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\begin{document}

\title{Changes in nitrogen and phosphorus availability driven by secondary
succession in temperate forests shape soil fungal communities and
function}



\author[1]{xinze Geng}%
\author[1]{Jincheng Zuo}%
\author[1]{Yunhao Meng}%
\author[1]{Ping Zhu}%
\author[1]{Yanhui Zhuge}%
\author[1]{Nan Wu}%
\author[1]{Xinfu Bai}%
\author[2]{Guangyan Ni}%
\author[1]{Yuping Hou}%
\affil[1]{Ludong University}%
\affil[2]{South China Botanical Garden}%


\vspace{-1em}



  
  \date{June 18, 2024}


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\selectlanguage{english}
\begin{abstract}
Soil fungal community plays an important role in forest ecosystems, and
forest secondary succession is a crucial driver of soil fungal
community. However, the driving factors of fungal community and function
during temperate forest succession and their potential impact on
succession processes are poorly understood. In this study, we
investigated the dynamics of the soil fungal community in three
temperate forest secondary successional stages (shrublands, coniferous
forests, and deciduous broadleaf forests) using high-throughput DNA
sequencing coupled with functional prediction via the FUNGuild database.
We found that fungal community richness, \selectlanguage{greek}α-\selectlanguage{english}diversity, and evenness
decreased significantly during the succession process. Soil available
phosphorus and nitrate nitrogen decreased significantly after initial
succession occurred, and redundancy analysis showed that both were
significant predictors of soil fungal community structure. Among
functional groups, fungal saprotrophs as well as pathotrophs represented
by plant pathogens were significantly enriched in the early-successional
stage, while fungal symbiotrophs represented by ectomycorrhiza were
significantly increased in the late-successional stage. The abundance of
both saprotroph and pathotroph fungal guilds was positively correlated
with soil nitrate nitrogen and available phosphorus content.
Ectomycorrhizal fungi were negatively correlated with nitrate nitrogen
and available phosphorus content and positively correlated with ammonium
nitrogen content. These results indicated that the dynamics of fungal
community and function reflected the changes in nitrogen and phosphorus
availability caused by the secondary succession of temperate forests.
The fungal plant pathogen accumulated in the early-successional stage
and ectomycorrhizal fungi accumulated in the late-successional stage may
have a potential role in promoting forest succession. These findings
contribute to a better understanding of the response of soil fungal
communities to the secondary forest succession process and highlight the
importance of fungal communities during temperate forest succession.%
\end{abstract}%



\sloppy


\textbf{Changes in} \textbf{nitrogen and phosphorus availability driven
bysecondary succession in temperate forests shape soil fungal
communities and function} 11\textbf{Abbreviations:}AN, soil available
nitrogen; AK, available potassium; AP, soil available phosphorus;
NH\textsubscript{4}\textsuperscript{+}-N, ammonium nitrogen; NMDS,
non-metric multidimensional scaling;
NO\textsubscript{3}\textsuperscript{-}-N, nitrate nitrogen; OTU,
operational taxonomic unit; RDA, redundancy analysis; SOC, soil organic
carbon; TC, total carbon; TK, total potassium; TN, soil total nitrogen;
TP, total phosphorus

Xinze Geng\textsuperscript{1}, Jincheng Zuo\textsuperscript{1}, Yunhao
Meng\textsuperscript{2}, Yanhui Zhuge\textsuperscript{2}, Ping
Zhu\textsuperscript{2}, Nan Wu\textsuperscript{2}, Xinfu
Bai\textsuperscript{2}, Guangyan Ni\textsuperscript{3}, and Yuping
Hou\textsuperscript{1}

\textsuperscript{1}\emph{College of Life Sciences, Ludong University,
Yantai 264025, China} ;\textsuperscript{2} \emph{School of Resources and
Environmental Engineering, Ludong University, Yantai, China} ;
\textsuperscript{3}\emph{Key Laboratory of Vegetation Restoration and
Management of Degraded Ecosystems and Guangdong Provincial Key
Laboratory of Applied Botany,South China Botanical Garden, Chinese
Academy of Sciences, Guangzhou 510650, China}

