Science of the Total Environment 856 (2023) 158904

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Effects of nitric acid rain stress on soil nitrogen fractions and fungal
communities in a northern subtropical forest, China
⁎
Meijia Zhou a, Haibo Hu a, , Jinlong Wang a, Xia Wang a, Ziwei Tian a, Wenbing Deng a, Chaoming Wu b, Li Zhu b,
Qianwen Lu c, Yuanyuan Feng a
a
Co-Innovation Center for Sustainable Forestry in Southern China, Jiangsu Province Key Laboratory of Soil and Water Conservation and Ecological Restoration, Nanjing Forestry
University, 159 Longpan Road, Nanjing, Jiangsu 210037, China
b
Wuxi branch, Bureau of investigation on hydrologic water resources, Wuxi, Jiangsu 214100, China
c
University of Connecticut, Department of Plant Sciences and Landscape Architecture, Storrs, CT 06269, United States of America

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Nitric acid rain (NAR) decreased soil

amino-sugar nitrogen (N) and amino-
acid N contents.
• NAR increased the α-diversity indices of
soil fungal communities.
• NAR caused an increase in the relative
abundance of Ascomycota and
Mortierellomycota.
• Soil amino-acid N was the main environ-
mental factor affecting the fungal commu-
nities.

A R T I C L E I N F O A B S T R A C T

Editor: Elena Paoletti Acid rain has severely negatively impacted terrestrial ecosystems and biogeochemical cycles. However, the potential im-
pacts of nitric acid rain (NAR) on soil nitrogen (N) fractions and fungal community diversity in northern subtropical forest
Keywords: soils remain largely unevaluated. In this study, treatments of NAR at pH = 4.5 (AR4.5), pH = 3.5 (AR3.5), and pH = 2.5
Nitric acid rain (AR2.5) were randomly sprayed in a typical Quercus acutissima Carruth. stand in northern subtropical China. The soil N
Quercus acutissima Carruth. forest
fractions and soil fungal communities were analyzed after a 12-month experimental period. The results revealed
Soil nitrogen fractions
Fungal community structure
that compared to the control, the soil total N (TN), microbial biomass N (MBN), hydrolysable ammonium N (HAN),
amino-sugar N (ASN) and amino-acid N (AAN) contents decreased significantly by 19.61–13.07 %, 20.10–9.04 %,
60.41–28.87 %, 74.10–62.25 %, and 65.69–45.64 % under stronger acidity inputs (i.e., AR2.5 and AR3.5), respectively.
Besides, the AR2.5 and AR3.5 treatments increased the α-diversity indices of soil fungal communities and altered the soil
fungal community structure. Moreover, the NAR treatments represented an increase in the relative abundance of
Ascomycota and Mortierellomycota and a decrease in that of Basidiomycota. Mortierella, Penicillium, and Tomentella
can be used as indicator genera for changes in soil fungal community structures under NAR stress. Furthermore, AAN
was the main environmental factor affecting soil fungal community at the phylum and genus levels. Cumulatively,
findings from this research provide valuable insight into NAR's effects on N cycling and microbial communities in forest
soils.

⁎ Corresponding author.
E-mail address: hhb@njfu.com.cn (H. Hu).

