Nutrient availability is a dominant predictor of soil bacterial and fungal community composition after nitrogen addition in subtropical acidic forests

Juyan Cui; Xiaochun Yuan; Qiufang Zhang; Jiacong Zhou; Kaimiao Lin; Jianguo Xu; Yaozhong Zeng; Yue Wu; Lei Cheng; Quanxin Zeng; Kongcan Mei; Yuehmin Chen

doi:10.1371/journal.pone.0246263

. 2021 Feb 23;16(2):e0246263. doi: 10.1371/journal.pone.0246263

Nutrient availability is a dominant predictor of soil bacterial and fungal community composition after nitrogen addition in subtropical acidic forests

Juyan Cui ^1,^2,³, Xiaochun Yuan ^1,^2,⁴, Qiufang Zhang ^1,², Jiacong Zhou ^1,², Kaimiao Lin ⁴, Jianguo Xu ⁵, Yaozhong Zeng ^1,², Yue Wu ^1,², Lei Cheng ^1,², Quanxin Zeng ^1,², Kongcan Mei ^1,², Yuehmin Chen ^1,^2,^6,^*

Editor: Julian Aherne⁷

PMCID: PMC7901772 PMID: 33621258

Abstract

Nutrient addition to forest ecosystems significantly influences belowground microbial diversity, community structure, and ecosystem functioning. Nitrogen (N) addition in forests is common in China, especially in the southeast region. However, the influence of N addition on belowground soil microbial community diversity in subtropical forests remains unclear. In May 2018, we randomly selected 12 experimental plots in a Pinus taiwanensis forest within the Daiyun Mountain Nature Reserve, Fujian Province, China, and subjected them to N addition treatments for one year. We investigated the responses of the soil microbial communities and identified the major elements that influenced microbial community composition in the experimental plots. The present study included three N treatments, i.e., the control (CT), low N addition (LN, 40 kg N ha^-1 yr^-1), and high N addition (HN, 80 kg N ha^-1 yr^-1), and two depths, 0−10 cm (topsoil) and 10−20 cm (subsoil), which were all sampled in the growing season (May) of 2019. Soil microbial diversity and community composition in the topsoil and subsoil were investigated using high-throughput sequencing of bacterial 16S rDNA genes and fungal internal transcribed spacer sequences. According to our results, 1) soil dissolved organic carbon (DOC) significantly decreased after HN addition, and available nitrogen (AN) significantly declined after LN addition, 2) bacterial α-diversity in the subsoil significantly decreased with HN addition, which was affected significantly by the interaction between N addition and soil layer, and 3) soil DOC, rather than pH, was the dominant environmental factor influencing soil bacterial community composition, while AN and MBN were the best predictors of soil fungal community structure dynamics. Moreover, N addition influence both diversity and community composition of soil bacteria more than those of fungi in the subtropical forests. The results of the present study provide further evidence to support shifts in soil microbial community structure in acidic subtropical forests in response to increasing N deposition.

Introduction

Industrialization and urbanization have increased the quantity of nitrogen (N) and phosphorus entering terrestrial systems [1], especially in China’s warm and humid climatic zones [2]. The annual atmospheric N deposition in China increased from 7.6 to 20 Tg in 1978−2010 [3], attracting the attention of researchers regard the potential effects of N deposition on terrestrial ecosystems. Excessive N input negatively impacts the structure and functioning of terrestrial ecosystems, causing soil acidification [4, 5], decline in tree productivity [6–8], reduced plant diversity [9, 10], and reduced understory vegetation species richness [11, 12]. N deposition could also alter ectomycorrhizal fungal [13] and soil bacterial community structure [14, 15].

Soil bacteria and fungi play pivotal roles in terrestrial ecosystems, mediating biogeochemical processes (e.g., carbon [C] and N cycling) [14, 16] and promoting aboveground plant health and productivity [2, 17, 18]. Several meta-analyses have examined the effects of N addition on soil microbial biomass and community composition [19–28]. Overall, simulated N deposition appears to suppress microbial biomass [2]; however, the effects varied across biomes. N addition reduced soil microbial biomass C (MBC) in temperate forests and grasslands, but significantly increased MBC in tropical/subtropical forests [27]. This increase in microbial biomass following N addition is thought to be mediated by increasing C or N resource availability [28].

N addition can also induce changes in soil microbial α-diversity and microbial community composition [29–31]. In a previous meta-analysis, N additions increased the Shannon indices and reduced bacterial Chao1 indices, although the effect on soil bacterial richness was greater than that on fungal richness [28]. Similarly, in a subtropical forest, simulated N deposition significantly decreased microbial α-diversity [29, 30, 32]. However, N additions do not always alter in the evenness and richness of soil bacterial and fungal communities. Short-term N addition significantly altered soil microbial community structure by increasing fungi/bacteria ratio (F/B) in tropical/subtropical forest soil, although long-term N addition did not induce such changes [29, 33]. Nevertheless, some studies have reported no change in the F/B ratio in response to short-term N enrichment [34], while others have demonstrated that N enrichment could reduce the F/B ratio in subtropical forests [28, 35]. According to the copiotrophic hypothesis, increasing N enrichment could decrease the abundance of oligotrophic groups but increase the abundance of copiotrophic groups [14, 15, 32, 36–38]. Therefore, the effects of N enrichment on microbial communities are variable, and the mechanisms by which N addition influence the soil microbial community composition needs to be elucidated.

N addition can influence soil physicochemical properties, which, in turn, influence soil microbial structure [39]. Numerous studies have explored the major factors that could explain the changes observed in sensitive soil microbial communities within terrestrial ecosystems following N enrichment [5]. For example, soil pH is considered a key factor influencing soil bacterial community composition in terrestrial ecosystems [38, 40]. However, fungal community composition appears to be less impacted by pH, because fungi usually exhibit a greater range of optimal pH than that in bacteria [40, 41].

The absolute abundances of major microbial groups present in the soil is positively correlated with soil C and N concentrations [42]. N addition could directly influence soil bacterial community composition by altering the availability of compounds such as modified ammonium N (NH₄⁺-N) [5, 28, 43]. Furthermore, soil bacterial community composition could be influenced by a slight decline in pH caused by an increase in soil NH₄⁺-N in extremely acidic soil (pH < 4.5) [5]. Although such mechanisms have been previously studied, few comprehensive studies have examined the wide array of soil physicochemical properties associated with changes in bacterial and fungal communities following N enrichment in subtropical forest ecosystems.

Currently, subtropical forests in Southeast China are experiencing extensive N deposition, accompanied by signs of N saturation, leading to soil acidification [44]. In the present study, we investigated the responses of bacterial and fungal communities to N addition and the underlying mechanisms of such responses. We addressed the following questions in this study: (1) How do bacterial and fungal communities respond to N additions? (2) How do biotic and abiotic elements in the soil modulate the responses of bacterial and fungal communities? (3) What are the potential mechanisms that are responsible for the observed changes in microbial community structure? We hypothesized that N addition could reduce the bacterial and fungal diversity, and, thereby alter soil microbial community composition in subtropical forests. We also hypothesized that soil pH and nutrients would regulate the responses of microbial communities through the direct or indirect effects of N amendments.

Materials and methods

Site description and experimental design

The study area is located in the scenic Jiuxian Mountains within the Daiyun Mountain Nature Reserve in Dehua County, Fujian Province, in southeast China (118°06ʹ3–5″E, 25°42ʹ22–27″N). The reserve is located in the transition zone between central subtropical and southern subtropical forests [45]. This nature reserve, which has the southernmost distribution in China, has the highest degree of biodiversity per unit area in China, and comprises the largest area of the best preserved natural Pinus taiwanensis community (Fig 1). P. taiwanensis is a unique alpine tree species in China that plays a valuable role in ecological restoration. The area is also the largest germ plasm gene pool of P. taiwanensis in China, which are mainly distributed at an altitude of 1000−1800 m. The climate type is subtropical maritime monsoon climate, with cold winters and hot summers. The climate can be simultaneously hot and rainy, with four distinct seasons, mean annual temperature of 20°C, a mean annual precipitation of 1800 mm, and a mean relative humidity of 80%. The soil is an Ultisol formed from sandstone and classified as red soil according to Chinese soil classification. Total N deposition in the region is approximately 38 kg N ha^-1 yr^-1 [44].

Fig 1 — (A) Map of China. (B) Map of Fujian Province. (C) Map of Dehua county. The blue pentagram is the sampling area.

In the P. taiwanensis forest, we randomly selected 12 experimental plots to perform the simulated N addition treatment in May 2018. No permits were required for the experiments since this study was part of an on-going collaborative scientific effort with the Daiyunshan Nature Reserve. All experimental plots are close to the top of the mountain, face the same direction, and have similar slopes and elevations. In each experimental plot, 12 subplots (10 × 10 m) were randomly assigned three different levels of N addition, including the control (CT), low N (LN), and high N (HN) (0, 40, and 80 kg N ha^-1 yr^-1, respectively), with four replicates for each N level. Each plot was surrounded by a 5-m wide buffer zone. We used urea [CO(NH₂)₂] as the N source, which was added into the experimental plots from March to September every year. The required amounts of urea were dissolved in 8 L purified water, and CT plots received an equivalent volume of water without urea.

Soil sampling and analysis

In May 2019, soil samples were randomly collected from 5−8 cores at depths of 0−10 cm (topsoil) and 10−20 cm (subsoil) from each plot after removing the surface litterfall. Soil samples were passed through a 2-mm sieve after removing the litter, roots, and stones, and divided into three parts. One portion of each sample was immediately processed to measure soil moisture content (SMC), mineral N, and dissolved organic C (DOC), and then stored at -80°C for the soil molecular biology analyses. Another portion was air-dried for soil physicochemical parameter analyses. The air-dried soil portion was passed through a 0.149-mm sieve to measure total soil C and N.

Soil pH was determined using a glass electrode (STARTER 300; OHAUS, USA). Samples were shaken for 0.5 h in a 1:2.5 soil:water solution (w/v). Soil moisture content was assessed following oven-drying for 48 h at 105°C to constant mass. The soil organic C (SOC) to total N ratio (C:N) was measured using an elemental analyzer (Elementar Vario EL III; Elementar, Langenselbod, Germany). Total P content was determined after digesting the samples with H₂SO₄ and HClO₄ (at a 4:1 ratio) using a continuous flow analyzer (Skalar san++, Skalar, Breda, Netherlands). After 2 M KCl extracts of fresh soil samples were prepared, soil suspensions were centrifuged at 4000 rpm for 10 min, filtered through Whatman 42 filter papers, and analyzed for NH₄⁺-N and NO₃^—N using the Continuous Flow Analytic System (Skalar san++; Skalar, Breda, the Netherlands). Available N (AN) was calculated as the sum of ammonium-N and nitrate-N. DOC was extracted with deionized water in a 1:4 soil:water solution (w/v) by shaking for 0.5 h, centrifuging at 4000 rpm for 0.5 h, and then filtering through 0.45-μm Millipore filters. The extracts were analyzed on a TOC-VCPH/CPN analyzer (Elementar Analysensysteme GmbH, Germany) to determine the DOC concentrations. The soil cation exchange capacity (CEC) was measured using the ammonium acetate method [46].

Soil MBC and N (MBN) were measured using the chloroform-fumigation extraction method [47]. The correction factor of MBC was 0.38 [48], while that of MBN was 0.45 [49].

Quantitative PCR (qPCR) analysis of soil microbial abundance

Soil DNA was extracted from 0.25-g soil samples using PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA) according to the manufacturers instruction. Genomic DNA purity and quality were checked on 0.8% agarose gels. The quantitative PCR (qPCR) method was used to measure the relative abundance of the bacterial 16S rDNA genes and fungal internal transcribed spacers (ITS) that were amplified using the primers detailed in S1 Table [50].

For each soil sample, a 10-digit barcode sequence was added to the 5ʹ end of the forward and reverse primers (provided by Allwegene Company, Beijing). PCR was carried out on a Mastercycler Gradient (Eppendorf, Germany) using 25-μl reaction volumes, containing 12.5 μl 2× Taq PCR MasterMix, 3 μl bovine serum albumin (2 ng μl^-1), 2-μl primers (5 μM), 2-μl template DNA, and 5.5-μl ddH₂O. The cycling parameters were as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 10 min. Three PCR products per sample were pooled to mitigate reaction-level PCR biases. PCR products were purified using a QIAquick Gel Extraction Kit (QIAGEN, Germany) and quantified using real time PCR. The fungal-to-bacterial (F/B) ratio was calculated using the 16S rDNA and ITS gene copy numbers.

Illumina Miseq sequencing and bioinformatics analysis

Deep sequencing was performed on a Miseq platform at Allwegene Company (Beijing). After the run, image analysis, base calling, and error estimation were performed using Illumina Analysis Pipeline v2.6 (Illumina, San Diego, CA, US).

Raw data were first split based on barcodes and then screened. Sequences were removed from consideration if they were shorter than 200 bp, had a low-quality score per sequence (≤ 20), contained ambiguous bases, or did not exactly match the primer sequences and barcode tags. The data were split then overlapped and spliced using PEAR (Paired-End read merger) software. Qualified reads were separated using the sample-specific barcode sequences and trimmed with Illumina Analysis Pipeline Version 2.6, while chimeras were removed. Subsequently, the dataset was analyzed using QIIME v1.8.0 (http://qiime.org). Sequences were clustered into operational taxonomic units (OTUs) at a similarity level of 97% [51] to generate rarefaction curves and to calculate the richness and diversity indices. The ribosomal database project classifier tool (release 10.3) was used to classify all sequences into different taxonomic groups [52].

Statistical analysis

All data are presented as means in the tables and figures. Statistical analyses were performed using IBM SPSS Statistics 20 (IBM Corp., Armonk, NY, US), and data were tested for normality and homoscedasticity before statistical analyses. One-way analysis of variance (ANOVA) was used to determine the differences in soil properties and soil microbial parameters, and multiple comparisons were conducted using the least significant difference (LSD) test at p < 0.05. Moreover, correlation analysis was performed with the Pearson’s test (two-tailed) at two significance levels, i.e., p < 0.05 and p < 0.01.

To examine similarity between different samples, we used clustering and Principal Components Analysis (PCA) on the OTUs from each sample in R [53]. We used PCA because it is suitable for ordination using only species composition, and is an unconstrained method. The evolutionary distances between microbial communities from each sample were calculated using the Tayc coefficient and represented as an unweighted pair group method with arithmetic mean (UPGMA) clustering tree describing the dissimilarity (1—similarity) between multiple samples [54]. Multivariate statistical analysis, known as linear discriminant analysis (LDA), was performed to calculate the effect sizes in order to identify the species with significant differences in abundance among the soil samples.