Corresponding Author

\emph{Yuping Hou}

\emph{Tel: +86 0535-668 5003}

\emph{mail address: hou\_yuping@163.com}

\textbf{Xinze Geng and Jincheng Zuo should be considered joint first
author.}

\textbf{Abstract}

Soil fungal community plays an important role in forest ecosystems, and
forest secondary succession is a crucial driver of soil fungal
community. However, the driving factors of fungal community and function
during temperate forest succession and their potential impact on
succession processes are poorly understood. In this study, we
investigated the dynamics of the soil fungal community in three
temperate forest secondary successional stages (shrublands, coniferous
forests, and deciduous broadleaf forests) using high-throughput DNA
sequencing coupled with functional prediction via the FUNGuild database.
We found that fungal community richness, \selectlanguage{greek}α-\selectlanguage{english}diversity, and evenness
decreased significantly during the succession process. Soil available
phosphorus and nitrate nitrogen decreased significantly after initial
succession occurred, and redundancy analysis showed that both were
significant predictors of soil fungal community structure. Among
functional groups, fungal saprotrophs as well as pathotrophs represented
by plant pathogens were significantly enriched in the early-successional
stage, while fungal symbiotrophs represented by ectomycorrhiza were
significantly increased in the late-successional stage. The abundance of
both saprotroph and pathotroph fungal guilds was positively correlated
with soil nitrate nitrogen and available phosphorus content.
Ectomycorrhizal fungi were negatively correlated with nitrate nitrogen
and available phosphorus content and positively correlated with ammonium
nitrogen content. These results indicated that the dynamics of fungal
community and function reflected the changes in nitrogen and phosphorus
availability caused by the secondary succession of temperate forests.
The fungal plant pathogen accumulated in the early-successional stage
and ectomycorrhizal fungi accumulated in the late-successional stage may
have a potential role in promoting forest succession. These findings
contribute to a better understanding of the response of soil fungal
communities to the secondary forest succession process and highlight the
importance of fungal communities during temperate forest succession.

\textbf{KEYWORDS:} Community potential function, ectomycorrhizal, fungal
community structure, fungal pathogen, secondary succession, temperate
forest

\textbf{INTRODUCTION}

Forest ecosystems play an important role in global biochemical cycles,
but natural disturbances and human activities have resulted in the
expansion of secondary forest succession, which has become more common
at the global scale (Chazdon et al., 2016; Millar and Stephenson, 2015;
Pugh et al., 2019). It is therefore becoming increasingly important to
investigate secondary forest succession in temperate forest ecosystems.
Forest succession can significantly affect the composition and function
of soil fungal communities (Cline and Zak, 2015; Zhou et al., 2017). As
important participants in ecosystem soil processes, changes in the
function of fungi may have feedback on above-ground vegetation, thereby
affecting succession processes (Geisen et al., 2022; Knoblochov\selectlanguage{ngerman}á et al.,
2017; Liao et al., 2018). However, much remains unknown about the
changing patterns of fungal communities during temperate forest
succession and their potential impacts.

Forest succession can indirectly affect soil properties by changing the
input of plant litter and root exudates, and by significantly affecting
soil microbial community structure and function. This effect has been
widely demonstrated in studies on bacterial communities (Dai et al.,
2021; Zhang et al., 2022b). Soil pH, soil organic carbon (SOC), soil
total nitrogen (TN), soil available nitrogen (AN), and soil available
phosphorus (AP) have had significant influences on bacterial communities
during secondary forest succession (Dai et al., 2021; Qu et al., 2020;
Zhang et al., 2021). Qu et al. (2020) found that the increase in soil pH
during forest secondary succession changed the structure of soil
bacterial community and caused the dominant functional groups involved
in the carbon cycle to be replaced by the groups involved in the
nitrogen and sulfur cycles. The increase in soil carbon and nitrogen
contents during secondary succession resulted in a significant decrease
in the abundance of nitrification, and aerobic ammonia oxidation
bacteria adapted to the oligotrophic environment had a considerable
impact on nutrient cycling in forest soil (Zhang et al., 2023). Compared
with bacteria, soil fungi are more sensitive to forest secondary
succession, which is often accompanied by changes in the soil microbial
community from bacteria-dominated to fungi-dominated (He et al., 2022;
Li et al., 2022; Susyan et al., 2011). In forest ecosystems, the effect
of the soil fungal community on ecosystem function is stronger than that
of bacteria (Jiang et al., 2021). However, the effects of soil
properties on the structure and function of soil fungal communities
change during the succession of secondary forests. Therefore, further
research is needed to deepen our understanding of the driving factors of
the soil microbial community in secondary succession of temperate
forests.