http://dx.doi.org/10.1016/j.scitotenv.2022.158904
Received 10 June 2022; Received in revised form 14 September 2022; Accepted 17 September 2022
Available online 19 September 2022
0048-9697/© 2022 Published by Elsevier B.V.
M. Zhou et al.
1. Introduction
With combined emissions of fossil fuel combustion and transportation
exhausts, the sulfur oxide (SOX) and nitrogen oxide (NOX) concentrations
in the ambient atmosphere have increased significantly, resulting in acid
rain (AR) pollution, which have become a global scale environmental
hotspot (Liu et al., 2019; Li et al., 2021; Liu et al., 2021; Zhou et al.,
2022). In China, for instance, over 6.4 % of the territory suffers from AR
(Wang et al., 2020). The pollution problem of sulfuric AR has been allevi-
ated (Duan et al., 2016), with the development of a series of measures to
control the emission of SO2 pollutants. However, due to the development
of social economy (Liu et al., 2021b) and the surge in the number of
motor vehicles (Li et al., 2021), nitrate concentration in AR rises with the
increase of NOX and NH3 emissions (Larssen et al., 2006). This phenome-
−
non leads to a decrease in the ratio of SO2− 4 /NO3 of precipitation (Zhao
et al., 2009; Fang et al., 2011; Wang et al., 2012), and a change in the
type of AR, from sulfuric AR to NAR (Li et al., 2021). Therefore, AR pollu-
tion will change from sulfuric AR to NAR (Li et al., 2021). Moreover, as a
large amount of NO− 3 contained in the NAR is imported into the forest eco-
system, the soil acidification problem is more serious than that of the sulfu-
ric AR (Van Breemen and Van Dijk, 1988; Wang et al., 2002). The main
reason is that in the process of soil obligate adsorption, SO2−
4 will generate
OH− and consume H+, which reduces the leaching of salt-based ions and
makes the soil have a certain buffering capacity against AR (Wang et al.,
2002). Some studies have shown that a large amount of NO− 3 input will
accelerate the leaching loss of basal-based cations in soil (Xiao, 2001), re-
sulting in the increase of A13+ concentration, reducing the buffer capacity
of soil against NAR (Van Breemen and Van Dijk, 1988; Bergkvist and
Folkeson, 1992), which directly affects soil nutrients, N cycle and soil mi-
crobial community. Given the increasing risks of NAR pollution, its impacts
on global ecosystems and biogeochemical cycles warrant intense research.
Nitrogen (N) is a critical component of biogeochemical cycles (Wang
et al., 2014). Among them, soil organic N accounts for >90 % of TN, and
is the source/reservoir of mineral N in soil (Zhong et al., 2019). The content
of organic N fractions of soil is of great significance for N mineralization
(Liu et al., 2018). Soil organic N fractions primarily include hydrolysable
ammonium N (HAN), amino-sugar N (ASN), amino-acid N (AAN), and
acid hydrolysable unknown N (HUN) (Goh and Edmeades, 1979). Of
these, ASN was almost exclusively derived from residues in the microbial
cell wall (Wu et al., 2018). The AAN was predominantly taken up by soil mi-
croorganisms, and a significant fraction of AAN could be remineralized to
ammonium N (Finzi and Berthrong, 2005). Moreover, a significant positive
correlation was found between nitrate fixation rate, heterotrophic nitrifica-
tion rate and fungal biomass (Zhu et al., 2013). As reported, fungi also
played an important role in N2O emissions through fungal denitrification
under strongly acidic soils (Wei et al., 2014) Thus, soil microbial commu-
nity dynamics and its distribution could affect soil N transformation
(Heenan et al., 2004). However, the relationships and interactions between
changes in various N fractions and fungal communities in forest soils
remain unclear.
Soil microbes comprise the most biodiverse community in the bio-
sphere. Soil fungi have strong eosinophilic properties, and the development
of bacteria and actinomycetes is inhibited and the activity of soil fungi is
enhanced in the acidic forest soil environment (Zhang et al., 2019). As
reported, Basidiomycota and Ascomycota were the primary decomposing
communities in forest soils, which could decompose refractory organic mat-
ter (Beimforde et al., 2014). Liu et al. (2017) observed that soil fungi were
sensitively affected by NAR stress. Due to the acidophilic characteristics of
fungi (Zhang et al., 2019), the input of NAR improved the diversity index of
the soil fungal community, as well as altered the structure (Ji et al., 2018;
Zhang et al., 2019; Wang et al., 2020). Besides, soil fungi play an important
role in regulating soil N cycle. Studies have found fungal diversity was