Two-way ANOVA was used to compare the results in the soil fractions (topsoil and subsoil) and fertilizer treatments. Pearson’s correlation analysis was performed to assess the relationships between soil properties and microbial α-diversity indices. Chao1, Simpson, Shannon, phylogenetic diversity whole tree (PD whole tree), and observed species indices were calculated using QIIME v1.8.0 to estimate the α-diversity levels. Partial least squares discriminant analysis (PLS-DA), which is a multivariate statistical analysis method for discriminant analysis, estimated the β-diversity in soil bacterial and fungal communities. Pearson’s correlation analysis was used to address the relationships among soil properties, microbial abundance, and microbial biomass. In all tests, a p < 0.05 was considered statistically significant. To identify the relationship between changes in the soil microbial community composition and soil environmental factors, redundancy analysis (RDA) and Mantel test analysis were performed using CANOCO 5.0 (Ithaca, NY, USA) and R (3.6.2), respectively.

Results

Responses of soil properties and microbial biomass to nitrogen additions

Among the soil physicochemical properties we examined, soil pH, SMC, CEC, SOC, total N (TN), TP, and C:N were not affected by N additions (Table 1). Meanwhile, soil NO₃^—N concentration in the topsoil ranged from 4.8 mg kg^-1 in the CT plots to 9.65 mg kg^-1 in the HN plots, and increased significantly under high-N addition, while soil NH₄⁺-N concentration in the topsoil ranged from 74.28 mg kg^-1 in the CT plots to 51.23 mg kg^-1 in the LN plots and decreased significantly under LN treatments. Furthermore, LN addition reduced soil AN concentration and HN addition reduced soil DOC concentration in both the topsoil and the subsoil (p < 0.05). However, changes in MBN in response to N addition were only observed in the topsoil, while N addition did not affect MBC in the topsoil or the subsoil.

Table 1. Responses of physicochemical properties of soil at different depths to nitrogen addition in the Daiyun Mountain Nature Reserve in southeastern China.

	Topsoil (0–10 cm)				Subsoil (10–20 cm)
	CT	LN	HN	p	CT	LN	HN	p
pH	4.22(0.04)	4.32(0.05)	4.35(0.10)	0.43	4.57(0.04)	4.6(0.03)	4.58(0.03)	0.80
SMC (%)	74(9.29)	66(3.20)	59(3.60)	0.31	41(2.53)	39(4.02)	39(3.77)	0.92
CEC (cmol kg^-1)	12.58(2.27)	9.47(1.83)	10.63(0.97)	0.49	12.27(1.65)	8.61(0.69)	8.79(0.90)	0.09
NH₄⁺ (mg kg^-1)	74.28(8.22)^a	51.23(2.91)^b	62.77(5.42)^ab	0.06	30.32(3.98)^a	16.19(3.55)^b	29.61(3.05)^a	0.02
NO₃^- (mg kg^-1)	4.80(1.56)^b	5.69(1.60)^ab	9.65(0.89)^a	0.07	6.22(0.29)	5.49(0.72)	6.45(0.55)	0.47
AN (mg kg^-1)	79.08(7.28)^a	56.92(7.79)^b	72.42(10.25)^ab	0.03	36.54(4.23)^a	21.68(2.97)^b	36.06(2.80)^a	0.04
DOC (mg kg^-1)	325.75(46.54)^a	245.37(11.86)^ab	206.87(18.36)^b	0.05	63.92(7.84)^a	46.07(4.38)^ab	28.51(4.56)^b	0.01
SOC (g kg^-1)	55.73(2.68)	55.99(4.41)	47.02(3.16)	0.17	26.25(2.96)	27.27(3.92)	30.98(3.77)	0.63
TN (g kg^-1)	4.06(0.47)	3.75(0.36)	3.12(0.19)	0.22	1.81(0.21)	1.62(0.24)	2.20(0.27)	0.27
TP (g kg^-1)	0.25(0.03)	0.22(0.02)	0.25(0.01)	0.51	0.17(0.02)	0.14(0.01)	0.19(0.03)	0.21
C:N	14.98(0.50)	15.01(0.40)	15.11(0.64)	0.98	14.55(0.09)	14.82(1.11)	14.11(0.58)	0.75
MBC	543.98(45.42)	682.61(119.04)	616.18(127.69)	0.66	283.83(53.21)	360.33(98.88)	437.41(59.25)	0.38
MBN	97.09(6.09)^c	201.99(8.58)^a	145.27(9.75)^b	0.00	50.31(13.23)	61.78(9.67)	81.91(7.73)	0.15

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The mean values of soil properties in control (CT), low (LN), and high (HN) nitrogen addition treatments in topsoil (0–10 cm) and subsoil (10–20 cm) soil samples are shown. Different letters represent significant differences (one-way ANOVA, p < 0.05, LSD post hoc analysis) between different levels of nitrogen addition.

Bacterial and fungal abundances were determined by quantification of copy numbers of bacterial 16S rDNA or fungal ITS using qPCR. The highest number of 16S rDNA gene copies in both the topsoil and the subsoil were observed in the LN plots, while the lowest numbers were observed in the topsoil and the subsoil of the HN plots (S2 Table). F/B values were greater in the topsoil than in the subsoil (S2 Table). Additionally, F/B values were positively correlated with soil MBN (R² = 0.58, p < 0.01) and NO3—N (R² = 0.51, p < 0.05) levels, and negatively correlated with soil pH values (R² = -0.61, p < 0.01) (S3 Table).

Effects of N addition on bacterial and fungal diversity

Overall, 3,760 OTUs were obtained from 1,185,330 high quality and chimera-free clean tags by Miseq sequencing of 16S rDNA gene amplicons, with an average of 19,719−89,416 clean tags per sample. Similarly, 4,584 OTUs were obtained from a total of 1,171,127 high quality and chimera-free clean tags by Miseq ITS sequencing, with an average of 23,183−86,895 clean tags per sample. The rarefaction curves showed that these are reasonable amounts of sequencing data (S1A and S1B Fig) with a Good’s coverage of 97.13−98.75%, which indicated that sequence reads were sufficient to capture the bacterial and fungal α-diversity (S4 Table).

Soil bacterial and fungal α-diversity was affected differently by N addition between the topsoil and the subsoil (Table 2; S5 Table). N addition decreased bacterial α-diversity (Chao1, observed species, PD whole tree, Shannon indices) in the subsoil of the HN plots relative to that of the CT plots (S5 Table; p < 0.05). However, bacterial richness indices, including Chao1, observed species, and PD whole tree, in samples of the topsoil of LN plots were all slightly higher than in those of the topsoil of CT and HN plots, though the differences were not statistically significant (p > 0.05) (S5 Table). The interaction between N addition and soil layer significantly affected α-diversity of bacteria (observed species, PD whole tree, Shannon, and Simpson) (Table 2). The fungal α-evenness (Shannon, Simpson) and α-richness indices (Chao1 and PD whole tree), were significantly higher in the topsoil than in the subsoil. However, the indices declined in the topsoil but increased in the subsoil with N addition (S5 Table).

Table 2. Effects of nitrogen deposition, soil layer, and their interactions on soil bacterial (B) and fungal (F) α diversity analyzed by two-way ANOVA.

Diversity indices	Soil layer		Nitrogen treatment		Interaction
Diversity indices	F	p	F	p	F	p
B_Chao1	2.98	0.10	2.69	0.09	1.22	0.32
B_goods_coverage	3.17	0.09	4.60	0.02^*	0.72	0.50
B_observed_species	2.02	0.17	1.66	0.22	3.54	0.05^*
B_PD_whole_tree	0.001	0.97	1.57	0.24	5.80	0.01^**
B_Shannon	9.37	0.007^**	0.70	0.51	5.67	0.01^**
B_Simpson	11.85	0.003^**	0.37	0.70	3.51	0.05^*
F_Chao1	24.24	0.00^***	1.39	0.27	1.65	0.22
F_goods_coverage	23.8	0.00^***	3.58	0.04^*	1.14	0.34
F_observed_species	12.85	0.002^**	1.10	0.36	1.48	0.25
F_PD_whole_tree	13.14	0.002^**	1.10	0.35	1.81	0.19
F_Shannon	1.33	0.263	0.05	0.95	0.76	0.48
F_Simpson	1.31	0.267	0.35	0.71	0.67	0.52

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*p < 0.05

**p < 0.01

***p < 0.001.

Pearson’s correlation coefficients indicated that soil microbial α-diversity was correlated with soil properties (Fig 2A & 2B). Changes in soil bacterial α-diversity (Shannon and Simpson index) induced by N addition were most closely related to soil pH (R² = -0.47, p < 0.05; R² = -0.49, p < 0.05) and NO₃^—N, with a strong positive correlation with NO₃^—N (R² = 0.47, p < 0.05; R² = 0.40, p < 0.05) observed. Changes in soil bacterial α-richness (Chao1 index) exhibited negative and positive correlations with soil pH and SOC, respectively (R² = -0.44, p < 0.05; R² = 0.44, p < 0.05). However, it was weakly correlated with C:N, CEC, TP, and AN. The pH of the soil was negatively correlated with fungal Chao1, observed species, and PD whole tree indices, and positively correlated with the Good’s coverage index (p < 0.01). Soil properties (SMC, NH₄⁺, AN, SOC, TN, DOC) exhibited significant positive correlations with fungal diversity indices (Chao1, observed species, PD whole tree), while they exhibited a negative correlation with Good’s coverage index (p < 0.01). However, the correlations between Shannon and Simpson indices, and soil properties, were weak. Moreover, PLS-DA, which estimates the distances among multiple samples (β-diversity), revealed that soil bacterial and fungal communities differed significantly among N addition treatments (S2A–S2D Fig).

Relative abundance of dominant microbial taxa and species variation

The relative abundances of dominant bacterial and fungal phyla in the topsoil and subsoil are shown in Fig 3. The variation in bacterial and fungal species under different treatments is illustrated in S3 Fig in cladograms constructed based on LDA analysis.

In LN plots, the bacterial phyla of Proteobacteria, Acidobacteria, and Actinobacteria constituted 80.25% and 74.22% of the total sequences in the topsoil and the subsoil, respectively, followed by Firmicutes (3.47% and 2.2%, respectively), Planctomycetes (4.3% and 6.62%, respectively), and Chloroflexi (3.23% and 8.24%, respectively). In the HN plots, the bacterial phyla of Proteobacteria, Acidobacteria, and Actinobacteria constituted 76.95% and 77.25% of the total sequences in the topsoil and subsoil, respectively, followed by Firmicutes (4.89% and 0.74%, respectively), Planctomycetes (4.22% and 5.97%, respectively), and Chloroflexi (2.87% and 9.62%, respectively) (Fig 3). Among fungi, the phylum Ascomycota was the most abundant in the LN plots, constituting 46.9% and 43.29% of the sequences in the topsoil and the subsoil, respectively. Basidiomycota (41.8% and 37.5%, respectively) were also present in both soil layers. In the HN plots, Ascomycota (50.09% and 39.59%, respectively) and Basidiomycota (37.5% and 51.65%, respectively) were the most abundant phyla in the topsoil and the subsoil (Fig 3).

The relative abundance of the bacterial phyla Firmicutes and Chloroflexi increased with an increase in N addition, especially in the HN plots. At the class level, six groups varied with N addition. These include Subgroup 2 and Acidobacteria from phylum Acidobacteria, Ktedonobacteria and JG37-AG-4 from phylum Chloroflexi, Clostridia from phylum Firmicutes, and Acidimicrobiia from phylum Actinobacteria (S3A Fig).

Two fungal families exhibited changes in abundance levels in both soil layers after N addition. One was the family Myxotrichaceae, from phylum Ascomycota, and the other was unidentified. At the genus level, the relative abundance of Oidiodendron and Cortinarius, belonging to the Ascomycota and Basidiomycota phyla, respectively, were significantly decreased in both soil layers of HN plots (S3B Fig).

Bacterial and fungal community structure and correlations with environmental parameters

The results of two-dimensional RDA carried out at the OTU level (Fig 4) indicated that soil DOC was the most important parameter influencing soil bacterial community composition, and their relationship was closely correlated with the first RDA axis (Fig 4A).

RDA also revealed that soil AN and MBN from the first two constrained axes explained 36.92% of the variation in the fungal community, with the first and second axes explaining 24.54% and 12.38% of the variation, respectively. Moreover, RDA clearly showed that the soil AN and MBN concentrations were the most significant contributors to the variation in fungal communities (p < 0.01).

The Mantel test analysis of soil physicochemical properties and microbial community structure revealed that soil bacterial community structure was not significantly related to most parameters of the topsoil and the subsoil. However, bacterial community structure was significantly correlated with SMC in the subsoil (p < 0.01), and fungal community structure was significantly correlated with MBN as well as AN in the topsoil, which were consistent with the RDA results (Table 3).

Table 3. Mantel test analysis of soil physicochemical properties and soil microbial biomass and community composition in topsoil and subsoil under different nitrogen addition treatments.

environment variable	Topsoil (0–10 cm)				Subsoil (10–20 cm)
	Bacterial community composition		Fungal community composition		Bacterial community composition		Fungal community composition
	r	p	r	p	r	p	r	p
pH	0.005	0.378	0.162	0.165	-0.186	0.930	-0.063	0.700
SMC	-0.046	0.475	0.295	0.112	0.4382	0.010^**	0.101	0.256
NH₄⁺-N	-0.137	0.784	0.253	0.093	0.016	0.424	0.127	0.238
NO₃^--N	0.004	0.448	0.202	0.139	0.217	0.136	-0.136	0.739
AN	-0.048	0.543	0.307	0.041^*	0.145	0.206	0.105	0.301
DOC	-0.155	0.738	0.207	0.130	0.1401	0.253	-0.130	0.684
SOC	0.016	0.380	0.295	0.096	0.201	0.130	-0.122	0.761
TN	0.033	0.351	0.383	0.080	0.222	0.160	-0.129	0.722
TP	-0.187	0.853	-0.076	0.572	0.112	0.250	-0.149	0.736
MBC	-0.013	0.449	0.070	0.299	-0.148	0.821	-0.207	0.913
MBN	-0.142	0.853	0.306	0.018^*	-0.206	0.878	0.349	0.049^*

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SMC, soil moisture content; CEC, soil cation exchange capacity; NH4⁺-N, ammonium N; NO3^—N, nitrate N; AN, available N; DOC, dissolved organic carbon; SOC, soil organic carbon; TN, total nitrogen; TP, total phosphorus; MBC, microbial biomass carbon; MBN, microbial biomass nitrogen.