Soil fungal pathogens and mutualists are key driving factors of plant
community succession (Nara and Hogetsu, 2004; Richard et al., 2009; Van
der Putten and Peters, 1997). The accumulation of soil fungal pathogens
during the early succession of dune vegetation significantly inhibited
the growth of conspecific seedlings, promoting the replacement of early
by late-successional succession (Van der Putten et al., 1993). Soil
fungal pathogens can significantly increase seedling mortality near
their parent trees and could be an important contributor to tropical
forest population regulation (Bagchi et al., 2010; Liang et al., 2016).
Symbiotic fungi can also influence the succession process through
positive feedback with plants (Nara, 2006; Roy-Bolduc et al., 2016). The
accumulation of ectomycorrhizal and arbuscular mycorrhizal fungi are
important mechanisms for host species to establish monodominant
communities (Bennett et al., 2017; Laliberté et al., 2015; Liu et al.,
2021). Although soil fungi play an important role in plant community
dynamics, much of the potential impact of functional changes of fungal
communities on temperate forest succession remains yet to be elucidated.

This study was conducted on Kunyu Mountain, a temperate forest nature
reserve located in northern China. We selected three typical forest
types representing the succession process, namely shrublands, coniferous
forests, and deciduous broadleaf forests. We used high-throughput
sequencing techniques combined with fungal functional taxon prediction
(FUNGuild 1.0) for the analysis. We aimed to answer the following
questions: (1) How do the soil fungal communities and function structure
respond to temperate forest succession? (2) What are the potential
impacts of changes in soil fungal community function on forest
succession processes?

\textbf{MATERIALS AND METHODS}

\textbf{Study area}

The study sites were located in Kunyu Mountain Nature Reserve, Shandong,
China (121°3'70''-121°51'0''E, 37°12'20''-37°18'50''E) in a warm
temperate continental monsoon climate. The annual average temperature
was 11.9\selectlanguage{english}@C, and annual rainfall was 650--900 mm (Wang et al., 2009). The
soil in the study area is classified as Eutric Cambisols according to
the scheme of the Food and Agriculture Organization (FAO).

The main plant communities in the study area were natural \emph{Pinus
densiflora} forests, scrub and meadows, and deciduous broad-leaved
forests dominated by oak species. In the 21\textsuperscript{st} century,
the native \emph{Pinus densiflora} forests have been destroyed by insect
pests. The selected area was closed for a long time as a former red pine
logging site, which led to randomly distributed forest patches
recovering from the soil seed bank in some unmanaged sites (Sun et al.,
2011). This process forms a typical natural forest community which can
represent the early, middle, and late-successional stages of secondary
succession, providing a ``common garden'' experiment at the landscape
scale for this study. We selected the most representative plant
communities in the three secondary successional stages of the forest on
Kunyu Mountain based on the descriptions in \emph{Vegetation of
Shandong} (Wang and Zhou, 2000). These were shrubs (\emph{Grewia biloba}
~G. Don, \emph{Rhus chinensis} Mill.) in the early-successional stage,
coniferous forest (\emph{Pinus densiflora} ~Sieb. et Zucc.) in the
middle-successional stage, and oak deciduous broadleaf forest
(\emph{Quercus acutissima} Carr., \emph{Quercus variabilis} Blume) in
the late-successional stage.

\textbf{Sample collection}

The soil samples were collected on 28 April 2021. There were three
monodominant forest stands per forest type, each with a size of 100 \selectlanguage{ngerman}×
100 m. Five individuals with similar diameter at breast high were chosen
from each plot. Three soil subsamples were collected at a depth of 0--10
cm after litter removal, observing 1 m distance from the central tree in
three directions at 120° angles. All five individual subsamples were
homogenized to a single sample (Qu et al., 2020). Care was taken during
tree selection to maintain at least a 10 m distance from non-target tree
species, and each targeted tree was located farther than 20 m from the
forest edge. A distance of at least 10 m was maintained between sampled
trees to ensure spatial independence.

All samples were put on ice and transported back to the laboratory.
After removing impurities (stones, roots), each sample was divided into
three subsamples. (1) One subsample was immediately air-dried for
determining soil pH, TN, total carbon (TC), total phosphorus (TP), total
potassium (TK), soil organic carbon (SOC), available phosphorus (AP),
and available potassium (AK). (2) A -20 \selectlanguage{english} storage subsample was used to
analyze nitrate nitrogen (NO\textsubscript{3}\selectlanguage{english}\textsuperscript{-}-N) and
ammonium nitrogen (NH\textsubscript{4}\textsuperscript{+}-N) within 2
weeks. (3) A -80 \selectlanguage{english} storage subsample was used to extract DNA.