associated with a range of forest ecosystem soil conditions (e.g., soil pH,
SOM, and N fractions) (Fierer et al., 2009; Wan et al., 2015). Specifically,
Ectomycorrhizal fungi can utilize both organic and inorganic N in the
soil, thus effectively improving the uptake of N sources (Xiong et al.,
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Science of the Total Environment 856 (2023) 158904
2020). Arbuscular mycorrhizal fungi can promote the decomposition of
organic matter (Hodge et al., 2001), and can absorb and utilize ammonia
N, nitrate N and some simple amino acids from the soil through extra-
root hyphae (Leigh et al., 2009). Couple studies have shown that different
forms of organic N can be absorbed by extra-root hyphae (Hawkins et al.,
2000). To date, most previous studies have focused on the response of
soil nutrients (Zheng et al., 2022) and microbial communities to AR (Liu
et al., 2018; Li et al., 2021a; Liu et al., 2022b). However, the changes in
soil N fractions and fungal communities and their interaction under NAR
stress remain unclear.
Quercus acutissima Carruth. (a typical tree species in the northern
subtropics) stand was selected for this study, wherein three acidity gra-
dients (pH = 4.5, 3.5, and 2.5) were employed as NAR treatments
(AR4.5, AR3.5, and AR2.5) for a one-year simulated NAR spraying
test. The objectives of the study were (1) to measure the impacts of
NAR stress on the soil N fractions in a Q. acutissima Carruth. forest;
(2) to compare changes in soil fungal diversity index and community
structures in responses to NAR, and (3) to identify how soil N fractions
interact with fungal communities under NAR stress. The results ob-
tained herein can provide an improved theoretical foundation for
exploring the reactions of soil N cycles and fungal communities to
NAR stress in a northern subtropical forest ecosystem.
2. Materials and methods
2.1. Experimental site
The experiment was carried out at the Yangtze River Delta Forest
Ecological Station (32° 7′ 49″ N, 119° 12′ 7″ E) in Zhenjiang. The region
was dominated by a subtropical monsoon climate with a mean annual pre-
cipitation of 1184.3 mm between 2011 and 2021, an annual mean temper-
ature of 15.1 °C, and an annual mean pH value of rainfall is ~5.15. The
annual AR frequency is approximately 55.8 % (Li et al., 2021). The main
tree species in the region is Quercus acutissima Carruth., whereas the shrub
layers include Rosa multiflora Thunb., Fortunearia sinensis Rehd. et Wils.,
Callicarpa cathayana H. T. Chang, and Ilex cornuta Sims, etc., and the herb
layer includes Parthenocissus tricuspidata (Sieb. & Zucc.) Planch., Ophiopogon
japonicus (Linn. f.) Ker-Gawl., and Trachelospermum jasminoides (Lindl.)
Lem., etc. The experimental sample site was located in a Q. acutissima
Carruth. artificial pure forest, with shrubs scattered in the plot. During the
experimental duration (2020−2021), the average Q. acutissima Carruth.
tree height was 15 m. The trees were ~70 years old, with a stand density
of 430 trees ha−1, and average diameters of 28.6 cm at 1 m.
2.2. Experimental treatments
From November 2020, three sample Q. acutissima Carruth. artificial
pure forest plots were selected in the study area, with each plot being
24 m long × 3 m wide and the distance between each plot was 6 m.
Among them, four subplots (3 m × 3 m) of different treatments were estab-
lished in each plot randomly for a total of 12 experimental subplots to sim-
ulate NAR (without blocking natural rainfall). Four subplots in the same
plot corresponded to four treatments, namely AR4.5 (pH = 4.5), AR3.5
(pH = 3.5), AR2.5 (pH = 2.5), and CK (pH = 6.5). The distance between
the subplots was 3 m, which were separated by PVC boards (30 cm high ×
0.5 cm thick) to prevent the lateral seepage of NAR (Fig. 1). Specifically,
there was one tree in the center of every subplot and no shrubs. Herbaceous
plants were evenly distributed within every subplot. The NAR solution was
composed of a 1:5 M mass ratio of 0.5 mol L−1 H2SO4 and 0.5 mol L−1
HNO3 (Lv et al., 2014; Liu et al., 2018). According to the rainfall data of
the ecological station, 2/3 of the average monthly rainfall in the sample
plot from 2011 to 2021 was established as the total annual spraying quan-
tity, which accounted for ~5.56 % of the annual rainfall (Liu et al., 2017;
Liu et al., 2022). Subsequently, the monthly spraying amount was
converted by the ratio of monthly average rainfall (Table 1). Due to the
influence of the plum rain season, rainfall is mainly concentrated from
M. Zhou et al. Science of the Total Environment 856 (2023) 158904