*p < 0.05

**p < 0.01.

Discussion

Effect of nitrogen addition on bacterial and fungal gene abundance and diversity

Previous studies have showed that N enrichment has positive [55, 56], neutral [47, 57], or negative [58, 59] impacts on soil microbial biomass (MBC/MBN) in tropical or subtropical forest ecosystems. Several studies have also reported that the F/B ratio increases in tropical or subtropical forest ecosystems under short-term N enrichment [33, 59, 60]. However, a recent meta-analysis illustrated that N enrichment enhanced MBC and reduced F/B in tropical or subtropical forests [28]. This suggests that there is no overall consensus regarding the effects of N addition on soil microbial diversity and biomass. In the present study, MBN was significantly increased by N addition (Table 1; p < 0.01). As soil microbes are mainly C limited [61], an increase in labile C input is expected to multiply microbial biomass. Hence, owing to CN coupling, the increase in MBN in our study is consistent with such expectations.

Previous studies have also demonstrated that N addition decreases microbial abundance and diversity [5, 24, 27, 38, 62]. In contrast, bacterial and fungal abundances in the present study were increased in the LN plots, although the increases were not significant. However, bacterial abundance estimated based on copy numbers of 16S rDNA and fungal abundance estimated based on ITS gene copy numbers were reduced in the HN plots, which is consistent with the findings of recent studies [27, 63, 64]. Pearson’s correlation analysis revealed that the abundance of bacterial 16S rDNA gene and the F/B ratio were strongly correlated with soil pH in the present study, suggesting that soil pH may be a key factor influencing microbial diversity (S3 Table). Bacterial abundance was considerable influenced by edaphic pH, potentially due to the low tolerance of most bacterial groups to variation in pH (mostly within 4−7 pH) [27, 31, 40, 65]. Soil parameters that can be drastically affected by C and N supply to the soil play a vital role in determining soil bacterial diversity in global terrestrial ecosystems [66, 67]. Previous studies have shown that soil pH is a major factor influencing microbial diversity in ecosystems [35, 42, 68, 69]. However, in the present study, DOC exerted the strongest effect on soil bacterial and fungal diversity (Fig 2), and AN significantly affected soil fungal diversity. Additionally, bacterial 16S rDNA gene abundance was closely related to AN, suggesting that N availability is a major factor regulating the abundance of soil bacteria [5, 70, 71].

Influence of nitrogen addition on bacterial and fungal community structure

In the present study, high-throughput sequencing analysis revealed that Acidobacteria, Proteobacteria, Actinobacteria, Firmicutes, and Chloroflexi are the dominant bacterial phyla present in the soils of tropical/subtropical forests, consistent with results from previous studies [5, 72]. The relative abundance of Proteobacteria and Actinobacteria decreased in the topsoil of the LN and HN plots compared to in the controls, while that of Acidobacteria and Chloroflexi increased upon N addition. These findings are not consistent with the copiotrophic hypothesis as advocated by Fierer et al. [71]. However, the relative abundance of Proteobacteria and Actinobacteria increased in the subsoil of HN plots compared to those of the controls, while that of Acidobacteria and Firmicutes decreased upon N addition. Our results with regard to the relative abundance of different phyla in the subsoil (10−20 cm) are consistent with the copiotrophic hypothesis. Moreover, no apparent divergence was observed in the relative abundance of Proteobacteria, Acidobacteria, Chloroflexi, Actinobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes between the LN and HN plots. This may be explained by the fact that soil pH remained at around 4.22−4.35 in the topsoil (Table 1), and most microbes are inhibited when pH is below 4.5 [73–75]. Since short-term N addition did not cause significant soil acidification in our experiment, we may conclude that pH was not the major factor regulating soil microbial community structure in the present study.

Actinobacteria and Proteobacteria are known to display more rapid growth rates under high C availability conditions, while Acidobacteria are oligotrophic bacteria that exist in nutrient-deficient and strongly acidic environments, and are capable of degrading recalcitrant and complex C compounds [76]. Zhang et al. [66] reported high Acidobacteria abundance under very low SOC conditions (2.66 g kg^-1). In addition, Acidobacteria have been reported to exhibit negative correlations with soil C availability [37]. The Ktedonobacteria is a distinctive class of prokaryotes that exhibits morphology similar to actinomycetes, and is thought participate in C cycling. The Ktedonobacteria class of bacteria was first reported by Cavaletti et al. [77], and classified within the phylum Chloroflexi [78], which is a diverse group of bacteria.

The class Acidimicrobiia belongs to the phylum Actinobacteria, which plays a pivotal role in the soil nutrient cycle by generating extracellular enzymes and forming symbiotic interactions with plants [79–81]. These extracellular enzymes can decompose plant litter, thus regulating C availability in the soil [82]. Several bacterial groups isolated from Actinobacteria also have a capacity to fix N and remove P from the soil [82–84]. Fungal communities are less affected by N addition than bacterial communities are, potentially because bacteria exhibit more copiotrophic characteristics [15, 29]. Soil fungal communities are a functionally diverse groups [85] that mediate numerous ecological processes and influence plant growth and soil health [86, 87]. ITS sequencing analysis results indicated that Ascomycota and Basidiomycota were the dominant fungal phyla in the acidic forest soil, supporting the copiotrophic hypothesis. Since soil nutrients decrease with an increase in soil depth, the relative abundance of Basidiomycota, which is the representative phylum of oligotrophic taxa, was lower than that of Ascomycota in the topsoil (Fig 3). Previous studies have shown that N addition enhances the relative abundance of copiotrophic phyla such as Ascomycota and reduces that of oligotrophic phyla such as Basidiomycota, which is consistent with the prediction of the copiotrophic hypothesis [34, 88–90]. The family Myxotrichaceae and the genus Oidiodendron participate in the decomposition of cellulose [91]. Although no significant impact of N addition on the soil fungal community structure was observed (Fig 3), RDA and Mantel test analysis results revealed that soil fungal community composition was related to AN and MBN in the soil (Fig 4, Table 3). The results suggest that N addition potential affects species diversity directly via increase of N availability.

Other physicochemical properties may also influence soil microbial community structure. For example, RDA analysis results suggested that soil bacterial community composition was significantly related to DOC concentrations, but not significantly correlated with soil pH in the subtropical acidic forests (Fig 4A). DOC is the most preferred nutrient by the vast majority of bacteria. Urea is the primary mode of N application, and its hydrolysis can consume H⁺ and increase pH. NH₄⁺ is often formed after urea is applied to the soil, which counteracts the H⁺ enrichment caused by NO₃^- leaching; this may why soil pH remained unchanged after short-term N application in the present study. N addition could also influence soil microbe structure indirectly by altering soil C availability, C:N, and soil pH [19, 92]. When N addition accelerates C consumption, C supply becomes a limiting factor and the rate of lignin decomposition reduces [93]. This leads to a decline in C storage in the soil, which facilitates the metabolic activities of other heterotrophic microorganisms [94]. According to the concept of resource allocation, adequate N supply can activate C-related microbial growth, resulting in the rapid putrefaction of plant cellulose-rich litter [30, 95, 96]. The N saturation hypothesis proposes that N addition may diminish the microbial demand for additional N in terrestrial ecosystems, resulting in C or P limitation for soil microorganisms [97–99]. RDA and Mantel test analysis results showed that soil fungal community composition was correlated with soil AN and MBN (p < 0.01) (Fig 4B, Table 3), probably due to the dominant role of fungi in the rapid mineralization of N as well as microbial retention of available N [100, 101].

In the present study, PLS-DA results demonstrated that fungi and bacteria inhabited disparate ecological niches and were partitioned across different treatments (S2 Fig). We believe that shifts in available C and N, rather than the alteration of soil pH, caused the changes in microbial diversity in response to N addition. The finding is consistent with those of recent studies showing that nutrient availability is mainly responsible for the responses of the soil microbial community structure to N addition [24, 25, 102].

Conclusion

Overall, in the present study, we showed that the composition, and diversity of soil bacterial and fungal communities were weakly influenced by short-term N addition. High-N addition reduced soil bacterial diversity in the subsoil and increased the relative abundance of oligotrophic bacteria in the soils of extremely acidic subtropical forests. Notably, N addition had positive impacts on some bacterial groups (e.g., Ktedonobacteria and Acidobacteria) involved in C cycling. Soil microbial community composition was also associated with soil physicochemical properties such as DOC, AN, NH₄⁺, and NO₃^-. N addition influence soil microbial community structure mainly by increasing nutrient availability rather than via edaphic acidification. Soil microbes are critical components of biogeochemical cycles, and possess the capacity to transform soil nutrients (non-available N and C) into usable forms (available N and C). Microbial community responses to N addition are long-lived and gradual processes, which may vary over time. Hence, the effects of chronic N addition on microbial communities and their interactions with biogeochemical cycles in such subtropical forest ecosystems should be examined further. Moreover, studies on the effects of N addition on biogeochemical cycles would also greatly benefit from linking such functions with microbial community structure.

Supporting information

S1 Fig

Rarefaction curves of bacterial (A) and fungal (B) α-diversity. The amount of sequencing data is deemed to be reasonable when the curves are flat and more data will produce only a small number of new species.

(ZIP)

Click here for additional data file.^{(551.4KB, zip)}

S2 Fig

Partial Least Squares Discrimination Analysis (PLS-DA) of the soil bacterial (A, B) and fungal (C, D) community structure in the topsoil and subsoil under different N addition treatments. The variation in community composition was determined based on the abundance of OTUs. CT, LN, and HN, represent the control, low, and high nitrogen addition treatments, respectively.

(ZIP)

Click here for additional data file.^{(1.3MB, zip)}

S3 Fig

Cladograms depicting bacterial (A) and fungal (B) species variation under different N addition treatments. LDA analysis was performed to obtain these cladograms.

(TIF)

Click here for additional data file.^{(4.2MB, tif)}

S1 Table. Primer sequences used in this study.

(DOCX)

Click here for additional data file.^{(15KB, docx)}

S2 Table. Effects of different N addition treatments at different soil depths based on 16S rDNA gene copy numbers, ITS gene copy numbers, and fungi-to-bacteria ratios.

(DOCX)

Click here for additional data file.^{(15.6KB, docx)}

S3 Table. Pearson’s correlations of microbial abundance with soil properties and microbial biomass.

(DOCX)

Click here for additional data file.^{(16.8KB, docx)}

S4 Table. Good’s coverage values of each group.

(DOCX)

Click here for additional data file.^{(15.7KB, docx)}

S5 Table. Effects of N addition on soil bacterial and fungal α-diversity indices.

(DOCX)

Click here for additional data file.^{(17.7KB, docx)}

Acknowledgments

We thank Prof. Zhigao Sun and Prof. Quanlin Zhong for encouraging the development of this study and to Yating Chen and Liangtai Zhou for their help in field work.

Data Availability

Data are available from the NCBI Sequence Read Archive (accession number PRJNA666625).

Funding Statement

The research was supported by the National Natural Science Foundation of China (No. 31670620) and Natural Science Foundation of Fujian Province(No. 2019J05163).

