treatment. In contrast, the
relative abundance of \emph{Paraglomus} reduced by 96.71\% in HN
treatment compared to CK.

The AM fungal community composition and relative abundance at the
species level was shown in Fig.4. The dominant species in the CK
was\emph{Glomus} -Glo3-VTX00074, \emph{Acaulospora} -VTX00272 in the LN
treatment, \emph{Diversispora} -unclassified in MN treatment,
and\emph{Glomus} -MO-G5-VTX00219 in HN treatment. It can be seen that
the N addition had a significant effect on AM fungal community
composition in Korean pine plantation. \emph{Glomus} -MO-G18-VTX00064 is
a special species for the CK and did not appear in other
treatments.\emph{Glomus} -group-B-\emph{Glomus} -acnaGlo7-VTX00057 was a
special and unique specie for the LN treatment. \emph{Glomus}
-MO-G22-VTX00125,\emph{Glomus} -Wirsel-OTU13-VTX00140 were special and
unique species for the MN treatment.

\textbf{3.3.4 Comparative and analysis of morphological and molecular
identification results of AM fungi}

The result of morphological identification showed that there were 9
genus and 20 species of AM fungi after wet sieving, while
high-throughput sequencing amplified and sequenced the DNA extracted
from the mixed soil from each small sample, and 4 genera and 11 virtual
species were identified. The genus \emph{Diversispora} detected by
high-throughput sequencing means was not found in the morphological
identification, while morphological identification revealed six genera
which were not identified by high-throughput. The change trend of AM
fungal diversity calculated by the two methods was the same, and the AM
fungal diversity index used in the subsequent analysis was the result of
morphological identification.

\textbf{3.4 Relationship between soil physicochemical properties and AM
fungal community composition under N addition conditions}

The RDA analysis of AM fungi and soil physicochemical properties showed
that six soil physicochemical factors could explain 82.15\% of the
variation in AM fungal communities as shown in Fig. 5. Soil pH and
NO\textsubscript{3}\textsuperscript{-}-N\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}AP\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}NH\textsubscript{4}\textsuperscript{+}-N
and TN content significantly influenced the distribution of AM fungal
species.

\textbf{3.5 Relationship between changes in environmental factors and
herb diversity under N addition conditions}

Long-term N addition reduced the diversity of understory herbaceous
plants. The correlation between the \selectlanguage{greek}α \selectlanguage{english}diversity index of herbs and
biochemical factors was analyzed by Pearson correlation. The diversity
of understory herbaceous plants was significantly and positively
correlated with the diversity of AM fungi (\emph{P} \textless{}0.05;
Fig.6), and the colonization rate of AM fungi (R\textsuperscript{2}
=0.66). The diversity of herbs was significantly and positively
correlated with total nitrogen content (\emph{P} \textless{}0.05),
significantly and negatively correlated with
NH\textsubscript{4}\textsuperscript{+}-N content (\emph{P}
\textless{}0.01), and significantly and positively correlated with pH
(R\textsuperscript{2} =0.67). Additionally, herbaceous plant fine root
biomass was significantly and positively correlated with diversity
(\emph{P} \textless{}0.01).

A structural equation model (SEM) was developed to illustrate the effect
of each factor on herb diversity under N addition conditions (Fig. 7),
The P-value of \selectlanguage{greek}χ\selectlanguage{english}2 was higher than 0.05 indicated that the model
structure was reasonable. The SEM throughput analysis largely
(R\textsuperscript{2} \textgreater{} 90\%) explained the effects of
biotic as well as abiotic factors on herb diversity. N addition
indirectly affected AM fungal diversity and plant diversity though soil
pH value, ammonium N, and AP content. Among them, AM fungal diversity
was the strongest pathway affecting plant diversity.

\textbf{4. Discussion}

\textbf{4.1 Effect of N addition on soil properties}

Soil acidification is caused by nitrogen deposition, and soil pH
decreases linearly with increasing N application (Tian \& Niu, 2015). In
this study, long-term HN treatment (80 kg N
ha\textsuperscript{-1}year\textsuperscript{-1}) reduced soil pH, while
long-term low and medium N treatments had no significant effect on soil
pH. The reason may be that soils in terrestrial ecosystems are more
sensitive to N inputs and forest ecosystems are more stable than other
types of ecosystems.

MN treatment had a significant effect on Soil TP content, while AP
content decreased to varying degrees with N addition. This may be
because soil acidification activates aluminum ions and iron ions in the
soil, causing more phosphorus to be adsorbed by the soil, resulting in a
decrease in AP content. When plants and microorganisms absorb and
utilize AP, they get a corresponding supplement from the phosphorus
element of other components, which leads to the reduction of AP content
in different degrees.

Soil TN content was stable, ammonium content increased linearly with N
application, and nitrate content fluctuated with N application. The
result of Qin et al. (2022) showed long-term nitrogen application can
change the soil ammonium and nitrate content. Besides, total N increased
significantly with the increase of N addition. The stability of whole
nitrogen content in this experiment may be because the forest ecosystem
is more stable and can resist a certain concentration of N addition.