\textbf{Soil chemical analysis}

Soil chemical analysis was performed following previously described
methods (Bao, 2000). Soil pH was measured using an electrode pH meter in
1:2.5 (w/v) soil water suspensions. Soil total carbon and nitrogen were
determined using dry combustion with an elemental analyzer (Vario EL;
Elementar Analysensysteme, Langenselbold, Germany). The SOC
concentration was measured using potassium dichromate dilution
calorimetry. The NO\textsubscript{3}\selectlanguage{english}\textsuperscript{-}-N and
NH\textsubscript{4}\textsuperscript{+}-N contents were determined after
extraction with KCl solution using a flow analyzer (A16786; Alliance
Instruments, Frepillon, France). To measure TP and TK, the samples were
first digested with sodium hydroxide in a muffle furnace (450 \selectlanguage{english}). TP and
TK were then determined using a spectrophotometer and a flame photometry
detector, respectively. AP content was determined using the sodium
bicarbonate extraction--molybdenum-antimony colorimetric method. AK
content was determined using the ammonium acetate extraction-flame
photometry method.

\textbf{DNA extraction and high-throughput DNA sequencing}

We isolated the total genomic DNA from 250 mg of each homogenized soil
sample using the EZNA(r) Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA)
and quantified concentrations in a NanoDrop2000 (Thermo Fisher
Scientific, Waltham, MA, USA). The primers ITS1/ITS4 were used to
amplify the ITS1 region (Lekberg et al., 2021; White et al., 1990). The
purified amplicons were pooled in equimolar ratios and paired-end
sequencing was performed using the Illumina MiSeq PE300/NovaSeq PE250
platform (Illumina, San Diego, CA, USA). We clustered operational
taxonomic units (OTUs) with 97\% similarity cutoff using UPARSE 7.1. The
taxonomy of each sequence was analyzed against the UNITE ITS sequence
database. We used the FUNGuild database to assign ecological guilds to
OTUs and only retained confidence rankings of ``probable'' or ``highly
probable'' guilds (Nguyen et al., 2016).

\textbf{Statistical analysis}

The Chao1 index, Shannon evenness index, and Shannon--Wiener index were
used as diversity estimates. One-way analysis of variance (ANOVA) and
false discovery rate correction were used to evaluate the differences in
the soil nutrients, \selectlanguage{greek}α-\selectlanguage{english}diversity, fungal community function, and the
relative abundance of the dominant phyla and genera among different
forest types in the succession stage. Non-metric multidimensional
scaling (NMDS) plots to represent the relationships between fungal
community compositions in different forest types. Their successional
stages were prepared using Bray--Curtis distances obtained with the
``vegan'' package in R. Subsequently, Adonis (999 permutations) was
performed in R to evaluate the significant differences in soil fungal
community composition among successional stages. Redundancy analysis
(RDA) was used to evaluate the relationships between the soil fungal
community and soil nutrients. Spearman analysis was used to analyze the
correlation between the sequence abundance of fungal functional groups
and soil nutrients. All statistical analyses were performed with R
program version 4.2.1 (R Core Team, 2022). Differences with\emph{P}
\textless{}0.05 were regarded as statistically significant.

\textbf{RESULTS}

\textbf{Soil properties and fungal community diversity during forest
succession}

There were differences among the forest sites representing different
successional stages in soil pH, SOC, TP, TN, AP, and AK contents (Figure
1). Soil NO\textsubscript{3}\textsuperscript{-}-N was significantly
higher in the forest type of early-successional stage than in other
stages of succession (\emph{P} \textless{}0.01). Soil pH and AP were the
highest in \emph{G. biloba} forest at the early successional stage, and
significantly higher than those in middle and late-stage forest types
(\emph{P} \textless{}0.05, Figure 1). TP in the middle-stage forest was
significantly lower than at other stages of succession (\emph{P}
\textless{}0.01). SOC and AK content was significantly enriched in the
soil of \emph{R. chinensis} in early succession (\emph{P}
\textless{}0.05, Figure 1).