References

1.Shukla P.R.; Skea J.; Calvo Buendia E.; Masson-Delmotte V.; Pörtner H.O.; Roberts D.C.; et al. Land–climate interactions. In: Climate Change and Land: an IPCC special report on climate change, desertifcation, land degradation, sustainable land management, food security, and greenhouse gas fluxes in terrestrial ecosystems. 2019. [Google Scholar]
2.Xie L.; Zhang Q.; Cao J.; Liu X.; Xiong D.; Kong Q.; et al. Effects of warming and nitrogen addition on the soil bacterial community in a subtropical Chinese Fir plantation. Forests 2019; 10: 861. [Google Scholar]
3.Cui S.; Shi Y.; Groffman P.M.; Schlesinger W.H.; Zhu Y. Centennial-scale analysis of the creation and fate of reactive nitrogen in China (1910–2010). Proc. Natl. Acad. Sci. USA. 2013; 110: 2052–2057. 10.1073/pnas.1221638110 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Lu X.; Mao Q.; Gilliam F.S.; Luo Y.; Mo J. Nitrogen deposition contributes to soil acidification in tropical ecosystems. Glob. Chang. Biol. 2014; 20: 3790–3801. 10.1111/gcb.12665 [DOI] [PubMed] [Google Scholar]
5.Nie Y.; Wang M.; Zhang W.; Ni Z.; Hashidoko Y.; Shen W. Ammonium nitrogen content is a dominant predictor of bacterial community composition in an acidic forest soil with exogenous nitrogen enrichment. Sci. Total Environ. 2018; 624: 407–415. 10.1016/j.scitotenv.2017.12.142 [DOI] [PubMed] [Google Scholar]
6.De Vries W.; Posch M.; Simpson D.; Reinds G.J. Modelling long-term impacts of changes in climate, nitrogen deposition and ozone exposure on carbon sequestration of European forest ecosystems. Sci. Total Environ. 2017; 1097–1116, 605–606. 10.1016/j.scitotenv.2017.06.132 [DOI] [PubMed] [Google Scholar]
7.Schmitz A.; Tanja G.M.; Sanders; Bolte A.; Bussotti F.; Dirnböck T.; et al. Responses of forest ecosystems in Europe to decreasing nitrogen deposition. Environ. Pollut. 2019; 244: 980–994. 10.1016/j.envpol.2018.09.101 [DOI] [PubMed] [Google Scholar]
8.Giammarchi F. P., Panzacchi M.; Tonon V.G. Tree growth and water-use efficiency do not react in the short term to artificially increased nitrogen deposition. Forests 2020; 11, 47. [Google Scholar]
9.Stevens C.J.; Dise N.B.; Mountford J.O.; Gowing D.J. Impact of nitrogen deposition on the species richness of grasslands. Science 2004; 303:1876–1879. 10.1126/science.1094678 [DOI] [PubMed] [Google Scholar]
10.Lu X.; Mo J.; Gilliam F.S.; Zhou G.; Fang Y. Effects of experimental nitrogen additions on plant diversity in an old-growth tropical forest. Glob. Chang. Biol. 2010; 16: 2688–2700. [Google Scholar]
11.Dirnböck T.; Grandin U.; Bernhardt-Romermann M.; Beudert B.; Canullo R.; Forsius M.; et al. Forest floor vegetation response to nitrogen deposition in Europe. Glob. Chang. Biol. 2014; 20: 429–440. 10.1111/gcb.12440 [DOI] [PubMed] [Google Scholar]
12.van Dobben H.F.; De Vries W. The contribution of nitrogen deposition to the eutrophication signal in understorey plant communities of European forests. Ecol. Evol. 2017; 7: 214–227. 10.1002/ece3.2485 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.van der Linde S.; Suz L.M.; Orme C.D.L.; Cox F.; Andreae H.; Asi E.; et al. Environment and host as large-scale controls of ectomycorrhizal fungi. Nature 2018; 558: 243–248. 10.1038/s41586-018-0189-9 [DOI] [PubMed] [Google Scholar]
14.Fierer N.; Lauber C.L.; Ramirez K.S.; Zaneveld J.; Bradford M.A.; Knight R. Comparative metagenomic, phylogenetic and physiological analyses of soil microbial communities across nitrogen gradients. ISME J. 2012; 6: 1007–1017. 10.1038/ismej.2011.159 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Ramirez K.S.; Craine J.M.; Fierer N. Consistent effects of nitrogen amendments on soil microbial communities and processes across biomes. Glob. Chang. Biol. 2012; 18: 1918–1927. [Google Scholar]
16.López-Lozano N.E.; Heidelberg K.B.; Nelson W.; Garcia-Oliva F.; Eguiarte L.E. Microbial secondary succession in soil microcosms of a desert oasis in the Cuatro Cienegas Basin, Mexico. Peer J. 2013; 1–47. 10.7717/peerj.47 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Van Der Heijden M.G.; Bardgett R.D.; Van Straalen N.M. The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecol. Lett. 2008; 11: 296–310. 10.1111/j.1461-0248.2007.01139.x [DOI] [PubMed] [Google Scholar]
18.Zhou J.; Liu X.; Xie J.; Lyu M.; Zheng Y.; You Z.; et al. Nitrogen addition affects soil respiration primarily through changes in microbial community structure and biomass in a subtropical natural forest. Forests 2019; 10, 435. [Google Scholar]
19.Treseder K.K. Nitrogen additions and microbial biomass: a meta-analysis of ecosystem studies. Ecol. Lett. 2008; 11: 1111–1120. 10.1111/j.1461-0248.2008.01230.x [DOI] [PubMed] [Google Scholar]
20.Janssens I.A.; Dieleman W.; Luyssaert S.; Subke J.A.; Reichstein M.; Ceulemans R.; et al. Reduction of forest soil respiration in response to nitrogen deposition. Na. Geo. 2010; 3: 315–322. [Google Scholar]
21.Liu L.; Greaver T.L. A global perspective on belowground carbon dynamics under nitrogen enrichment. Ecol. Lett. 2010; 13: 819–828. 10.1111/j.1461-0248.2010.01482.x [DOI] [PubMed] [Google Scholar]
22.Lu M.; Zhou X.H.; Luo Y.Q.; Yang Y.H.; Fang C.M.; Chen J.K.; et al. Minor stimulation of soil carbon storage by nitrogen addition: a meta-analysis. Agr. Ecosyst. Environ. 2011; 140: 234–244. [Google Scholar]
23.Lu M.; Yang Y.H.; Luo Y.Q.; Fang C.M.; Zhou X.H.; Chen J.K.; et al. Responses of ecosystem nitrogen cycle to nitrogen addition: a meta-analysis. New Phytol. 2011; 189: 1040–1050. 10.1111/j.1469-8137.2010.03563.x [DOI] [PubMed] [Google Scholar]
24.Geisseler D., Scow K.M. Long-term effects of mineral fertilizers on soil microorganismse: a review. Soil Biol. Biochem. 2014; 75: 54–63. [Google Scholar]
25.Geisseler D.; Lazicki P.A.; Scow K.M. Mineral nitrogen input decreases microbial biomass in soils under grasslands but not annual crops. Appl. Soil Ecol. 2016; 106: 1–10. [Google Scholar]
26.Yue K.; Peng Y.; Peng C.H.; Yang W.Q.; Peng X.; Wu F.Z. Stimulation of terrestrial ecosystem carbon storage by nitrogen addition: a meta-analysis. Sci. Rep. 2016; 6, 19895 10.1038/srep19895 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Wang C.; Liu D.; Edith Bai. Decreasing soil microbial diversity is associated with decreasing microbial biomass under nitrogen addition. Soil Biol. Biochem. 2018; 120: 126–133. [Google Scholar]
28.Zhou Z.; Wang C.; Zheng M., Jiang L.; Luo Y. Patterns and mechanisms of responses by soil microbial communities to nitrogen addition, Soil Biol. Biochem. 2017; 115: 433–441. [Google Scholar]
29.Wang C., Lua X., Moria T, Mao Q, Zhou K, Zhou G.,et al. Responses of soil microbial community to continuous experimental nitrogen additions for 13 years in a nitrogen-rich tropical forest. Soil Biol. Biochem. 2018; 121: 103–112. [Google Scholar]
30.Wu J., Liu W., Zhang W., Shao Y., Duan H., Chen B., et al. Long-term nitrogen addition changes soil microbial community and litter decomposition rate in a subtropical forest. Appl.Soil Ecol. 2019; 142: 43–51. [Google Scholar]
31.Wang H., Liu S.R., Zhang X., Mao Q, Li X., You Y., et al. , Nitrogen addition reduces soil bacterial richness, while phosphorus addition alters community composition in an old-growth N-rich tropical forest in southern China. Soil Biol. Biochem. 2018; 127: 22–30. [Google Scholar]
32.Li H.; Xu Z.; Yang S.; Li X.; Top E.M.; Wang R.; et al. Responses of soil bacterial communities to nitrogen deposition and precipitation increment are closely linked with aboveground community variation. Microb. Ecol. 2016; 71: 974–989. 10.1007/s00248-016-0730-z [DOI] [PubMed] [Google Scholar]
33.Liu L.; Zhang T.; Gilliam F.S.; Gundersen P.; Zhang W.; Chen H.; et al. Interactive effects of nitrogen and phosphorus on soil microbial communities in a tropical forest. PLoS One 2013; 8, e61188 10.1371/journal.pone.0061188 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Li J.; Li Z.; Wang F.; Zou B.; Chen Y.; Zhao J.; et al. Effects of nitrogen and phosphorus addition on soil microbial community in a secondary tropical forest of China. Biol. Fert. Soils, 2015; 51: 207–215. [Google Scholar]
35.Tian D.; Jiang L.; Ma S.; Fang W.; Schmid B.; Xu L.; et al. Effects of nitrogen deposition on soil microbial communities in temperate and subtropical forests in China. Sci. Total Environ. 2017; 607: 1367–1375. 10.1016/j.scitotenv.2017.06.057 [DOI] [PubMed] [Google Scholar]
36.Ramirez K.S.; Lauber C.L.; Knight R.; Bradford M.A.; Fierer N. Consistent effects of nitrogen fertilization on soil bacterial communities in contrasting systems. Ecology, 2010; 91: 3463–3470. 10.1890/10-0426.1 [DOI] [PubMed] [Google Scholar]
37.Fierer N.; Bradford M.A.; Jackson R.B. Toward an ecological classification of soil bacteria. Ecology 2007; 88: 1354–1364. 10.1890/05-1839 [DOI] [PubMed] [Google Scholar]
38.Ling N.; Chen D.; Guo H.; Wei J.; Bai Y.; Shen Q.; et al. Differential responses of soil bacterial communities to long-term N and P inputs in a semi-arid steppe. Geoderma, 2017; 292: 25–33. [Google Scholar]
39.Albornoz F. E.; Teste F. P.; Lambers H.; Bunce M.; Murray D. C.; White N. E.; et al. Changes in ectomycorrhizal fungal community composition and declining diversity along a 2‐million‐year soil chronosequence. Mol.Ecol. 2016; 25: 4919–4929. 10.1111/mec.13778 [DOI] [PubMed] [Google Scholar]
40.Lauber C.L.; Hamady M.; Knight R.; Fierer N. Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl. Environ. Microb. 2009; 75 (15): 5111–5120. 10.1128/AEM.00335-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Rousk J.; Bååth E.; Brookes P.C.; Lauber C.L.; Lozupone C.; Caporaso J.G.; et al. Soil bacterial and fungal communities across a pH gradient in an arable soil. ISME J. 2010; 4 (10): 1340–1351. 10.1038/ismej.2010.58 [DOI] [PubMed] [Google Scholar]
42.Stone M.M.; Kan J.; Plante A.F. Parent material and vegetation influence bacterial community structure and nitrogen functional genes along deep tropical soil profiles at the Luquillo Critical Zone Observatory. Soil Biol. Biochem. 2015; 80: 273–282. [Google Scholar]
43.Zeng J.; Liu X.; Song L.; Lin X.; Zhang H.; Shen C.; et al. Nitrogen fertilization directly affects soil bacterial diversity and indirectly affects bacterial community composition. Soil Biol. Biochem. 2016; 92: 41–49. [Google Scholar]
44.Zhu J.; He N.; Wang Q.; Yuan G.; Wen D.; Yu G.; et al. The composition, spatial patterns, and influencing factors of atmospheric wet nitrogen deposition in Chinese terrestrial ecosystems. Sci. Total Environ. 2015; 511: 777–785. 10.1016/j.scitotenv.2014.12.038 [DOI] [PubMed] [Google Scholar]
45.Ji Zhao, Chuankang Chen. Chinese geography. 1st ed Higher Education Press; 2004. [Google Scholar]
46.Fan Y.X., Zong X.J., Lin F., Liu C.C., Wang M.H., Chen G.S., et al. Responses of soil phosphorus fractions after nitrogen addition in a subtropical forest ecosystem: insights from decreased Fe and Al oxides and increased plant roots. Geoderma 2019, 337: 246–255. [Google Scholar]
47.Turner BL.; Wright JS. The response of microbial biomass and hydrolytic enzymes to a decade of nitrogen, phosphorus, and potassium addition in a lowland tropical rain forest. Biogeochemistry, 2014; 117: 115–130. [Google Scholar]
48.Vance E.D.; Brookes P.C.; Jenkinson D.S. An extraction method for measuring soil microbial biomass C. Soil Biol. Biochem. 1987; 19: 703–707. [Google Scholar]
49.Brookes P.C.; Landman A.; Pruden G.; Jenkinson D.S. Chloroform fumigation and the release of soil-nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. Biochem. 1985; 17: 837–842. [Google Scholar]
50.Munyaka P. M.; Eissa N.; Bernstein C. N. Antepartum Antibiotic Treatment Increases offspring susceptibility to experimental colitis: A role of the gut microbiota. PLoS One. 2015; 10 (11): e0142536 10.1371/journal.pone.0142536 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Edgar R.C. UPARSE: Highly accurate OTU sequences from microbial amplicon reads, Nature Methods. 2013. 10.1038/nmeth.2604 [DOI] [PubMed] [Google Scholar]
52.Cole J. R.; Wang Q.; Cardenas E.; Fish J.; Chai B.; Farris R. J.; et al. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 2009; 37: 141–145. 10.1093/nar/gkn879 [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Wang Y.; Sheng H.; He Y.; Wu J.; Jiang Y.; Tam N. F.; et al. Comparison of the levels of bacterial diversity in freshwater, intertidal wetland, and marine sediments by using millions of illumina tags. Appl. Environ. Microbiol. 2012; 78 (23): 8264 10.1128/AEM.01821-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Jiang X.T., Peng X., Deng G.; Sheng H.; Wang Y.; Zhou H.; et al. Illumina sequencing of 16S rRNA tag revealed spatial variations of bacterial communities in a mangrove wetland. Microb. Ecol. 2013; 66: 96–104. 10.1007/s00248-013-0238-8 [DOI] [PubMed] [Google Scholar]
55.Cusack D.F.; Silver W.L.; Torn M.S.; Burton S.D.; Firestone M.K. Changes in microbial community characteristics and soil organic matter with nitrogen additions in two tropical forests. Ecology 2011; 92: 621–632. 10.1890/10-0459.1 [DOI] [PubMed] [Google Scholar]
56.Li Y.Q.; Xu M.; Zou X.M. Effects of nutrient additions on ecosystem carbon cycle in a Puerto Rican tropical wet forest. Glob. Chang. Biol. 2006; 12: 284–293. [Google Scholar]
57.Fanin N.; Hättenschwiler S.; Schimann H.; Fromin N.; Bailey J.K. Interactive effects of C, N and P fertilization on soil microbial community structure and function in an Amazonian rain forest. Funct. Ecol. 2015; 29: 140–150. [Google Scholar]
58.Peng Y.; Chen G.; Chen G.; Li S.; Peng T.; Qiu X.; et al. Soil biochemical responses to nitrogen addition in a secondary evergreen broad-leaved forest ecosystem. Scientific Reports 2017; 7, 2783 10.1038/s41598-017-03044-w [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Li Y.; Sun J.; Tian D.; Wang J.; Ha D.; Qu Y.; et al. Soil acid cations induced reduction in soil respiration under nitrogen enrichment and soil acidification. Sci. Total Environ. 2018; 615: 1535–1546. 10.1016/j.scitotenv.2017.09.131 [DOI] [PubMed] [Google Scholar]
60.Li J.; Li Z.; Wang F.; Zou B.; Chen Y.; Zhao J.; et al. Effects of nitrogen and phosphorus addition on soil microbial community in a secondary tropical forest of China. Biol. Fertil. Soils 2015; 51: 207–215. [Google Scholar]
61.Fierer N.; Strickland M.S.; Liptzin D.; Bradford M.A.; Cleveland C.C. Global patterns in belowground communities. Ecol. Letters 2009; 12: 1238–1249. 10.1111/j.1461-0248.2009.01360.x [DOI] [PubMed] [Google Scholar]
62.Zhou Z.H.; Wang C.K. Reviews and syntheses: soil resources and climate jointly drive variations in microbial biomass carbon and nitrogen in China’s forest ecosystems. Biogeosciences, 2015; 12: 6751–6760. [Google Scholar]
63.Wang Q.; Wang C.; Yu W.W.; Turak A.; Chen D.; Huang Y.; et al. Effects of Nitrogen and Phosphorus Inputs on Soil Bacterial Abundance, Diversity, and Community Composition in Chinese Fir Plantations, Front. Microbiol. 2018; 9: 1543. [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Huang Y.; Kang R.; Mulder J.; Zhang T.; Duan L. Nitrogen saturation, soil acidification, and ecological effects in a subtropical pine forest on acid soil in southwest China. J. Geophys. Res-Biogeo. 2015; 120: 2457–2472 [Google Scholar]
65.Dai Z.; Su W.; Chen H.; Barberán A.; Zhao H.; Yu M.; et al. Long-term nitrogen fertilization decreases bacterial diversity and favors the growth of Actinobacteria and Proteobacteria in agro-ecosystems across the globe. Glob. Chang. Biol. 2018; 24 (8): 3452–3461. 10.1111/gcb.14163 [DOI] [PubMed] [Google Scholar]
66.Zechmeister-Boltenstern S.; Keiblinger K.M.; Mooshammer M.; PENuelas J.; Richter A.; Sardans J.; et al. The application of ecological stoichiometry to plant–-microbial–soil organic matter transformations. Ecol. Monogr. 2015; 85: 133–155. [Google Scholar]
67.Zhang C.; Liu G.B.; Xue S.; Wang G.L. Soil bacterial community dynamics reflect changes in plant community and soil properties during the secondary succession of abandoned farmland in the Loess Plateau. Soil Biol. Biochem. 2016; 97: 40–49. [Google Scholar]
68.Zhang X.; Liu W.; Zhang G.; Jiang L.; Han X. Mechanisms of soil acidification reducing bacterial diversity. Soil Biol. Biochem. 2015; 81: 275–281. [Google Scholar]
69.Zhang H.; Wang L.; Liu H.; Zhao J.; Li G.; Wang H.; et al. Nitrogen deposition combined with elevated precipitation is conducive to maintaining the stability of the soil fungal diversity on the Stipa baicalensis steppe. Soil Biol. Biochem. 2018; 117: 135–138. [Google Scholar]
70.Zhou J.; Guan D. W.; Zhou B. K.; Zhao B. S.; Ma M. C.; Qin J.; et al. Influence of 34-years of fertilization on bacterial communities in an intensively cultivated black soil in northeast China. Soil Biol. Biochem. 2015; 90: 42–51. [Google Scholar]
71.Wang Q. F.; Jiang X.; Guan D.; Wei D.; Zhao B.; Ma M.; et al. Long-term fertilization changes bacterial diversity and bacterial communities in the maize rhizosphere of Chinese Mollisols. Appl. Soil Ecol. 2018; 125: 88–96. [Google Scholar]
72.Cui J.; Wang J.; Xu J.; Xu C.; Xu X. Changes in soil bacterial communities in an evergreen broad-leaved forest in east China following 4 years of nitrogen addition. J. Soils Sediments, 2017; 17: 2156–2164. [Google Scholar]
73.Högberg M.N.; Högberg P.; Myrold D.D. Is microbial community composition in boreal forest soils determined by pH, C-to-N ratio, the trees, or all three? Oecologia 2007; 150: 590–601. 10.1007/s00442-006-0562-5 [DOI] [PubMed] [Google Scholar]
74.Rousk J.; Brookes P.C.; Bååth E. Fungal and bacterial growth responses to N fertilization and pH in the 150-year ‘Park Grass’ UK grassland experiment. Fems Microbiol.Ecol. 2011; 76: 89–99. 10.1111/j.1574-6941.2010.01032.x [DOI] [PubMed] [Google Scholar]
75.Chen D. M.; Lan Z. C.; Bai X.; Grace J.B.; Bai Y. F. Evidence that acidification-induced declines in plant diversity and productivity are mediated by changes in belowground communities and soil properties in a semi-arid steppe. J. Ecol. 2013; 101: 1322–1334. [Google Scholar]
76.Ai C.; Liang G.; Sun J.; Wang X.; He P.; Zhou W.; et al. Reduced dependence of rhizosphere microbiome on plant-derived carbon in 32-year long-term inorganic and organic fertilized soils. Soil Biol. Biochem. 2015; 80: 70–78. [Google Scholar]
77.Cavaletti L.; Monciardini P.; Bamonte R.; Schumann P.; Rohde M.; Sosio M.; et al. New lineage of filamentous, spore-forming, gram-positive bacteria from soil. Appl. Environ. Microbiol. 2006; 72: 4360–4369. 10.1128/AEM.00132-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
78.Yabe S.; Aiba Y.; Sakai Y.; Hazaka M.; and Yokota A. Thermosporothrix hazakensis gen. nov., sp. nov., isolated from compost, description of Thermosporotrichaceae fam. nov. within the class Ktedonobacteria Cavaletti et al. 2007 and emended description of the class Ktedonobacteria. Int. J. Syst. Evol. Microbiol. 2010; 60: 1794–1801. 10.1099/ijs.0.018069-0 [DOI] [PubMed] [Google Scholar]
79.Albertsen M.; Hansen L.B.S.; Saunders A.M.; Nielsen P.H.; Nielsen K.L. A metagenome of a full-scale microbial community carrying out enhanced biological phosphorus removal. ISME J. 2012; 6: 1094 10.1038/ismej.2011.176 [DOI] [PMC free article] [PubMed] [Google Scholar]
80.Sellstedt A.; Richau K.H. Aspects of nitrogen-fixing Actinobacteria, in particular free-living and symbiotic Frankia. FEMS Microbiol. Lett. 2013; 342: 179–186. 10.1111/1574-6968.12116 [DOI] [PubMed] [Google Scholar]
81.Vetrovsky T.; Steffen K.T.; Baldrian P. Potential of cometabolic transformation of polysaccharides and lignin in lignocellulose by soil Actinobacteria. PLoS One 2014; 9: e0089108 10.1371/journal.pone.0089108 [DOI] [PMC free article] [PubMed] [Google Scholar]
82.Anderson I.; Abt B.; Lykidis A.; Klenk H.P.; Kyrpides N.; Ivanova N. Genomics of aerobic cellulose utilization systems in actinobacteria. PLoS One 2012; 7: e0039331 10.1371/journal.pone.0039331 [DOI] [PMC free article] [PubMed] [Google Scholar]
83.Pujic P.; Bolotin A.; Fournier P.; Sorokin A.; Lapidus A.; Richau K.H.; et al. Genome sequence of the atypical symbiotic Frankia R43 strain, a nitrogen-fixing and hydrogen-producing actinobacterium. Genome Announcements 2015; 3: 01387–01315. 10.1128/genomeA.01387-15 [DOI] [PMC free article] [PubMed] [Google Scholar]
84.Nouioui I.; Ghodhbane-Gtari F.; del Carmen Monterro-Calasanz M.; Rohde M.; Tisa L.S.; Gtari M.; et al. Frankia inefficax sp. nov., an actinobacterial endophyte inducing ineffective, non nitrogen-fixing, root nodules on its actinorhizal host plants. Antonie van Leeuwenhoek 2017; 110: 313–320. 10.1007/s10482-016-0801-7 [DOI] [PubMed] [Google Scholar]
85.Paungfoo-Lonhienne C.; Yeoh Y.K.; Kasinadhuni N.R.P.; Lonhienne T.G.A.; Robinson N.; Hugenholtz P.; et al. Nitrogen fertilizer dose alters fungal communities in sugarcane soil and rhizosphere. Scientific Reports 2015; 5: 8678 10.1038/srep08678 [DOI] [PMC free article] [PubMed] [Google Scholar]
86.Klaubauf S.; Inselsbacher E.; Zechmeister-Boltenstern S.; Wanek W.; Gottsberger R.; Strauss J.; et al. Molecular diversity of fungal communities in agricultural soils from Lower Austria. Fungal Diversity 2010; 44: 65–75. 10.1007/s13225-010-0053-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
87.Chen W.; Xu R.; Wu Y.; Chen J.; Zhang Y.; Hu T.; et al. Plant diversity is coupled with beta not alpha diversity of soil fungal communities following N enrichment in a semi-arid grassland. Soil Biol. Biochem. 2018; 116: 388–398. [Google Scholar]
88.She W.; Bai Y.; Zhang Y.; Qin S.; Feng W.; Sun Y.; et al. Resource availability drives responses of soil microbial communities to short-term precipitation and nitrogen addition in a desert shrubland. Front. Microbiol. 2018; 9: 186 10.3389/fmicb.2018.00186 [DOI] [PMC free article] [PubMed] [Google Scholar]
89.Yao F.; Yang S.; Wang Z.; Wang X.; Ye J.; Wang X.; et al. Microbial taxa distribution is associated with ecological trophic cascades along an elevation gradient. Front. Microbiol. 2017; 8: 2071 10.3389/fmicb.2017.02071 [DOI] [PMC free article] [PubMed] [Google Scholar]
90.Niu S.; Classen AT.; Dukes JS.; Kardol P.; Liu L.; Luo Y.; et al. Global patterns and substrate based mechanisms of the terrestrial nitrogen cycle. Ecol. Letters 2016; 19(6): 697–709. 10.1111/ele.12591 [DOI] [PubMed] [Google Scholar]
91.DALPÉ Y., Ericoid mycorrhizal fungi in the Myxotrichaceae and Gymnoascaceae. New Phytologist 1989; 113(4): 523–527. [Google Scholar]
92.Serna-Chavez H.M.; Fierer N.; van Bodegom P.M. Global drivers and patterns of microbial abundance in soil. Global Ecol. and Biogeogr. 2013; 22: 1162–1172. [Google Scholar]
93.Magill A.H.; Aber J.D. Long-term effects of experimental nitrogen additions on foliar litter decay and humus formation in forest ecosystems. Plant Soil 1998; 203: 301–311. [Google Scholar]
94.Zhang T.; Luo Y.; Chen H. Y. H.; Ruan H. Responses of litter decomposition and nutrient release to N addition: A meta-analysis of terrestrial ecosystems. Appl. Soil Ecol. 2018; 128: 35–42. [Google Scholar]
95.Allison SD.; Weintraub MN.; Gartner TB.; Waldrop MP. Evolutionary-economic principles as regulators of soil enzyme production and ecosystem function. In: Shukla G,Varma A(eds) Soil enzymology, 1st edn Springer, Berlin, 2011; pp 229–243. [Google Scholar]
96.Carreiro MM.; Sinsabaugh RL.; Repert DA.; Parkhurst DF. Microbial enzyme shifts explain litter decay responses to simulated nitrogen deposition. Ecology 2000; 81, 2359–2365. [Google Scholar]
97.Aber J.D.; Nadelhoffer K.J.; Steudler P.; Melillo J.M. Nitrogen saturation in northern forest ecosystems. Bioscience 1989; 39: 378–386. [Google Scholar]
98.Gress S.E.; Nichols T.D.; Northcraft C.C.; Peterjohn W.T. Nutrient limitation in soils exhibiting differing nitrogen availabilities: what lies beyond nitrogen saturation? Ecology 2007; 88: 119 10.1890/0012-9658(2007)88[119:nlised]2.0.co;2 [DOI] [PubMed] [Google Scholar]
99.Kopáček J.; Cosby B.; Evans C.; Hruška J.; Moldan F.; Oulehle F.; et al. Nitrogen, organic carbon and sulphur cycling in terrestrial ecosystems: linking nitrogen saturation to carbon limitation of soil microbial processes. Biogeochemistry 2013; 115: 33–51. [Google Scholar]
100.Rousk J.; Frey S.D. Revisiting the hypothesis that fungal-to-bacterial dominance characterizes turnover of soil organic matter and nutrients. Ecol. Monogr. 2015; 85: 457–472. [Google Scholar]
101.Zhou J.; Jiang X.; Zhou B.; Zhao B.; Ma M.; Guan D.; et al. Thirty-four years of nitrogen fertilization decreases fungal diversity and alters fungal community composition in black soil in northeast China. Soil Biol. Biochem. 2016; 95: 135–143. [Google Scholar]
102.Farrer E.C.; Suding K.N. Teasing apart plant community responses to N enrichment: the roles of resource limitation, competition and soil microbes. Ecol. Letters 2016; 19: 1287–1296. [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0246263.r001