Fig. 1. Layout of sample plots. CK: control without acid rain; AR4.5: Acid rain at pH 4.5; AR3.5: Acid rain at pH 3.5; AR2.5: Acid rain at pH 2.5.

June to August (Liu et al., 2017), and the frequency of rainfall in other
months is very low. So NAR was sprayed twice a month from June to
August, and once a month in other months from December 2020 to Novem-
ber 2021. The prepared liquor was mixed into tap water to adjust to the
corresponding pH value and then sprayed on the soil of the plots. As a
control, the sample plot was sprayed with an identical amount of tap
water (pH = 6.5).
The collection of soil samples occurred on November 30, 2021, for
which a sterilized soil drill with a diameter of 15 cm was used to extract
samples from the surface layer (0– 10 cm) according to the five-point
sampling method (Wang et al., 2020). In detail, five drills were collected
per experimental subplot and mixed as a composited soil sample per ex-
perimental subplot. A sterile self-sealing bag was used to store soil sam-
ples, which were then stored in a -80 °C freezer for high-throughput
sequence analysis. For the determination of total soil N and N fractions,
the remaining soil samples were placed into sterile ziplock bags and
stored in another incubator.
2.3. Soil chemical analysis
During sampling, any visible gravel, roots, and other plant materials
were discarded, and then the soil sample was sieved through a 2 mm
mesh. The soil pH was determined at a 1:2.5 (soil: water, w/v) ratio using
a PB-10 pH meter (Sartorius GmbH, Göttingen, Germany). The soil TN was
detected with an elemental analyzer (Vario EL III, ELementar, Germany).
The MBN was determined using a TOC instrument (TOC-L CPH, Japan).
The soil N fractions were used in Bremner's distillation method. The soil
organic N was divided into non-acid hydrolysable N (NHN) and total hydro-
lysable N (THN) according to chemical forms (mainly including HAN, ASN,
AAN, and HUN) (Bremner, 1965).

Table 1
Spraying amounts of simulated acid rain at the test plots.
Month Jan. Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec.

average monthly rainfall(mm) 53.64 59.59 82.32 83.76 94.09 199.2 212.5 166.6 79.54 59.52 57.72 35.78
the monthly spraying amount(mm) 2.98 3.31 4.57 4.65 5.23 11.07 11.80 9.20 4.42 3.31 3.21 1.99
the amount of spraying at a plot (L plot−1) 26.82 29.79 41.16 41.88 47.04 99.59 106.2 82.80 39.77 29.76 28.86 17.89

Note: 2/3 of the average monthly rainfall from 2011 to 2021 was established as the total annual spraying amount per square meter, and 1/12 was taken as the monthly
spraying amount per square meter. The amount of spraying at a given plot was calculated by the monthly spraying amount × 9 m2 (the area of a single plot).