Forest secondary successional stages significantly altered the fungal
community richness (Chao1), diversity (Shannon), and evenness (Shannon
evenness). All three indicators showed a trend of decrease with
succession (Figure 2). The richness index was highest in the forest
types in the early-successional stage and significantly higher than in
the middle and late-successional stages (\emph{P} \textless{}0.01,
Figure 2A). The \selectlanguage{greek}α-\selectlanguage{english}diversity index decreased significantly during forest
succession (\emph{P} \textless{}0.01, Figure 2B). The evenness index
decreased in the late-successional stages, when it was significantly
lower than in the early and middle-successional stages (\emph{P}
\textless{}0.01, Figure 2C).

\textbf{Fungal community composition during forest succession}

All sequences (1,194,454) were classified according to fungal phylum and
were assigned to 2,994 OTUs across all samples. These OTUs belonged to
15 phyla, including 63 classes, 151 orders, and 667 genera. The most
abundant phyla across all samples were Basidiomycota (44.27\% of
sequences), followed by Ascomycota (39.11\%), Mortierellomycota
(10.28\%), Rozellomycota (1.63\%), and Olpidiomycota (1.34\%) (Figure
3A). The abundance of some phyla differed during the succession process,
but differences were not significant between forest types at the same
successional stage. The abundance of Basidiomycota increased
significantly (\emph{P} \textless{}0.001) during secondary succession,
whereas Ascomycota abundance was significantly lower (\emph{P}
\textless{}0.001) in the late-successional stages. Mortierellomycota
abundance was the highest in the early-successional stage and decreased
significantly after succession occurred (Figure 3C).

The most abundant genus was \emph{Russula} (Basidiomycota, 21.15\% of
sequences) followed by \emph{Mortierella} (Mortierellomycota,
10.2\%),\emph{Penicillium} (Ascomycota, 8.37\%), and
\emph{Saitozyma}(Basidiomycota, 3.79\%). A detailed genus list is shown
in Figure 3B.\emph{Russula} abundance increased with succession and was
significantly higher in the late-successional \emph{Q. acutissima}
forest than in other forest types except the \emph{Q. variabilis} forest
(\emph{P} \textless{}0.01). The abundance of \emph{Mortierella} was
highest in the early-successional shrublands and significantly higher
than in \emph{P. densiflora} and \emph{Q. acutissima} forest (\emph{P}
\textless{}0.05). The abundance of \emph{Penicillium} was significantly
lower in the late-successional stage (\emph{P} \textless{}0.01, Figure
3D). NMDS based on Bray--Curtis distances showed that soil fungal
communities were significantly separated between different forest types
and different successional stages. Subsequent Adonis confirmed the
differences among the communities (\emph{P} \textless{}0.05 for each of
the pairs, Figure 4).

\textbf{Fungal functional groups during forest succession}

Based on the annotation of fungal ITS sequences in the FUNGuild
database, the fungal communities were classified into 17 guilds, which
belonged to three trophic modes: symbiotroph, pathotroph, and
saprotroph. The relative abundance of trophic modes differed
significantly among forest types representing different successional
stages. Fungal symbiotrophs were significantly enriched in the
late-successional stage (\emph{P} \textless{}0.01), while fungal
pathotrophs were significantly enriched in the early stage (\emph{P}
\textless{}0.01). Fungal saprotrophs were significantly lower in the
\emph{Q. variabilis} forest at the late-successional stage than at the
early-successional stage (\emph{P} \textless{}0.05, Figure S1).

Within the symbiotrophic guilds, ectomycorrhizal fungi had the highest
relative abundance in the later stages (\emph{P} \textless{}0.01) and
showed an increasing trend during forest succession (Figure 5). In
contrast, the highest relative abundance of arbuscular mycorrhizal fungi
in the early-successional stage was significantly increased in \emph{G.
biloba} forest (\emph{P} \textless{}0.01). The relative abundance of
ericoid mycorrhiza increased significantly in the middle-successional
stage \emph{(P} \textless{}0.01). Within the pathotrophic guilds, animal
and plant pathogens were significantly more abundant in the
early-successional stage than in the middle and late-successional stage
(\emph{P} \textless{}0.01); however, there was no significant difference
between the middle and late stages (Figure 5). The distribution of soil,
leaf, and wood saprotrophs was significantly different among fungal
saprotrophs. All three groups were significantly enriched in the
early-successional stage (\emph{P} \textless{}0.01, Figure 5).