Decision Letter 0

Julian Aherne

17 Sep 2020

PONE-D-20-10592

Nutrient availability is a dominant predictor of soil bacterial and fungal community composition after nitrogen addition in subtropic al acidic forest

PLOS ONE

Dear Dr. Chen,

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: No

Reviewer #3: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study compared soil microbial community in forest soil under different N treatments. The authors found N addition only decreased bacteria α-diversity in subsoil (Note: contradictory statement L37 & L255) and soil nutrients rather than pH plays a larger role in determining community composition. While I think the data could be useful, the manuscript in its current form needs some revision before acceptation. I hope the suggestions below help the authors improve the manuscript.

-The introduction of the manuscript is well written. But I found some recent published long-term studies on N fertilization in tropical/subtropical forests were not included. Particularly, a paragraph focusing on microbial community shift caused by N addition in subtropical forests could be added. I attach a few references and hope they can be incorporated into introduction/discussion.

Wang et al., (2018) https://doi.org/10.1016/j.soilbio.2018.08.022

Wang et al., (2018) https://doi.org/10.1016/j.soilbio.2018.03.009

Wu et al., (2019) https://doi.org/10.1016/j.apsoil.2019.05.014

-Methods did not describe measurement of CEC.

-For discussion of diversity, an explanation of why only bacteria diversity was affected in subsoil is needed. Particularly I found attributing diversity change to pH (L334) WAS NOT reasonable as I found it contradictory that HN treatment decreased bacteria α-diversity in subsoil significantly (Table S5), but subsoil pH did not show significant difference in CK and HN plots. Instead, subsoil pH was significant different between CK and LN plots (Table 1). And in L395 authors stated pH was not changed making it more confusing.

-For discussion of community structure, does the community structure change somehow reflect observed functions? For instance, the observed decreased NH4 and increased NO3 in LN plots suggest potential high nitrification rate.

-L428-431 I found the concluding remark is quite general, a stronger take home message is preferred. Also a lot of studies have studied biogeochemical cycling functions under N addition, it would make a more significant contribution by connecting functions with community structure.

-The writing needs substantial work. I encourage the authors to thoroughly check the manuscript to avoid grammatical mistakes and incomplete sentences. I also suggest authors remake some tables and figures. It is very hard to read figure legends in its current layout (e.g., Fig 1-3, Fig S1-3). Fig.2 does not have legend and caption does not provide enough information to interpret figure. Fig.4 does not have a legend and data labels have inconsistent font, also these labels make the figure messy. In Table 1, use superscript letters to avoid incomplete post-hoc results. Also I am very concerned about the reported soil moisture content (SMC<1%), authors should double check their data.

-L787-789 correlation?

-Please include all data (prior to statistical analysis) in supplement or deposit to a public repository for review.

Reviewer #2: The paper by Cui et al. reveals soil microbial diversity and community structure under nitrogen (N) addition in a subtropical forest. The experiment is well conducted, but the present paper does not write well. "Introduction" and "Discussion" are not well setting out, and some conclusions are not suitable. Especially, the conclusion in "Abstract" that soil available N was significantly decreased under N addtion is not exact. In fact, soil available N was not decreased under high N addition according to their results. The duration of the experiment is relatively short (1 year) and the mean annual precipitation on this study site is relatively high (1800 mm). Soil N leaching may be high, and thus influenced results of soil available N based on one sampling time. I suggest the authors polish the English writing.