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M. Zhou et al.
2.4. Soil fungi DNA extraction and sequencing
The e.z n.a.® Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) was
used to extract microbial DNA from the soil samples. The PCR amplification
of the fungal ribosome internal transcribed spacer was done using primer
pairs. Each sample was identified with a 6-base barcode. The PCR reactions
were carried out in a mixture consisting of high-fidelity DNA polymerase,
template DNA, and each of the F/R primers. Amplicons were extracted
from 2 % agarose gel. This was then purified with the AxyPrep DNA Gel
Extraction Kit (Axygen Biosciences, U.S.). Quantification was performed
using Qubit® 3.0 (Life Invitrogen), and the amplicon library was paired-
end sequenced (2 × 250) using an Illumina Novaseq 6000 platform
(Nanjing GenePioneer Co. Ltd) following standard protocols.
2.5. Bioinformatics and statistics analyses
For soil fungal community analysis, we used PANDASEQ (2.11) for
sequence assembly; PRINSEQ (0.20.4) software for data quality control;
VSEARCH (2.15.0) for cluster analysis and dechimera; and QIIME (1.9.1)
for species annotation, distance calculation, and cluster analysis.
For analyzing soil N fractions and soil fungal diversity indices data, one
factorial analysis of variance (ANOVA) was used. Before ANOVA, assump-
tions of normality and homogeneity of variance were checked using
Shapiro-Wilk and Leven tests, respectively. All data met the requirement
of normality and constant variance prior to the analysis of variance. SPSS
21.0 was used for One-way ANOVA, multiple comparison analysis (Least
Significant Difference), and Pearson's correlation analysis. The significance
level was set at p < 0.05. The alpha diversity (Shannon, Simpson, and Chao1
indices) was calculated with MOTHUR software (V1.30.2). GraphPad Prism
9 was employed to plot the contents of N fractions and the top 13 phyla
abundance of soil fungi. R (V3.6.2) was employed to draw an Upset chart
and Chord plot, which were used to count the number of common and
unique OTUs in the four treatments and to plot the top 10 genus abundance
of soil fungi. The “vegan” package in R studio and principal coordinates
analysis (PCoA) accounting for the Bray-Curtis distance were used to exam-
ine the AR treatment effects on the soil fungal communities. LEfSe (LDA
Effect Size) analysis was used to find the difference in fungal species
between groups. The redundancy analysis (RDA) of soil N fractions and
the fungal communities was performed using Canoco5 software.
3. Results
3.1. Effects of NAR on soil pH, TN, MBN and N fractions
The results showed that the soil pH values decreased significantly with
the more strongly acidic sprays in contrast to the CK. Specifically, the
AR2.5 treatments had lower soil pH values than the CK treatments by 0.16
units, respectively. (p < 0.05, Fig. 2a). Moreover, the soil TN and MBN con-
tents exhibited a similar trend to the soil pH values. Compared with the CK,
Fig. 2. Soil pH (a), TN (b), and MBN (c) of all treatments. Lowercase letters indicate significant differences according to Duncan's multiple range test (p < 0.05, n = 4). TN:
total nitrogen; MBN: microbial biomass nitrogen. CK: control without acid rain; AR4.5: Acid rain at pH 4.5; AR3.5: Acid rain at pH 3.5; AR2.5: Acid rain at pH 2.5.
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Science of the Total Environment 856 (2023) 158904
the TN contents with AR2.5 and AR3.5 decreased by 19.61 % and 13.07 %,
respectively (p < 0.05, Fig. 2b). However, compared with the CK, the MBN
contents with AR2.5 and AR3.5 dropped significantly by 20.10 % and
9.04 %, respectively, (p < 0.05, Fig. 2c).
The difference in soil N fractions between CK and NAR treatments was
significant (Fig. 3). We found that the HAN, ASN, and AAN contents under
stronger acidity inputs (i.e., AR2.5 and AR3.5) were significantly reduced
by 60.41–28.87 %, 74.10–62.25 %, and 65.69–45.64 %, respectively, (p <
0.05, Fig. 3b, c, d) compared to the CK. Meanwhile, the AR4.5 treatment
had a significantly higher ASN content than CK by 50.53 % (p < 0.05,
Fig. 3c). However, HUN and NHN contents were not significantly different
compared with CK across all treatment groups (p > 0.05, Fig. 3e, f).
3.2. Correlation analysis of soil TN and N fractions
As shown in Fig. 4, the soil pH values was positively correlated with the
MBN (R = 0.624) and HAN (R = 0.622) (p < 0.05). Meanwhile, significant
consistently positive correlations between the MBN (R = 0.752), ASN (R =
0.743), AAN (R = 0.885), and NHN (R = 0.726) with the soil TN were
found (p < 0.01). Moreover, soil MBN exhibited a highly significant corre-
lation with the HAN (R = 0.762), as did soil ASN (R = 0.758) and AAN
(R = 0.732) (p < 0.01).
3.3. Changes in fungal community composition
There were 2588 fungal OTUs obtained in our study (Fig. 5a), of which
463 were identified during processing, representing 17.89 % of the total.
The distribution of fungal OTUs unique to the CK was the lowest, account-
ing for 4.98 % of the total, while the distribution of fungal OTUs unique
to the soil under the AR3.5 treatment was the highest, accounting for
~14.99 % of the total.
Across all treatments, 13 known fungal phyla were detected (Fig. 5b).
Among them, the relative abundances of Basidiomycota, Ascomycota, and
Mortierellomycota under the CK treatment were 90.04 %, 6.34 %, and
1.81 %, respectively, which were the dominant fungal phyla in the soil of
the Q. acutissima Carruth. forest (relative abundance >1 %). The NAR repre-
sented a decrease in the relative abundance of Basidiomycota and an increase
in that of Ascomycota and Mortierellomycota. Specifically, under the AR2.5,
AR3.5, and AR4.5 treatments, there was a significant decrease in Basidiomy-