\textbf{Relationships between soil chemical properties, fungal community
composition, and functional groups}

The first and second axes of the RDA analysis explained 55.74\% and
10.88\% (66.62\% combined) of the variation in fungal genus-level
communities, respectively (Figure 6 A). The results indicate that soil
NO\textsubscript{3}\textsuperscript{-}-N (\emph{P} \textless{}0.01) and
AP (\emph{P} \textless{}0.05) were the most significant soil nutrient
factors affecting fungal community composition. Both were positively
correlated with the fungal community composition in the
early-successional stage and negatively correlated with the fungal
community in the middle and late-successional stages. This could
effectively explain the separation of fungal communities in the three
successional stages (Figure 6 A).

Correlation analysis highlighted different relationships between fungal
functional guilds and soil nutrients (Figure 6 B). All saprotrophic
guilds were significantly positively correlated with
NO\textsubscript{3}\textsuperscript{-}-N and AP. Leaf saprotrophs were
significantly and positively correlated with total carbon, SOC, and TN.
Plant saprotrophs were significantly and positively correlated with TN.
Soil saprotrophs were significantly and positively correlated with TP
(Figure 6 B). All pathotrophic guilds were significantly positively
correlated with NO\textsubscript{3}\textsuperscript{-}-N and available
phosphorus and were significantly negatively correlated with
NH\textsubscript{4}\textsuperscript{+}-N. Plant pathogens were
significantly positively correlated with TP (Figure 6 B). Symbiotrophs
were significantly positively correlated with
NH\textsubscript{4}\textsuperscript{+}-N and negatively correlated with
NO\textsubscript{3}\textsuperscript{-}-N. Arbuscular mycorrhizal fungi
were significantly positively correlated with
NO\textsubscript{3}\textsuperscript{-}-N and AP. Meanwhile,
ectomycorrhizal fungi were significantly positively correlated with
NH\textsubscript{4}\textsuperscript{+}-N and negatively correlated with
NO\textsubscript{3}\textsuperscript{-}-N, TP, and AP. Ericoid mycorrhiza
was significantly negatively correlated with pH, TP, and AP (Figure 6
B).

\textbf{DISCUSSION}This study illustrates the response pattern of fungal
communities during the secondary succession of temperate forests, from
shrublands and coniferous forests to deciduous broadleaf forests. The
change in soil properties caused by succession was likely the reason
driving the changes in the fungal community. Soil AP and
NO\textsubscript{3}\textsuperscript{-}-N were strongly associated with
fungal community structure and function. In the early stage of
succession, the shrub biomes significantly accumulated fungal pathogens,
while in the late stage, the deciduous broad-leaved forest significantly
accumulated ectomycorrhizal fungi. The changes in fungal community
function may have a potential influence on the succession process of
temperate forests.

We found that secondary succession in temperate forests had significant
effects on soil nutrients, with AP and
NO\textsubscript{3}\textsuperscript{-}-N being significantly higher in
the early-successional than in later-successional stages. Nitrogen and
phosphorus availability showed a trend of decreasing during forest
succession. In previous studies on subtropical and tropical forest
ecosystems, succession was often accompanied by an increase in soil
nutrient availability (Bauters et al., 2022; Sullivan et al., 2019;
Zheng et al., 2020). In contrast, in temperate forest ecosystems, plants
in the middle and late-successional stages were predominantly
ectomycorrhizal trees from the Pinaceae and Fagaceae families. These
have been shown to have slowly decomposing litter and lower nutrient
cycling rates, which are characteristics that can explain the reduced
availability of nitrogen and phosphorus during temperate forest
succession (Lin et al., 2017; Phillips et al., 2013; Schilling et al.,
2016). The tendency for fungal community diversity to decrease across
successional stages may be explained by changes in soil nutrients
(Dini-Andreote et al., 2015; Zheng and Song, 2022). In the early stages
of secondary succession, disturbances often bring about the liberation
of nutrients. This can facilitate the rapid colonization of soils by a
diverse microbial population (Zhang et al., 2016). Plants appearing
during the secondary stage of succession have been shown to grow faster
and consume more nutrients (Lajtha, 2019). Therefore, as succession
proceeds, the use of nutrients by plants can lead to a decrease in soil
nutrient availability (Huang et al., 2012), thereby reducing the
diversity of the soil fungal community.