Abstract

Line 36, not exact.

Line 40, add a sentence about the results' indication.

Introduction

The "Introduction" is lack of continuity in logic, and does not show how scientific questions are proposed. Some information should be given in " Discussion".

Line 62-73, should be rewritten.

Material and methods

Line 107, subtropical. It is...

Line 109, "Pinus taiwanensis" should be in italic.

Line 211, the typeface is not consistent.

Results

Line 218-228, the results should show the data rather than description.

Line 223-224, the sentences should be rewritten. Is soil available N the sum of NH4+-N and NO3--N? Some values are the sum, and others are not in Table 1.

Line 229, as a main result, it is not suitable that tables and figures in this section are not present in the main text. In fact, I think the results of soil bacterial and fungal gene copy numbers can be deleted in the text. Because the gene copy numbers are determined by the PCR cycles. The values are not in situ absolute values.

Table 1, data should be shown as mean ± SE/SD.

Discussion

Line 314-320, what indications of these pevious studies for this study?

Line 325-331, the "Discussion" should not repeat results simply.

Reviewer #3: Cui et al. investigated soil microbial responses to one-year N addition treatments and identified the dominant factors. I believe the data is sufficient and the overall structure of the manuscript is clear. However, there is a big problem with writing. My major concerns are as follows. See the detailed comments in the attached pdf.

1, I can understand what the authors have written, but there are a lot of grammatic errors in this manuscript.

2, Although I like the three questions asked at the end of the Introduction, the section has not been well developed. I understand the structure of the section. Yet sentences within a paragraph were not tightly linked or well organized. Besides, the knowledge gap was not well developed and thus the significance of the work seems less appealing to me.

3, I encourage the authors to further explore the relationships among microbial communities and environmental parameters. Right now, they only used RDA to identify important variables for overall community composition. I cannot see the direction of the effects of these variables on specific microbial groups.

4, The whole Discussion was terribly written. It was superficial and discrete most of the time. I really hope the authors could rewrite the whole section.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachment

Submitted filename: PONE-D-20-10592_reviewer.pdf

Click here for additional data file.^{(1MB, pdf)}

PLoS One. 2021 Feb 23;16(2):e0246263. doi: 10.1371/journal.pone.0246263.r002

Author response to Decision Letter 0

26 Oct 2020

Response to Reviewer’s Comments

Editor’s Review

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response: The manuscript has been revised with for consistency with Plos One guidelines for style. We have also provided figures and supplementary information in separate files according to guidelines and placed figure legends and tables at the appropriate places within the text. The writing has been substantially improved to meet your quality standards.

https://peerj.com/articles/7631/

https://www.sciencedirect.com/science/article/abs/pii/S0048969717335684?via%3Dihub

We will carefully review your manuscript upon resubmission, so please ensure that your revision is thorough.

Response: Thank you for your comments. We have rewritten the introduction and discussion and revised passages that may be similar to text from previous publications.

3.In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

Response: The corresponding author, Professor Yuemin Chen, has established a stable relationship with Daiyunshan Nature Reserve for cooperation in scientific research, and accordingly established an expert workstation within this reserve. Additionally, a member of the Administration Bureau of the Daiyunshan Nature Reserve is a co-author on this paper. Therefore, no permits were required for access to the field site. The sentence is on Page 9, Lines 145-146.

4.In your Methods section, please provide additional location information, including geographic coordinates for the data set if available.

Response: We have added accurate information about the longitude and latitude for the location on Page 8, Lines 125-127.

Response: Thank you for your comments. Figure 1 is drawn by Zeng Yaozhong using cartographic software (ArcGIS 10.2, Esri, USA). The map data is DEM elevation data downloaded from the geospatial data cloud of China.

Website: http://www.gscloud.cn/home#page1/4

6.We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing.

Response: Thank you for your comments. We have uploaded the data as requested in NCBI Sequence Read Archive. The accession number that can be used to access these SRA data is PRJNA666625 and the SRP ID is SRP286064.

7.Please upload a new copy of Figure 1 as the detail is not clear. Please follow the link for more information: https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/

Response: Thank you for your comment. Figure 1 has been recreated to ensure that the details are clear.

Response to Reviewer’s Comments Reviewer # 1

The authors would like to thank you for the valuable suggestions. The manuscript has been revised with due attention to your comments. The writing has been substantially improved to meet your publication quality standards.

Specific Comments:

Wang et al., (2018) https://doi.org/10.1016/j.soilbio.2018.08.022

Wang et al., (2018) https://doi.org/10.1016/j.soilbio.2018.03.009.

Wu et al., (2019) https://doi.org/10.1016/j.apsoil.2019.05.014

Response: We have revised the introduction according to your comments and have cited the suggested references [29-31]. We have also added a paragraph focusing on microbial community shift caused by N addition in subtropical forests. These revisions can be found on Page 5-6, Lines 74-90.

-Methods did not describe measurement of CEC.

Response: We have added details regarding the measurement of CEC in the Materials and Methods section. Please see the revised text on Page 11, lines 181-182.

Response: Thank you for the valuable comments. We have revised this paragraph as suggested. We have mentioned that DOC exerted the strongest effect on soil bacterial and fungal diversity, while pH does not appear to be as closely related to bacterial diversity. Please see the revised text on Page 25, Lines 419-420.

Response: Thank you for the valuable comments. We have revised this statement as suggested and incorporated discussion regarding whether the changes in the nutrients may reflect differences in microbial community functions such as nitrification rate. Please see the revised text on Pages 28-29, Lines 478-499.

Response: We appreciate your suggestion. We have added a concluding paragraph giving a strong take-home message. Briefly, we mention that soil microbial community is affected by N addition, especially increasing oligotrophic bacteria and some microbial groups that play major roles in nutrient cycles. We have also highlighted the need for further studies connecting microbial community structure and functions and their interaction with biogeochemical cycles when subjected to N addition. Please see the revised text on Pages 29-30, Lines 508-524.

Response: Thank you for the valuable comments. We have remade some tables and figures and added detailed figure legends and data labels. We have also attempted to make figure 4 less messy and maintain consistency in font. We have also used superscript letters for depicting post-hoc results in Table 1. Please see the revised figures 1-4 and tables 1-3. Regarding the soil moisture content, we inaccurately reported proportions instead of percentages and have now rectified this error.

-L787-789 correlation?

Response: We have revised this statement as suggested by the reviewer. Please see the revised text on Page 18, Lines 300 and refer to Tables 2 and S3 Table.

-Please include all data (prior to statistical analysis) in supplement or deposit to a public repository for review.

Response: We have uploaded the data as requested in the NCBI Sequence Read Archive. Accession number of this SRA data is PRJNA666625 and SRP ID is SRP286064.

Response to Reviewer’s Comments Reviewer # 2

We thank the reviewer for the useful comments. We have extensively revised the manuscript according to your suggestions. We hope you will find the revised manuscript to be suitable for publication in Plos one.

Abstract

Line 36, not exact.

Response: Thank you for pointing this out. We have rewritten this sentence to clearly mention that soil dissolved organic C (DOC) and available N (AN) were significantly decreased after N additions in both topsoil and subsoil. Please see the revised text on Page 3, Lines 42-43.

Line 40, add a sentence about the results' indication.

Response: Thank you for your suggestion. We have added the sentence “This experiment provides further evidence in support of shifts in the diversity and structure of soil microbial communities in acidic subtropical forests in response to increasing nitrogen deposition.” Please see the revised text on Page 4, Lines 49-51.

Introduction

The "Introduction" is lack of continuity in logic, and does not show how scientific questions are proposed. Some information should be given in " Discussion". Line 62-73, should be rewritten.

Response: The authors would like to thank the reviewer for the valuable comments. The paragraph mentioned by the reviewer has been rewritten. We have added a paragraph focusing on microbial community shift caused by N addition in subtropical forests. Subsequently, we have mentioned the scientific questions and the accompanying hypotheses that we attempted to address. We hope that the revised version of the manuscript has better logical continuity.

Material and methods

Line 107, subtropical. It is...

Response: We have revised this statement as suggested. Please see the revised text on Page 8, Lines 128.

Line 109, "Pinus taiwanensis" should be in italic.

Response: Thank you for your advice. We have italicized all instances where this species is mentioned as suggested. Please see the revised text on Page 8, Lines 130-133.

Line 211, the typeface is not consistent.

Response: Thank you for identifying this inconsistency. We have ensured consistency in the font and spacing throughout the manuscript.

Results

Line 218-228, the results should show the data rather than description.

Response: Thank you for your comment. We have modified the results section accordingly to include data. Please see the revised text on Page 14, Lines 258-261.

Line 223-224, the sentences should be rewritten. Is soil available N the sum of NH4+-N and NO3--N? Some values are the sum, and others are not in Table 1.

Response: We have revised this statement as suggested. Available N is the sum of ammonium and nitrate and we have verified the values of these parameters. Please see the revised text on Page 10, 14 Lines 176-177, 262 and the new Table 1.

Response: This paragraph has been deleted in the text according to the reviewer’s comment. We have now used copy numbers only to provide an estimate of relative abundance of different species. Please see the revised text on Page 13, Lines 262-263.

Table 1, data should be shown as mean ± SE/SD.

Response: We have revised the data as suggested. Please see the new Table 1.

Discussion

Line 314-320, what indications of these previous studies for this study?

Response: These previous studies indicated that there is no consensus on the response of soil microbial biomass to nitrogen addition in subtropical and tropical forest ecosystems. We have added such a statement in the discussion section. Please see the revised text on Page 24, Lines 399-400.

Line 325-331, the "Discussion" should not repeat results simply.

Response: This descriptive sentence has been deleted from the text and the possible implications of the results have been discussed instead. Please see the revised text on Pages 24-25, Lines 410-422.

Response to Review Comments Reviewer # 3

The authors would like to thank you for the insightful comments. The manuscript has been substantially revised with careful consideration of your suggestions. I hope you will find the revised manuscript to be suitable for publication in Plos One.

1.I can understand what the authors have written, but there are a lot of grammatic errors in this manuscript.

Response: The authors would like to thank you for the valuable comments. The manuscript has been rewritten and reviewed to ensure grammatical correctness.

2. Although I like the three questions asked at the end of the Introduction, the section has not been well developed. I understand the structure of the section. Yet sentences within a paragraph were not tightly linked or well organized. Besides, the knowledge gap was not well developed and thus the significance of the work seems less appealing to me.

Response: Thank you for your constructive comments. We have added a paragraph focusing on microbial community shifts caused by N addition in subtropical forests. Subsequently, we have presented our scientific questions and corresponding hypotheses. We hope that the organization of the text and the continuity of ideas have been improved in this version of the manuscript. I have added to the end of the introduction (beginning of last paragraph) the following sentence: “Although the effects of N addition have been studied, it is not clear if the changes in microbial community are mediated by direct increases in N availability, changes in the nutrient composition or acidification of the soil.” Please see the revised text on Page 5-6, Lines 74-90 and Page 7, Lines 105-108.

3. I encourage the authors to further explore the relationships among microbial communities and environmental parameters. Right now, they only used RDA to identify important variables for overall community composition. I cannot see the direction of the effects of these variables on specific microbial groups.

Response: Thank you for your constructive comments. We have now used the Mantel test analysis to understand the association of the soil environmental factors with the composition of soil microbial communities. Please see the added test results on Page 22, Lines 379-384.

4.The whole Discussion was terribly written. It was superficial and discrete most of the time. I really hope the authors could rewrite the whole section.

Response: We have rewritten the entire discussion section as suggested by the reviewer. We hope there is greater depth and logical continuity in this revised version.

Minor Revisions:

1.line27: You could clarify sampling time here.

Response: We have revised this sentence to include the sampling time. Please see the revised text on Page 3, line 32.

2.Line29: It would be more clear to specify how long had the N addition lasted before sampling for microbial analyses?

Response: We have revised this statement as suggested. We sampled the soil sampling one year after N addition. Please see the revised text on Page 3, line 39.

3.Line35: Spell the full word when it first appears.

Response: We have ensured that all abbreviations have been expanded at first mention. We have clarified that DOC refers to soil dissolved organic carbon. Please see the revised text on Page 3, line 42-43.

4.Line37-39: This result is too general. Clarify what variable is important for bacterial and fungal communities, respectively.

Response: We found that DOC is most important for bacterial communities while AN is most important for fungal communities. We have modified the statement to include these details. Please see the revised text on Page 3, lines 45-47.

5.Line50: Why is this negative?

Response: Thank you for your question. Nitrogen addition has resulted in the reduction of understory vegetation. Therefore, this value is negative. This has been clarified. Please see the revised text on Page 5, lines 62.

6.Line60: Why would you use additionally here? Do you have another reason explaining why N addition could increase MBC in subtropical forests?

Response: We have removed the word “additionally” since it was not appropriate, as pointed out by the reviewer. Please see the revised text on Page 5, line 72.

7.Line62: You should give this paragraph a topic sentence.

Response: We have added the following sentence: N addition can also induce changes in soil microbial α-diversity and microbial community composition. Please see the revised text on Page 5, line 74.

8.Line79: This sentence is not clear. Do you mean absolute abundance of major microbial groups were positively correlated with nutrient concentrations?

Response: Yes, we have rewritten the sentence for clarity. Please see the revised text on Page 7, line 99-100.

9.Line82: This sentence should go to the beginning of the paragraph.

Response: We have moved this sentence as suggested. Please see the revised text on Page 6, line 91.

10.Line89: I am quite confused by the first two sentences. What is your point here? Besides, many studies have investigated microbial responses to nutrient addition.

Response: We have deleted these two sentences. Please see the revised text on Page 7.

11.Line97-101: I like these questions. They are well linked.

Response: Thank you for your appreciation.

12.Line108: citation?

Response: This sentence was quoted [45] from Chinese geography textbook. Author: Zhao Ji, Chen Chuankang. Press: Higher Education Press.

13.Line119: Do these plots have similar elevations, topography/slope, and facing directions? I believe these factors all affect microbial communities. I know it may be not easy to find a big area with all the above-mentioned factors uniform on mountains. But the authors should introduce these conditions.

Response: The experimental plots are close to the top of the mountain, face the same direction, and have similar slopes and elevations. We have mentioned this in the methods section. Please see the revised text on Page 9, Lines 147-148.

14.Line135: Please clarify that you used air-dried soils

Response: We have clarified this as suggested. Please see the revised text on Page 10, Lines 165.

15.Line139:C should come first.

Response: We have revised the statement as suggested. Please see the revised text on Page 10, Lines 168-169.

16.Line143: Why would you use 'exchangeable' before ammonium and nitrate?