cota relative abundance compared to CK treatments of 25.57 %, 34.72 %,
and 17.58 %, respectively. In contrast, the relative abundance of Ascomycota
increased considerably by 17.55 %, 32.2 %, and 14.21 %, respectively.
Meanwhile, Mortierella abundance increased by 3.23 % under the AR2.5
treatment in comparison to CK.
Fig. 5c showed the top 10 fungal genera in terms of relative abundance.
The relative abundances of Russula, Tomentella, and Mortierella under the CK
treatment were 79.40 %, 12.64 %, and 1.59 %, respectively, which could be
used as indicator genera for the northern subtropical Q. acutissima Carruth.
forest (relative abundance > 1 %). Penicillium, Mortierella, and Tomentella
M. Zhou et al.
Fig. 3. Soil THN (a), HAN (b), ASN (c), AAN (d), HUN (e), and NHN (f) of all treatments. Lowercase letters indicate significant differences according to Duncan's multiple range
test (p < 0.05, n = 4). THN: total hydrolysable nitrogen; HAN: hydrolysable ammonium nitrogen; ASN: amino-sugar nitrogen; AAN: amino-acid nitrogen; HUN: hydrolysable
unknown nitrogen; NHN: non-acid hydrolysable nitrogen. CK: control without acid rain; AR4.5: Acid rain at pH 4.5; AR3.5: Acid rain at pH 3.5; AR2.5: Acid rain at pH 2.5.
abundance significantly altered across all treatment groups in comparison to
CK treatment, which could be used as indicator genera for changes in the
soil fungal community structures under NAR stress. Specifically, compared
with the CK, the relative abundances of Tomentella under the AR2.5,
AR3.5, and AR4.5 treatments were significantly reduced by 12.40 %,
Fig. 4. Heat map of the correlation analysis between pH, TN, MBN, and nitrogen
fractions. The size of the circle indicates the size of the correlation coefficient. **
and * indicate significant difference at p < 0.01 and p < 0.05. TN: total nitrogen;
MBN: microbial biomass nitrogen; THN: total hydrolysable nitrogen; HAN:
hydrolysable ammonium nitrogen; ASN: amino-sugar nitrogen; AAN: amino-acid
nitrogen; HUN: hydrolysable unknown nitrogen; NHN: non-acid hydrolysable
nitrogen.
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12.64 %, and 12.64 %, respectively. However, the relative abundances of
Penicillium and Mortierella under the AR2.5 treatment were significantly
increased by 7.35 % and 13.59 %, respectively, over that of the CK.
3.4. Changes in fungal diversity
The fungal community diversity values under the various experimentally
simulated NAR treatments were depicted in Fig. 6. Compared with the CK,
the Chao1, Shannon, and Simpson indices under the AR2.5 and AR3.5 treat-
ments were significantly increased by 55.20–55.09 %, 88.81–79.42 %, and
46.01–47.34 %, respectively, (p < 0.05, Fig. 6a-c), which suggested a more
complex sample diversity. According to the Bray-Curtis distance algorithm
(Fig. 6d), primary coordinate analysis (PCoA) revealed that the first axis ex-
plained 55.13 % of the contribution rate, whereas the second axis explained
31.39 %, for a total variance contribution rate of 86.52 %. Surprisingly, the
differences in β-diversity between the AR2.5 and AR 3.5 treatments were
much larger than those between the AR4.5 and CK treatments. In addition,
the ANOSIM method (Fig. 6e) was employed to analyze the similarities
between the soil fungal communities under different treatments. This indi-
cated that the differences between the groups were significantly greater
than those within the groups (R = 1, p = 0.001).
3.5. LEfSe analysis of soil fungal communities
As shown in Fig. 7, when the LDA threshold was 3.0, there were 105 dif-
ferent species of fungi under different treatments. Moreover, the species
number of soil fungi between CK and NAR treatments were significantly
different. With the increasing pH, the fungal species number was improved,
and it was significantly higher in the AR2.5 treatments than in the CK.
3.6. Responses of soil N fractions to fungal communities
Correlation analysis results revealed that the soil pH values was sig-
nificantly negatively correlated with the Shannon indices (p < 0.05,
Table 2). Consistent significantly negative correlations between the
M. Zhou et al.
Fig. 5. OTU numbers(a), community composition at the phyla of soil fungi(b), and community composition at the genus of soil fungi(c) of all treatments. CK: control without
acid rain; AR4.5: Acid rain at pH 4.5; AR3.5: Acid rain at pH 3.5; AR2.5: Acid rain at pH 2.5.
Shannon (R = − 0.725) and Simpson (R = − 0.726) indices with the
soil TN were observed (p < 0.01). Meanwhile, the soil MBN was signif-
icantly negatively correlated with Chao1 index (p < 0.01). The soil
HAN and Chao1 (R = − 0.755), Shannon (R = − 0.742), and Simpson
indices (R = − 0.777) were extremely significantly negatively corre-
lated (p < 0.01). The ASN was significantly negatively correlated with
the Shannon (R = − 0.824) and Simpson indices (R = − 0.763) (p <
0.01). Further, the AAN and Chao1 (R = − 0.776), Shannon (R = −
0.809), and Simpson indices (R = − 0.841) were negatively correlated
significantly (p < 0.01).
The RDA analysis of soil N fractions and the top ten soil fungi in abun-
dance at the phylum and genus levels in the Q. acutissima Carruth. forest
(Fig. 8) revealed that the interpretation rate of the first two ranking axes
attained 72.52 % and 87.12 %, with the first axis explaining 51.51 % and
66.13 %, and the second axis explaining 21.01 % and 20.99 % of the vari-
able. Meanwhile, Fig. 8a showed that Glomeromycota and Basidiomycota
had a positive correlation with the soil pH, TN, MBN, HAN, and AAN on
the left side of the first axis. The AAN (F = 5.2, p = 0.002) was the signifi-
cant factor that affected the phylum of soil fungal community structure, with
an explanation rate of 34.2 % and a contribution rate of 46.7 %, respectively.
Moreover, Fig. 7b showed that the soil pH, TN, MBN, HAN, and AAN showed