NO\textsubscript{3}\textsuperscript{-}-N and AP were the main predictors
of soil fungal community structure during this succession process, and
were significantly correlated with fungal functional groups. As
important decomposers of soil organic matter, saprophytic fungi are
sensitive to changes in soil properties. The abundance of saprotrophic
guilds was positively correlated with
NO\textsubscript{3}\textsuperscript{-}-N and AP, and the high abundance
of saprotrophs led to a faster turnover of soil nutrients and
facilitated the maintenance of high nutrient availability (Liu et al.,
2022; Wu et al., 2019). High nitrogen and phosphorus availability also
promoted the colonization of fungal saprotrophs in the soil (Zhang et
al., 2022a). Different guilds of saprotrophs showed different responses
to total nutrients. This may reflect variation in the preference of
different saprotroph guilds in temperate forest soils for total
nutrients in the substrate.

Furthermore, the contents of NO\textsubscript{3}\textsuperscript{-}-N
and AP during the succession process were not only negatively correlated
with the abundance of ectomycorrhizal fungi, but also positively
correlated with those of arbuscular mycorrhizal fungi. This can be
explained by the difference in plant nutrient acquisition between these
two groups (Cheeke et al., 2016; Genre et al., 2020; Tedersoo et al.,
2012). Ectomycorrhizal fungi have the ability to access nitrogen and
phosphorus from organic material and transfer them to the host plant
(Smith and Read, 2008; Tedersoo and Bahram, 2019), whereas arbuscular
mycorrhizal fungi have relatively limited capacity for enzymatic
degradation and mainly take up nutrients in mineral form (Chen et al.,
2018; Tisserant et al., 2013). Therefore, soils with high nitrogen and
phosphorus availability are more conducive to colonization by arbuscular
mycorrhizal fungi. Given that ectomycorrhizal fungi incur a considerable
carbon cost from cooperation, host plants in high nutrient availability
environments often reduce their symbiosis with these fungi (Guo et al.,
2021; Nilsson et al., 2005; Peng et al., 2022). Ectomycorrhizal fungi
are thus more abundant in nutrient-deficient environments than in
nutrient-rich environments (Bai et al., 2019), and this finding is
consistent with those of our study.

One emerging pattern in natural systems analysis is that pathogens often
thrive in resource-rich environments (Revillini et al., 2016; Reynolds
et al., 2003). A series of studies in grassland ecosystems and
agroecosystems have also shown that high nitrogen and phosphorus
availability often leads to an increase in fungal pathogens (Ebeling et
al., 2021; Lekberg et al., 2021). We observed that the abundance of
pathotrophic guilds was positively correlated with
NO\textsubscript{3}\textsuperscript{-}-N and AP. This suggests that
higher soil nutrient availability may be the reason for the accumulation
of fungal pathogens in the early stage of succession. However, the
correlation between plant pathogen fungi and soil nutrients is the
opposite of that with ectomycorrhizal fungi, as the association of plant
roots with ectomycorrhizal fungi also protects the host from pathogens
(Bennett et al., 2017; Liang et al., 2020; Tedersoo et al., 2020). The
correlation between plant pathogens and soil nutrients may thus be
indirectly driven by ectomycorrhizal fungi.

We found that during secondary succession in temperate forests, early
successional forest types accumulated fungal plant pathogens, while
late-successional forest types accumulated ectomycorrhizal fungi. The
positive driving role of microbial pathogens in community succession and
the accumulation of pathogenic fungi in the soil can cause reduced
seedling recruitment and survival around a conspecific adult. This in
turn may facilitate forest succession (Dom\selectlanguage{ngerman}ínguez-Begines et al., 2020;
Van der Putten et al., 1993). The accumulation of ectomycorrhizal fungi
is an important mechanism for maintaining dominant communities of host
trees in temperate forests (Chen et al., 2019; Liang et al., 2020). As
an important plant symbiont, ectomycorrhizal fungi can expand the
absorption range of the root system and transport nutrients from organic
matter to the host (Genre et al., 2020; Tedersoo et al., 2020). Given
that nutrient availability decreased with succession in this study,
ectomycorrhizal fungi may play a more important role in nutrient
acquisition in late-successional forest types. Therefore, we suggest
that changes in fungal functions during secondary succession in
temperate forests potentially have a driving role in the succession
process.