Response: We have deleted the word “exchangeable.” Please see the revised text on Page 10, Lines 175.

17.Line174: This paragraph is a little too simple compared the qPCR paragraph. I am not encouraging you to digging into every details, but at least we should see the key details here.

Response: We have revised this paragraph as suggested by including relevant details. Please see the revised text on Page 12, Lines 209-213.

18.Line175: Do you mean quality score per sequence? Clarify.

Response: Yes, we did mean quality score per sequence. We have clarified this. Please see the revised text on Page 12, Lines 210-211.

19.Line180: Did you rarefy the OTU table?

Response: Yes, we have done so. The rarefaction curves are presented in S1 Fig.

20.Line181: Version number?

Response: The version number of this software is 10.3. We have included this detail as suggested. Please see the revised text on Page 12, Lines 219.

21.Line184: Some repeated analyses in the first and third paragraphs are confusing me.

Response: We have revised these paragraphs and removed redundant statements as suggested. Please see the revised text on Pages 12-13, Lines 222-229 and Page 13 Lines 239-243.

22.Line192: Why PCA instead of more common ordination analyses in microbial ecology such as NMDS/RDA/CCA?

Response: PCA is a method of ordination using only species composition, and is one of the unconstrained methods of ordination. CCA and RDA are two constricted ordination methods that use both species and environmental factors to ordinate data. We have provided an explanation for using this in the methods section on Page 13, Lines 231-232.

23.Line193: Do you mean relative abundance of OTUs? If yes, clarify.

Response: Here, we used the relative abundance of OTUs to calculate species composition and abundance of the microbes. We have provided the raw data in the S2 Table.

24.Line219: Did you mention all these properties in the Materials and methods section? For example, total P and CEC.

Response: We have added the methods that we used for estimating total P and CEC in the methods section. Please see the revised text on Page 10-11, Lines 170-182.

25.Line224: Do you mean the sum of ammonium and nitrate? Please clarify.

Response: Yes. Available N is the sum of ammonium and nitrate. Please see the revised text on Page 10, Lines 176.

26.Line243: you said negative?

Response: Yes. We have revised the sentence and added the minus sign. Please see the revised text on Page 15, Lines 271.

27.Line247: It seems like you did not rarefy sequences before calculate alpha diversity indices. Can you justify this?

Response: Thank you for your comment. The Allwegene sequencing company provided us with the following alpha diversity indices: Chao1 index, Observed species index, PD whole tree index, Shannon, Simpson index. The sequences were rarefied prior to calculating these indices. The total number of rarefaction is 18402. The rarefaction curves are presented in S1 Fig.

28.Line268: What about the associations among fungal alpha diversity indices and soil properties? You showed the results in Fig. 2B but you totally neglected these results.

Response: We have added sentences explaining these associations. The pH was negatively correlated with fungal Chao1, Observed species, and PD whole tree indices, and positively correlated with the good coverage index. Soil properties (SMC, NH4+, AN, SOC, TN, DOC) were negatively correlated with fungal diversity indices (Chao1, Observed species, PD whole tree) and positively correlated with the good coverage index. We also observed weak associations of Shannon and Simpson indices with soil properties. Please see the revised text on Page 19, Lines 308-314.

29.Line776: Fig 4. Are these environmental parameters all significant? If not, please remove the insignificant ones.

Response: Some of these parameters are statistically significant. We have removed the insignificant environmental parameters and re-drawn Figure 4. Please see the revised Figure 4.

30. Line804: Fig. S3. By lefse, you mean LDA in the Materials and methods section? Please keep consistency throughout the manuscript.

Response: Yes, we have clarified this method of analysis and consistently used LDA as suggested. Please see the revised text on Page 47, Lines 877-878.

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PLoS One. doi: 10.1371/journal.pone.0246263.r003

Decision Letter 1

Julian Aherne

30 Dec 2020

PONE-D-20-10592R1

Nutrient availability is a dominant predictor of soil bacterial and fungal community composition after nitrogen addition in subtropic al acidic forest

PLOS ONE

Dear Dr. Chen,

==============================

The revised manuscript is much improved but still requires minor revisions, specifically focusing on the three points from Reviewer #2, who notes that "The results do not strongly support their main conclusions". Further, as noted by Reviewer #1, the manuscript still requires additional language editing.

==============================

Please submit your revised manuscript by Feb 13 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Julian Aherne

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The manuscript requires minor revisions, specifically focusing on three points noted by Reviewer #2, who notes that "The results do not strongly support their main conclusions". Further, as noted by Reviewer #1, the manuscript still requires additional language editing.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: Authors have substantially improved the quality of the manuscript. But the writing still has not met publication criteria, I suggest authors seek further editorial advice.

Some sentences I found need revision:

L28,31 change UNDERGROUND to BELOWGROUND

L205 MiSeq is used, but in results, authors used HiSeq, clarify.

L353,430 What statistical analysis was used to compare community structure change under N treatments.

L402-403 ” As soil microbes are mainly C limited [61], the increase of labile C input is expected to multiply microbial biomass.” The context was talking MBN, why authors bring up C? Labile C?

L477 use N loss to explain lack of response in fungi community does not make sense, it contradicts bacteria change.

L499 this sentence lacks logic and evidence. Either elaborate it, or remove it.

Fig 1. The study area should not have red shading, as the fig already has red color for elevation.

Reviewer #2: The results do not strongly support their main conclusions. 1) "DOC and AN significantly decreased after N addition to both topsoil and subsoil" is not accurate. 2) bacterial alpha-diversity in subsoil only decreased under high N addition treatment. 3) "soil DOC is the most important environmental factor for bacterial community composition while AN is the most important factor for fungal communities" are not precise. Furthermore, the r value is very small (0.1-0.3).

Reviewer #3: The authors put a lot of effort into improving the manuscript. Most of my concerns have been well addressed. I do not have further content-related comments.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

PLoS One. 2021 Feb 23;16(2):e0246263. doi: 10.1371/journal.pone.0246263.r004

Author response to Decision Letter 1

14 Jan 2021

Response to Reviewer’s Comments Reviewer # 1

Minor Revisions:

-L28,31 change UNDERGROUND to BELOWGROUND

Response: Thank you for the valuable comments. We have revised this statement as suggested. Please see the revised text on Page 3, Lines 29 and 32.

-L205 MiSeq is used, but in results, authors used HiSeq, clarify.

Response: Thank you for the comment. We have revised this statement as suggested. Please see the revised text on Page 17, Lines 286 and 288.

-L353,430 What statistical analysis was used to compare community structure change under N treatments.

Response: Thank you for the valuable question. One-way analysis of variance (ANOVA) was used to determine the differences in soil microbial relative abundance, and multiple comparisons were conducted using the least significant difference (LSD) test at a significance level of p < 0.05. Please see the revised text on Page 13, Lines 270–230.

-L402-403 “ As soil microbes are mainly C limited [61], the increase of labile C input is expected to multiply microbial biomass.” The context was talking MBN, why authors bring up C? Labile C?

Response: Thank you for the valuable question. C input led to the increase of MBC, but there was no statistical significance. Owing to CN coupling, MBN increases significantly. We have revised this statement as suggested. Please see the revised text on Page 24, Lines 411–413.

-L477 use N loss to explain lack of response in fungi community does not make sense, it contradicts bacteria change.

Response: Thank you for the valuable comments. This sentence cited the other authors’ views. We did not find the available evidence to support this view; hence, the sentence has been deleted in the text.

-L499 this sentence lacks logic and evidence. Either elaborate it, or remove it.

Response: Thank you for the valuable comments. The sentence has been deleted in the text as per the reviewer’s comment.

-Fig 1. The study area should not have red shading, as the fig already has red color for elevation.

Response: We have modified Fig 1 as suggested by the reviewer. Please see the revised text on Page 9, Line 142.

Response to Reviewer’s Comments Reviewer # 2

Specific Comments:

1) "DOC and AN significantly decreased after N addition to both topsoil and subsoil" is not accurate.

Response: Thank you for pointing this out. We have rewritten this sentence to clearly mention this and modified the result section. Please see the revised text on Page 3, 14, Lines 44–45, 265–267.

2)Bacterial alpha-diversity in subsoil only decreased under high N addition treatment.

Response: Thank you for your suggestion. We have made the necessary changes in the abstract and result sections. Please see the revised text on Page 3, 17, 30, Lines 46–47, 299–301, 515-517.

3) Soil DOC is the most important environmental factor for bacterial community composition while AN is the most important factor for fungal communities" are not precise. Furthermore, the r value is very small (0.1-0.3).

Response: The authors would like to thank the reviewer for the valuable comments. The paragraph mentioned by the reviewer has been rewritten. We have added MBN as the predictor in fungal community shift caused by N addition in subtropical forests. We hope that the revised version of the manuscript has better logical continuity. Please see the revised text on Page 3-4, 22, Lines 47–50, 385–393.

Response to Review Comments Reviewer # 3

The authors would like to thank you for the positive comments. The manuscript has been slightly revised with careful consideration of other reviewers’ suggestions. I hope you will find the revised manuscript to be suitable for publication in Plos One.

Specific Comments:

The authors put a lot of effort into improving the manuscript. Most of my concerns have been well addressed. I do not have further content-related comments.

Response: The authors would like to thank you for the valuable and constructive comments.

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PLoS One. doi: 10.1371/journal.pone.0246263.r005

Decision Letter 2

Julian Aherne

18 Jan 2021

Nutrient availability is a dominant predictor of soil bacterial and fungal community composition after nitrogen addition in subtropical acidic forests

PONE-D-20-10592R2

Dear Dr. Chen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Julian Aherne

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have revised the manuscript in response to the reviewers comments. The manuscript is suitable for publication.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0246263.r006

Acceptance letter

Julian Aherne

3 Feb 2021

PONE-D-20-10592R2

Nutrient availability is a dominant predictor of soil bacterial and fungal community composition after nitrogen addition in subtropical acidic forests

Dear Dr. Chen:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Julian Aherne

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig

(ZIP)

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S2 Fig

(ZIP)

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S3 Fig

Cladograms depicting bacterial (A) and fungal (B) species variation under different N addition treatments. LDA analysis was performed to obtain these cladograms.

(TIF)

Click here for additional data file.^{(4.2MB, tif)}

S1 Table. Primer sequences used in this study.

(DOCX)

Click here for additional data file.^{(15KB, docx)}

S2 Table. Effects of different N addition treatments at different soil depths based on 16S rDNA gene copy numbers, ITS gene copy numbers, and fungi-to-bacteria ratios.

(DOCX)

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S3 Table. Pearson’s correlations of microbial abundance with soil properties and microbial biomass.

(DOCX)

Click here for additional data file.^{(16.8KB, docx)}

S4 Table. Good’s coverage values of each group.

(DOCX)

Click here for additional data file.^{(15.7KB, docx)}

S5 Table. Effects of N addition on soil bacterial and fungal α-diversity indices.

(DOCX)

Click here for additional data file.^{(17.7KB, docx)}

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Data Availability Statement

Data are available from the NCBI Sequence Read Archive (accession number PRJNA666625).

[pone.0246263.ref001] 1.Shukla P.R.; Skea J.; Calvo Buendia E.; Masson-Delmotte V.; Pörtner H.O.; Roberts D.C.; et al. Land–climate interactions. In: Climate Change and Land: an IPCC special report on climate change, desertifcation, land degradation, sustainable land management, food security, and greenhouse gas fluxes in terrestrial ecosystems. 2019. [Google Scholar]

[pone.0246263.ref002] 2.Xie L.; Zhang Q.; Cao J.; Liu X.; Xiong D.; Kong Q.; et al. Effects of warming and nitrogen addition on the soil bacterial community in a subtropical Chinese Fir plantation. Forests 2019; 10: 861. [Google Scholar]

[pone.0246263.ref003] 3.Cui S.; Shi Y.; Groffman P.M.; Schlesinger W.H.; Zhu Y. Centennial-scale analysis of the creation and fate of reactive nitrogen in China (1910–2010). Proc. Natl. Acad. Sci. USA. 2013; 110: 2052–2057. 10.1073/pnas.1221638110 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref004] 4.Lu X.; Mao Q.; Gilliam F.S.; Luo Y.; Mo J. Nitrogen deposition contributes to soil acidification in tropical ecosystems. Glob. Chang. Biol. 2014; 20: 3790–3801. 10.1111/gcb.12665 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref005] 5.Nie Y.; Wang M.; Zhang W.; Ni Z.; Hashidoko Y.; Shen W. Ammonium nitrogen content is a dominant predictor of bacterial community composition in an acidic forest soil with exogenous nitrogen enrichment. Sci. Total Environ. 2018; 624: 407–415. 10.1016/j.scitotenv.2017.12.142 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref006] 6.De Vries W.; Posch M.; Simpson D.; Reinds G.J. Modelling long-term impacts of changes in climate, nitrogen deposition and ozone exposure on carbon sequestration of European forest ecosystems. Sci. Total Environ. 2017; 1097–1116, 605–606. 10.1016/j.scitotenv.2017.06.132 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref007] 7.Schmitz A.; Tanja G.M.; Sanders; Bolte A.; Bussotti F.; Dirnböck T.; et al. Responses of forest ecosystems in Europe to decreasing nitrogen deposition. Environ. Pollut. 2019; 244: 980–994. 10.1016/j.envpol.2018.09.101 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref008] 8.Giammarchi F. P., Panzacchi M.; Tonon V.G. Tree growth and water-use efficiency do not react in the short term to artificially increased nitrogen deposition. Forests 2020; 11, 47. [Google Scholar]

[pone.0246263.ref009] 9.Stevens C.J.; Dise N.B.; Mountford J.O.; Gowing D.J. Impact of nitrogen deposition on the species richness of grasslands. Science 2004; 303:1876–1879. 10.1126/science.1094678 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref010] 10.Lu X.; Mo J.; Gilliam F.S.; Zhou G.; Fang Y. Effects of experimental nitrogen additions on plant diversity in an old-growth tropical forest. Glob. Chang. Biol. 2010; 16: 2688–2700. [Google Scholar]