positive associations predominantly with the Russula and Tomentella. The
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Science of the Total Environment 856 (2023) 158904
AAN (F = 4.9, p = 0.008), HAN (F = 3, p = 0.022), and THN (F = 2.9,
p = 0.012) significantly affected fungal community composition at the
phylum level (p < 0.05). Taken together, AAN was the main environmental
factor affecting the fungal community.
4. Discussion
4.1. Effects of NAR stress on forest soil N fractions
N transformation in forest soils is a critical process in the material
cycling of forest ecosystems and is of great significance to terrestrial
ecosystems (Jones and Kielland, 2012). After spraying AR on forest soil, it
directly affected the soil nutrient and N cycle (Zheng et al., 2022). In this
study, we found the soil TN content decreased significantly under stronger
acidic inputs (i.e., AR2.5 and AR3.5 treatments), compared to the control
(Fig. 2b), and TN was positively correlated with pH (Fig. 4), which was
consistent with previous studies (Xu et al., 2015; Li et al., 2021a; Liu
et al., 2021a). In the study of Xu et al. (2015), when pH = 2.5, TN and
SOM contents decreased the most, indicating that soil N supply potential
decreased. Moreover, the soil HAN, AAN, and ASN contents in the N frac-
tions exhibited a similar trend to the soil TN contents (Figs. 3b-d), which
was semblable to the results of Tian et al. (2017). Besides, the HAN, AAN,
M. Zhou et al.
Fig. 6. Effects of different treatments on soil fungal community. (a) Chao1 index. (b) Shannon index. (c) Simpson index. (d) Principal Coordinate Analysis (PCoA) and (e) Bray
Curtis Anosim of the effects on the soil fungi community. Lowercase letters indicate significant differences according to Duncan's multiple range test (p < 0.05, n = 4). CK:
control without acid rain; AR4.5: Acid rain at pH 4.5; AR3.5: Acid rain at pH 3.5; AR2.5: Acid rain at pH 2.5.
and ASN contents were positively correlated with the soil pH, TN, and MBN
contents (Fig. 4), which were similar to the findings of Atanasova (2008)
and Wang et al., 2012. This may have been due to key sources and struc-
tures of the N fractions, which were intimately related to the soil TN and
MBN.
There may be three reasons for the change of TN and N fractions under
stronger acidic inputs (i.e., AR2.5 and AR3.5 treatments) compared with
the control. One is that the release of TN in soil caused by NAR is primarily
due to the loss of dissolved organic N (Yamada et al., 2002). Many studies
have examined that the massive input of H+ and NO− 3 in NAR will lead
to the increase of soil acidity and A13+ content, which will reduce the
buffer capacity of soil against NAR (Aitken, 1992; Bergkvist and Folkeson,
1992; Wang et al., 2002) and aggravate the loss of soil dissolved organic
N. Therefore, when pH = 2.5, soil TN and N fractions decreased greatly.
The second is due to the leaching of potent NAR, which induced the frag-
mentation of soil macro aggregates and diminished the total soil porosity.
This likely degraded the physical protection of SOM, which translated to
the loss of organic N, and reduced soil N supply potential (Evrendilek
et al., 2004). The third is that NO−3 carried by NAR is a substrate for deni-
trification and an inhibitor of N2O reductase (Liu et al., 2020), which can
also affect the soil N cycle. In future research, attention should be paid to
the influence of NAR on different soil layers to explore the whereabouts
of N.
Moreover, soil MBN is an essential component of soil N, which drives
soil organic N and inorganic N transformation and energy cycles. Soil resi-
dent N is partially derived from the biological N integration of N-fixing
microorganisms. These microorganisms are generally suitable for activities
in a neutral environment (Yu et al., 2017). Under NAR stress conditions, the
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Science of the Total Environment 856 (2023) 158904
activities of N-fixing microorganisms are greatly inhibited. This may be one
of the reasons behind the decreased MBN content in soils under the AR2.5
and AR3.5 treatments in this study (Fig. 2c). Moreover, as reported, soil
acidification can activate the release of harmful and toxic elements such
as soil aluminum, reduce plant root biomass, and inhibit the growth of
rhizosphere microorganisms, thereby decreasing the soil MBN content
(Wu et al., 2016).
4.2. Effects of NAR stress on forest soil fungal community structures and diversity
This study (Fig. 5b) showed that the dominant fungal phyla in the
Q. acutissima Carruth. forest were Basidiomycota, Ascomycota, and
Mortierellomycota, which was semblable to the results of (Sui et al.,
2016). Meanwhile, the simulated NAR treatments boosted the relative abun-
dance of Ascomycota and reduced that of Basidiomycota in our study. On
one hand, it may have been due to soil diseases caused by NAR stress,
with a higher relative abundance of Ascomycota in affected soils (Yuan
et al., 2020). On the other hand, this may have been due to the lower soil
pH, which increased the proportion of NHN to TN in the soil, as well as
more abundant refractory N-containing compounds (Wu et al., 2018).
Since Ascomycota decomposes refractory organic matter (Beimforde et al.,
2014), its relative abundance will increase accordingly.
We observed that Russula, Tomentella, and Mortierella could be employed
as indicator genera of fungi in northern subtropical Q. acutissima Carruth.
forests (Fig. 5c), which aligned with earlier forest soil fungal community
structure analysis results. Previous studies revealed that Tomentella is an
ectomycorrhizal fungus that can utilize both organic and inorganic N in
the soil, thereby effectively improving the absorption of N sources (Xiong
M. Zhou et al. Science of the Total Environment 856 (2023) 158904