\textbf{CONCLUSION}

Our results suggest the presence of shifts in the structure and function
of soil fungal communities during temperate forest secondary succession.
Soil NO\textsubscript{3}\selectlanguage{english}\textsuperscript{-}-N and AP contents are
important environmental filters in this process. Changes in fungal
function in temperate forest secondary succession may potentially have a
driving role in the succession process. The accumulation of fungal plant
pathogens in early-successional forests may be detrimental to seedling
regeneration, thereby contributing to forest succession. At the same
time, the significant accumulation of ectomycorrhizal fungi in
late-successional forests may help to maintain the dominance of
established late-succession species, suggesting that the soil microbial
community shifts from promoting species turnover to promoting stability
as succession progresses. These findings provide useful information to
further our understanding of the response of soil fungal communities to
secondary forest succession, and highlight the importance of fungal
community function during forest succession.

\textbf{CONFLICT OF INTEREST}

The authors declare that there are not any potential conflicts of
interest.

\textbf{ACKNOWLEDGMENTS}

This study was supported by the National Natural Science Foundation of
China {[}grant numbers 31770581; 31971498{]}, National Key Research and
Development Program of China {[}grant number 2021YFC3100403{]},
Fundamental Research Projects of Science \& Technology Innovation and
Development Plan in Yantai City {[}grant number 2022JCYJ029{]}, and
Taishan Scholars Program of Shandong Province {[}grant number
tsqn201812097{]}. We also grateful to JC Xie, JQ Xu, XT Wang, YL Qu, and
HR Sun for laboratory support.

\textbf{AUTHOR CONTRIBUTIONS}

\textbf{Xinze Geng:} Investigation (equal); Methodology (equal); Writing
original draft (lead). \textbf{Jincheng Zuo:}Investigation (equal);
Methodology (equal); Writing-review

\& editing (equal); Visualization (lead). \textbf{Yunhao
Meng:}Investigation (equal). \textbf{Yanhui Zhuge:} Methodology
(equal);\textbf{Ping Zhu:} Writing-review \& editing (equal).
\textbf{Nan Wu:} Supervision (equal); \textbf{Guangyan Ni:}Methodology
(supporting); Investigation (supporting). \textbf{Xinfu
Bai:}Conceptualization (supporting); Data curation (equal); Validation
(supporting). \textbf{Yuping Hou:}Data curation (equal); Funding
acquisition (lead); Writing-review \& editing (equal).

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study will be uploaded to
Dryad after the manuscript is Submitted.

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ZHOU, Z., WANG, C., JIANG, L. \& LUO, Y. 2017. Trends in soil microbial
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\par\null

\textbf{Figure captions}

FIGURE 1 Soil properties during the forest succession process. Different
letters indicate significant differences (\emph{p} \textless{}0.05), and
the error bars represent the standard error. TC, total carbon; SOC, soil
organic carbon; TP, total phosphorus; TK, total potassium; TN, total
nitrogen; NH\textsubscript{4}\textsuperscript{+}, ammonium nitrogen;
NO\textsubscript{3}\textsuperscript{-}, nitrate nitrogen; AP, available
phosphorus; AK, available potassium.

FIGURE 2 Fungal community richness (A), a-diversity (B), and evenness
(C) during the forest succession process.

FIGURE 3 The composition and differences of fungal communities at the
phylum (A, C) and genus levels (B, D) during forest succession.

FIGURE 4 Non-metric multidimensional scaling (NMDS) of fungal community
composition between different successional stages (A) and different
forest types (B).

FIGURE 5 Bar chart showing the significantly different functional guilds
during the forest succession.

FIGURE 6 Redundancy analysis (RDA) biplot integrating soil nutrients
under different successional stages. (A) Parameters represented by
longer arrows made a stronger contribution to group separation; red
arrows represent a significant effect; black arrows represent no
significant effect. (B) Double matrix correlation heatmap of
differential fungal functional guilds and environmental factors with a
significant difference. The color key represents the range of different
R values. Red represents positive correlation, and blue represents
negative correlation. *\textsuperscript{,} **, and *** indicate
significant differences at 0.01 \textless{} \emph{p} \textless{} 0.05,
0.001 \textless{} \emph{p} \textless{} 0.01, and \emph{p} \textless{}
0.00. TC, total carbon; SOC, soil organic carbon; TP, total phosphorus;
TK, total potassium; TN, total nitrogen;
NH\textsubscript{4}\textsuperscript{+}-N, ammonium nitrogen;
NO\textsubscript{3}\selectlanguage{english}\textsuperscript{-}-N, nitrate nitrogen; AP,
available phosphorus; AK, available potassium.

FIGURE S1 Trophic modes of fungal functional groups during the forest
succession.\selectlanguage{english}
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