As the gradient of N addition concentration increased, there was a
tendency for the easily EE-GRSP content in the in-situ soil to increase,
and the T-GRSP content was significantly higher in the HN treatment than
CK, probably due to the significant acidification of the soil, and it
has been shown that acidic soil had high GRSP content (Agnihotri et al.,
2022). On the contrary, short-term N addition reduced the GRSP content
in the soil (Huang et al., 2022; Jia et al., 2021), the reason for this
contrasting result may be that short-term N additions cannot
significantly change soil acidity and alkalinity, but could lead to a
decrease in GRSP by changing soil dissolved organic carbon and N/P
ratio. The proportion of large soil aggregates and GRSP content was
significantly elevated in the HN treatment. Pearson correlation analysis
of in-situ soil GRSP content and MWD showed that both EE-GRSP and T-GRSP
were significantly and positively correlated with MWD values (Fig.3),
which is further evidence of the importance of AM fungi in soil
stability.

\textbf{4.2 Effect of N addition on the structure and diversity of AM
fungi community}

Different N addition content and local soil conditions affect plants and
soil microorganisms (Cao et al., 2020; Zeng et al., 2021). In this
study, both high-throughput sequencing and traditional spore
morphological identification were used to investigate the community
structure and diversity of woodland AM fungi. Morphological
identification has inconsistencies, limitations, and chances, but can be
improved by means such as increasing the sample size. High-throughput
sequencing technology has been widely used by researchers, but stills
has some limitations. Due to the variety of microorganisms, the current
database cannot cover all the microorganisms or lacks accurate species
information, resulting in the omission of some low-abundance species, so
we combine the two methods to accurately reflect the diversity and
structural characteristics of AM fungi (Chaudhary, Nolimal,
Sosa-Hernandez, Egan , \& Kastens, 2020). Both high-throughput
sequencing results and morphological identification results showed that
N addition altered AM fungi community composition and reduced AM fungal
alpha diversity.

It is clear from the RDA analysis (Fig. 5) that pH significantly
affected the community composition of AM fungi in Korean pine
plantation, and there was research reported that soil pH was one of the
most important abiotic factors affecting the ecological distribution of
AM fungi (Davison et al., 2021). The abundance of \emph{Glomus}increased
gradually (Fig. 4) with increasing N addition concentration and was
significantly higher in the HN treatment, the main reason for this
phenomenon being that \emph{Glomus} showed a better ecological advantage
in acidic soils. In the experiments of (Cao et al., 2020), short-term HN
treatment (80 kg N ha\textsuperscript{-1}year\textsuperscript{-1}) also
reduced fungal diversity and changed community composition. It is
believed that this phenomenon is caused by the change of P elements in
the soil due to N addition, which causes plants to select AM fungi with
high phosphorus absorption capacity

\textbf{4.3 Effect of N addition on understory herb plants}

Cases of nitrogen deposition-induced soil acidification leading to
reduced plant diversity have been reported (Kimmel et al., 2020).In this
study, we found that the diversity of understory herb plants in the
forest also decreased with decreasing soil pH value. In addition, excess
N can also cause nutrient imbalance. Large-scale input of exogenous N
can break the original stable stoichiometry, thus causing a nutrient
imbalance in the soil and plants, which can also lead to the loss of
some species of plants. N deposition indirectly affected plant species
diversity by altering the composition of soil microbial communities
(Bobbink et al., 2010). In the present study, it was also found that the
varies of understory plant diversity was linearly and positively
correlated with AM fungal diversity and AM fungal colonization rate
(Fig.6), this also verified that the function of AM fungi in maintaining
the stability of ecosystems and preserving biodiversity should not be
underestimated. In addition, plant fine root biomass was also
significantly reduced by long-term N addition, and a small amount of N
input increased plant root biomass (Frew, 2022), In contrast, excessive
N addition decreased the fine root biomass with increasing time of N
application. The SEM clearly shows that AM fungal diversity is the
strongest influence pathway affecting herb diversity under N addition
conditions (Fig. 7), which is further evidence of the ability of AM
fungi in maintaining understory herb diversity.

\textbf{5. Conclusion}

N addition in temperate Korean pine plantation ecosystems altered AM
fungal community structure, reduced AM fungal and herb diversity,
decreased soil pH, increased inorganic N concentration, and decreased
soil fast-acting phosphorus content. We suggested that the N application
first changed the soil characteristics and then changed the composition
and diversity of the soil AM fungal, and the changes in the AM fungal
community in turn affect the distribution of soil aggregates, and the
above-mentioned factors (biotic and abiotic) in the subsurface part
together affect the plant community and diversity. Considering the
importance of AM fungi in the ecosystem, future research should explore
the pathways by which AM fungi regulate the ecological functions under N
additional conditions to improve our understanding of the ecological
functions of AM communities under global environmental change
conditions.

\textbf{Data availability statement}

The original contributions presented in the study are included in the
article/supplementary material, further inquiries can be directed to the
corresponding author/s.

\textbf{Author contributions}

All authors made significant contributions to this study. FS and GJ
provided the conceptualization. FS and WW framed the methodology. WW and
YF wrote and prepared the original draft. XW, RW and XH did the review
and editing. MZ and KL oversaw the project administration. FS was
responsible for funding acquisition. All authors contributed to the
article and approved the submitted version

\textbf{Funding}

This work was financially supported by the National Natural Science
Foundation of China (31971527), the team project of the Heilongjiang
Natural Science Foundation (TD2019C002), the Key R\&D plan guidance
projects in Heilongjiang Province (GZ20210009).

\textbf{Acknowledgments}

We acknowledge the editor and reviewers for their comments and
suggestions to improve our manuscript.

\textbf{Declaration of Competing Interest}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

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