Fig. 7. Analysis of differences in soil fungal community structure at different treatments. CK: control without acid rain; AR4.5: Acid rain at pH 4.5; AR3.5: Acid rain at pH 3.5;
AR2.5: Acid rain at pH 2.5.

8
M. Zhou et al.
Table 2
Correlation analysis of soil nitrogen fractions and fungal communities.
pH TN MBN THN
Chao1 −0.561 −0.693* −0.722** −0.375
Shannon −0.579* −0.725** −0.668* −0.503
Simpson −0.575 −0.726** −0.676* −0.467
** and * indicate significant difference at p < 0.01 and p < 0.05. TN: total nitrogen; MBN: microbial biomass nitrogen; THN: total hydrolysable nitrogen; HAN: hydrolysable
ammonium nitrogen; ASN: amino-sugar nitrogen; AAN: amino-acid nitrogen; HUN: hydrolysable unknown nitrogen; NHN: non-acid hydrolyzable nitrogen.
et al., 2020). The results of this study observed that the relative abundance
of Mortierella and Penicillium increased remarkably. Besides, the relative
abundance of Tomentella decreased with lower soil pH, which may have
been due to its poor adaptability to strongly acidic environments, resulting
in decreased relative abundance, and thus affecting soil nitrogen cycling.
Furthermore, the soil fungal community structural diversity was im-
pacted by NAR stress. Results indicated that NAR significantly improved
the Chao1, Shannon, and Simpson indices of fungal communities in the
forest soil (Fig. 6a-c). Moreover, strong NAR significantly altered the β-
diversity of soil fungal communities (Fig. 6d). Wang et al. (2020) also
reported similar findings that have been associated with acidophilic
fungi. Simulated NAR induced a decrease in soil pH, which was the main
environmental factor that affected the soil fungi community structures.
Since soil fungi are dominant in acidic soils, their diversity is increased
(Zhang et al., 2019).
4.3. Influencing factors and intrinsic relationship between forest soil N fractions
and fungal communities under NAR stress
Various types of soil N are closely related to changes in fungal commu-
nity composition (Wang et al., 2014). The results of the RDA analysis
revealed that Glomeromycota and Basidiomycota of the fungal phylum
and Russula and Tomentella of the fungal genus showed positive associa-
tions predominantly with the soil pH, TN, MBN, HAN, and AAN, which
were similar to a previous study (Kivlin and Hawkes, 2016).
Soil pH is one of the key environmental factors that influence soil N
(Romanowicz et al., 2016), and it has been confirmed to be a major driver
of soil microbial changes (Zhu et al., 2022). We observed a significant pos-
itive correlation between the soil pH and fungal community (phylum and
genus), which indicated that soil pH was an important factor that affected
soil microbial biomass (Zhu et al., 2022).
Taken together, AAN was the main environmental factor affecting the
fungal community at the phylum and genus levels in organic N fractions.
Fig. 8. Redundancy Analysis (RDA) of the soil nitrogen fractions and phylum (a) and genus (b) of soil fungal community. TN: total nitrogen; MBN: microbial biomass nitrogen;
THN: total hydrolysable nitrogen; HAN: hydrolysable ammonium nitrogen; ASN: amino-sugar nitrogen; AAN: amino-acid nitrogen; HUN: hydrolysable unknown nitrogen;
NHN: non-acid hydrolysable nitrogen.
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Science of the Total Environment 856 (2023) 158904
HAN ASN AAN HUN NHN
−0.755** −0.695* −0.776** −0.060 −0.401
−0.742** −0.824** −0.809** −0.062 −0.338
−0.777** −0.763** −0.841** −0.018 −0.366
This may have been related to the fact that AAN is the major source of avail-
able N absorbed by microorganisms (Bardgett et al., 2003), which can also
directly assimilate and absorb small molecular amino acids (Chen et al.,
2015), thereby affecting the abundance of soil fungi. Besides, fungi can
produce nitrite from easily decomposed nitrogenous organic compounds
(such as amino acids) in soil (Martikainen, 2022).
4.4. Research limitations and implications
This study provided supportive data for understanding the environmen-
tal risks posed by the introduction of NAR into forest ecosystems. Some
perspectives for future research include: (1) The field test period of NAR
stress in this study was quite short; thus, longer-term positioning tests of
two years or more are required to verify this result; (2) Since NAR may
cause nitrate leaching into the soil, it is necessary to further consider the
depth and gradient of the soil sampling layer. (3) Soil microbes include
bacteria, fungi, and actinomycetes; thus, further research will be required
to elucidate the relationships between changes in microbial flora.
NAR affected the soil N cycle and N supply potential, which may affect
the utilization of soil available N by plants. Moreover, the diversity and
community structures of soil fungi were significantly affected by the NAR.
In summary, the environmental degradation and longer-term risks caused
by NAR to forest soil ecosystems deserve serious and sustained attention.
5. Conclusions
For this study, different intensities of acid rain were investigated for
their effects on the nitrogen (N) fractions and fungal communities of forest
soils by simulating nitric acid rain (NAR) in a northern subtropical Quercus
acutissima Carruth. forest. The results showed that strongly NAR reduced
the soil pH, total N (TN), and microbial biomass N (MBN), whereas the
contents of soil hydrolysable ammonium N (HAN), amino-sugar N (ASN),
and amino-acid N (AAN) in the soil N fractions decreased significantly
M. Zhou et al.
with lower soil pH. In addition, the simulated NAR treatments increased the
α-diversity indices of soil fungal communities and altered the soil fungal
community structure. The co-preferred phyla under the four treatments in-
cluded Basidiomycota, Ascomycota, and Mortierellomycota. Mortierella,
Penicillium, and Tomentella can be used as indicator genera for changes in
soil fungal community structures under NAR stress. AAN was the main
environmental factor affecting the fungal community at the phylum and
genus levels. This study provided a theoretical basis for exploring soil N
supply capacities and microbial community responses to NAR stress in
northern subtropical forest ecosystems in China. Future research should
focus more on the long-term mechanisms and implications of NAR on soil
microorganisms.
Data availability
Data will be made available on request.
Declaration of competing interest
The authors declare that they have no known competing financial inter-
ests or personal
relationships that could have appeared to influence the work reported
in this paper.
Acknowledgements
We gratefully thank the funding by Forest ecosystem localization re-
search project of Yangtze River Delta of State Forestry Administration
(2019132158), Technical support and cooperation project of Wuxi Water
Conservancy Bureau (2107116), Forestry Advantage discipline construc-
tion project in Jiangsu Province (164010641).
CRediT authorship contribution statement
M.J. Zhou: Methodology, Sample collection, Data analysis, Original
draft. H.B. Hu: Supervision, Manuscript reviewing and editing. J.L. Wang:
Sample collection, Data analysis. X. Wang: Manuscript reviewing and
editing. Z.W. Tian: Sample collection, Data analysis. W.B. Deng: Sample
collection. C.M. Wu: Manuscript reviewing. L. Zhu: Manuscript reviewing.
Q.W. Lu: Manuscript reviewing and editing. Y.Y. Feng: Methodology,
Manuscript reviewing and editing, Conceptualization.
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