[pone.0246263.ref011] 11.Dirnböck T.; Grandin U.; Bernhardt-Romermann M.; Beudert B.; Canullo R.; Forsius M.; et al. Forest floor vegetation response to nitrogen deposition in Europe. Glob. Chang. Biol. 2014; 20: 429–440. 10.1111/gcb.12440 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref012] 12.van Dobben H.F.; De Vries W. The contribution of nitrogen deposition to the eutrophication signal in understorey plant communities of European forests. Ecol. Evol. 2017; 7: 214–227. 10.1002/ece3.2485 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref013] 13.van der Linde S.; Suz L.M.; Orme C.D.L.; Cox F.; Andreae H.; Asi E.; et al. Environment and host as large-scale controls of ectomycorrhizal fungi. Nature 2018; 558: 243–248. 10.1038/s41586-018-0189-9 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref014] 14.Fierer N.; Lauber C.L.; Ramirez K.S.; Zaneveld J.; Bradford M.A.; Knight R. Comparative metagenomic, phylogenetic and physiological analyses of soil microbial communities across nitrogen gradients. ISME J. 2012; 6: 1007–1017. 10.1038/ismej.2011.159 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref015] 15.Ramirez K.S.; Craine J.M.; Fierer N. Consistent effects of nitrogen amendments on soil microbial communities and processes across biomes. Glob. Chang. Biol. 2012; 18: 1918–1927. [Google Scholar]

[pone.0246263.ref016] 16.López-Lozano N.E.; Heidelberg K.B.; Nelson W.; Garcia-Oliva F.; Eguiarte L.E. Microbial secondary succession in soil microcosms of a desert oasis in the Cuatro Cienegas Basin, Mexico. Peer J. 2013; 1–47. 10.7717/peerj.47 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref017] 17.Van Der Heijden M.G.; Bardgett R.D.; Van Straalen N.M. The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecol. Lett. 2008; 11: 296–310. 10.1111/j.1461-0248.2007.01139.x [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref018] 18.Zhou J.; Liu X.; Xie J.; Lyu M.; Zheng Y.; You Z.; et al. Nitrogen addition affects soil respiration primarily through changes in microbial community structure and biomass in a subtropical natural forest. Forests 2019; 10, 435. [Google Scholar]

[pone.0246263.ref019] 19.Treseder K.K. Nitrogen additions and microbial biomass: a meta-analysis of ecosystem studies. Ecol. Lett. 2008; 11: 1111–1120. 10.1111/j.1461-0248.2008.01230.x [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref020] 20.Janssens I.A.; Dieleman W.; Luyssaert S.; Subke J.A.; Reichstein M.; Ceulemans R.; et al. Reduction of forest soil respiration in response to nitrogen deposition. Na. Geo. 2010; 3: 315–322. [Google Scholar]

[pone.0246263.ref021] 21.Liu L.; Greaver T.L. A global perspective on belowground carbon dynamics under nitrogen enrichment. Ecol. Lett. 2010; 13: 819–828. 10.1111/j.1461-0248.2010.01482.x [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref022] 22.Lu M.; Zhou X.H.; Luo Y.Q.; Yang Y.H.; Fang C.M.; Chen J.K.; et al. Minor stimulation of soil carbon storage by nitrogen addition: a meta-analysis. Agr. Ecosyst. Environ. 2011; 140: 234–244. [Google Scholar]

[pone.0246263.ref023] 23.Lu M.; Yang Y.H.; Luo Y.Q.; Fang C.M.; Zhou X.H.; Chen J.K.; et al. Responses of ecosystem nitrogen cycle to nitrogen addition: a meta-analysis. New Phytol. 2011; 189: 1040–1050. 10.1111/j.1469-8137.2010.03563.x [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref024] 24.Geisseler D., Scow K.M. Long-term effects of mineral fertilizers on soil microorganismse: a review. Soil Biol. Biochem. 2014; 75: 54–63. [Google Scholar]

[pone.0246263.ref025] 25.Geisseler D.; Lazicki P.A.; Scow K.M. Mineral nitrogen input decreases microbial biomass in soils under grasslands but not annual crops. Appl. Soil Ecol. 2016; 106: 1–10. [Google Scholar]

[pone.0246263.ref026] 26.Yue K.; Peng Y.; Peng C.H.; Yang W.Q.; Peng X.; Wu F.Z. Stimulation of terrestrial ecosystem carbon storage by nitrogen addition: a meta-analysis. Sci. Rep. 2016; 6, 19895 10.1038/srep19895 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref027] 27.Wang C.; Liu D.; Edith Bai. Decreasing soil microbial diversity is associated with decreasing microbial biomass under nitrogen addition. Soil Biol. Biochem. 2018; 120: 126–133. [Google Scholar]

[pone.0246263.ref028] 28.Zhou Z.; Wang C.; Zheng M., Jiang L.; Luo Y. Patterns and mechanisms of responses by soil microbial communities to nitrogen addition, Soil Biol. Biochem. 2017; 115: 433–441. [Google Scholar]

[pone.0246263.ref029] 29.Wang C., Lua X., Moria T, Mao Q, Zhou K, Zhou G.,et al. Responses of soil microbial community to continuous experimental nitrogen additions for 13 years in a nitrogen-rich tropical forest. Soil Biol. Biochem. 2018; 121: 103–112. [Google Scholar]

[pone.0246263.ref030] 30.Wu J., Liu W., Zhang W., Shao Y., Duan H., Chen B., et al. Long-term nitrogen addition changes soil microbial community and litter decomposition rate in a subtropical forest. Appl.Soil Ecol. 2019; 142: 43–51. [Google Scholar]

[pone.0246263.ref031] 31.Wang H., Liu S.R., Zhang X., Mao Q, Li X., You Y., et al. , Nitrogen addition reduces soil bacterial richness, while phosphorus addition alters community composition in an old-growth N-rich tropical forest in southern China. Soil Biol. Biochem. 2018; 127: 22–30. [Google Scholar]

[pone.0246263.ref032] 32.Li H.; Xu Z.; Yang S.; Li X.; Top E.M.; Wang R.; et al. Responses of soil bacterial communities to nitrogen deposition and precipitation increment are closely linked with aboveground community variation. Microb. Ecol. 2016; 71: 974–989. 10.1007/s00248-016-0730-z [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref033] 33.Liu L.; Zhang T.; Gilliam F.S.; Gundersen P.; Zhang W.; Chen H.; et al. Interactive effects of nitrogen and phosphorus on soil microbial communities in a tropical forest. PLoS One 2013; 8, e61188 10.1371/journal.pone.0061188 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref034] 34.Li J.; Li Z.; Wang F.; Zou B.; Chen Y.; Zhao J.; et al. Effects of nitrogen and phosphorus addition on soil microbial community in a secondary tropical forest of China. Biol. Fert. Soils, 2015; 51: 207–215. [Google Scholar]

[pone.0246263.ref035] 35.Tian D.; Jiang L.; Ma S.; Fang W.; Schmid B.; Xu L.; et al. Effects of nitrogen deposition on soil microbial communities in temperate and subtropical forests in China. Sci. Total Environ. 2017; 607: 1367–1375. 10.1016/j.scitotenv.2017.06.057 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref036] 36.Ramirez K.S.; Lauber C.L.; Knight R.; Bradford M.A.; Fierer N. Consistent effects of nitrogen fertilization on soil bacterial communities in contrasting systems. Ecology, 2010; 91: 3463–3470. 10.1890/10-0426.1 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref037] 37.Fierer N.; Bradford M.A.; Jackson R.B. Toward an ecological classification of soil bacteria. Ecology 2007; 88: 1354–1364. 10.1890/05-1839 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref038] 38.Ling N.; Chen D.; Guo H.; Wei J.; Bai Y.; Shen Q.; et al. Differential responses of soil bacterial communities to long-term N and P inputs in a semi-arid steppe. Geoderma, 2017; 292: 25–33. [Google Scholar]

[pone.0246263.ref039] 39.Albornoz F. E.; Teste F. P.; Lambers H.; Bunce M.; Murray D. C.; White N. E.; et al. Changes in ectomycorrhizal fungal community composition and declining diversity along a 2‐million‐year soil chronosequence. Mol.Ecol. 2016; 25: 4919–4929. 10.1111/mec.13778 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref040] 40.Lauber C.L.; Hamady M.; Knight R.; Fierer N. Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl. Environ. Microb. 2009; 75 (15): 5111–5120. 10.1128/AEM.00335-09 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref041] 41.Rousk J.; Bååth E.; Brookes P.C.; Lauber C.L.; Lozupone C.; Caporaso J.G.; et al. Soil bacterial and fungal communities across a pH gradient in an arable soil. ISME J. 2010; 4 (10): 1340–1351. 10.1038/ismej.2010.58 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref042] 42.Stone M.M.; Kan J.; Plante A.F. Parent material and vegetation influence bacterial community structure and nitrogen functional genes along deep tropical soil profiles at the Luquillo Critical Zone Observatory. Soil Biol. Biochem. 2015; 80: 273–282. [Google Scholar]

[pone.0246263.ref043] 43.Zeng J.; Liu X.; Song L.; Lin X.; Zhang H.; Shen C.; et al. Nitrogen fertilization directly affects soil bacterial diversity and indirectly affects bacterial community composition. Soil Biol. Biochem. 2016; 92: 41–49. [Google Scholar]

[pone.0246263.ref044] 44.Zhu J.; He N.; Wang Q.; Yuan G.; Wen D.; Yu G.; et al. The composition, spatial patterns, and influencing factors of atmospheric wet nitrogen deposition in Chinese terrestrial ecosystems. Sci. Total Environ. 2015; 511: 777–785. 10.1016/j.scitotenv.2014.12.038 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref045] 45.Ji Zhao, Chuankang Chen. Chinese geography. 1st ed Higher Education Press; 2004. [Google Scholar]

[pone.0246263.ref046] 46.Fan Y.X., Zong X.J., Lin F., Liu C.C., Wang M.H., Chen G.S., et al. Responses of soil phosphorus fractions after nitrogen addition in a subtropical forest ecosystem: insights from decreased Fe and Al oxides and increased plant roots. Geoderma 2019, 337: 246–255. [Google Scholar]

[pone.0246263.ref047] 47.Turner BL.; Wright JS. The response of microbial biomass and hydrolytic enzymes to a decade of nitrogen, phosphorus, and potassium addition in a lowland tropical rain forest. Biogeochemistry, 2014; 117: 115–130. [Google Scholar]

[pone.0246263.ref048] 48.Vance E.D.; Brookes P.C.; Jenkinson D.S. An extraction method for measuring soil microbial biomass C. Soil Biol. Biochem. 1987; 19: 703–707. [Google Scholar]

[pone.0246263.ref049] 49.Brookes P.C.; Landman A.; Pruden G.; Jenkinson D.S. Chloroform fumigation and the release of soil-nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. Biochem. 1985; 17: 837–842. [Google Scholar]

[pone.0246263.ref050] 50.Munyaka P. M.; Eissa N.; Bernstein C. N. Antepartum Antibiotic Treatment Increases offspring susceptibility to experimental colitis: A role of the gut microbiota. PLoS One. 2015; 10 (11): e0142536 10.1371/journal.pone.0142536 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref051] 51.Edgar R.C. UPARSE: Highly accurate OTU sequences from microbial amplicon reads, Nature Methods. 2013. 10.1038/nmeth.2604 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref052] 52.Cole J. R.; Wang Q.; Cardenas E.; Fish J.; Chai B.; Farris R. J.; et al. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 2009; 37: 141–145. 10.1093/nar/gkn879 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref053] 53.Wang Y.; Sheng H.; He Y.; Wu J.; Jiang Y.; Tam N. F.; et al. Comparison of the levels of bacterial diversity in freshwater, intertidal wetland, and marine sediments by using millions of illumina tags. Appl. Environ. Microbiol. 2012; 78 (23): 8264 10.1128/AEM.01821-12 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref054] 54.Jiang X.T., Peng X., Deng G.; Sheng H.; Wang Y.; Zhou H.; et al. Illumina sequencing of 16S rRNA tag revealed spatial variations of bacterial communities in a mangrove wetland. Microb. Ecol. 2013; 66: 96–104. 10.1007/s00248-013-0238-8 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref055] 55.Cusack D.F.; Silver W.L.; Torn M.S.; Burton S.D.; Firestone M.K. Changes in microbial community characteristics and soil organic matter with nitrogen additions in two tropical forests. Ecology 2011; 92: 621–632. 10.1890/10-0459.1 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref056] 56.Li Y.Q.; Xu M.; Zou X.M. Effects of nutrient additions on ecosystem carbon cycle in a Puerto Rican tropical wet forest. Glob. Chang. Biol. 2006; 12: 284–293. [Google Scholar]

[pone.0246263.ref057] 57.Fanin N.; Hättenschwiler S.; Schimann H.; Fromin N.; Bailey J.K. Interactive effects of C, N and P fertilization on soil microbial community structure and function in an Amazonian rain forest. Funct. Ecol. 2015; 29: 140–150. [Google Scholar]

[pone.0246263.ref058] 58.Peng Y.; Chen G.; Chen G.; Li S.; Peng T.; Qiu X.; et al. Soil biochemical responses to nitrogen addition in a secondary evergreen broad-leaved forest ecosystem. Scientific Reports 2017; 7, 2783 10.1038/s41598-017-03044-w [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref059] 59.Li Y.; Sun J.; Tian D.; Wang J.; Ha D.; Qu Y.; et al. Soil acid cations induced reduction in soil respiration under nitrogen enrichment and soil acidification. Sci. Total Environ. 2018; 615: 1535–1546. 10.1016/j.scitotenv.2017.09.131 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref060] 60.Li J.; Li Z.; Wang F.; Zou B.; Chen Y.; Zhao J.; et al. Effects of nitrogen and phosphorus addition on soil microbial community in a secondary tropical forest of China. Biol. Fertil. Soils 2015; 51: 207–215. [Google Scholar]

[pone.0246263.ref061] 61.Fierer N.; Strickland M.S.; Liptzin D.; Bradford M.A.; Cleveland C.C. Global patterns in belowground communities. Ecol. Letters 2009; 12: 1238–1249. 10.1111/j.1461-0248.2009.01360.x [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref062] 62.Zhou Z.H.; Wang C.K. Reviews and syntheses: soil resources and climate jointly drive variations in microbial biomass carbon and nitrogen in China’s forest ecosystems. Biogeosciences, 2015; 12: 6751–6760. [Google Scholar]

[pone.0246263.ref063] 63.Wang Q.; Wang C.; Yu W.W.; Turak A.; Chen D.; Huang Y.; et al. Effects of Nitrogen and Phosphorus Inputs on Soil Bacterial Abundance, Diversity, and Community Composition in Chinese Fir Plantations, Front. Microbiol. 2018; 9: 1543. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref064] 64.Huang Y.; Kang R.; Mulder J.; Zhang T.; Duan L. Nitrogen saturation, soil acidification, and ecological effects in a subtropical pine forest on acid soil in southwest China. J. Geophys. Res-Biogeo. 2015; 120: 2457–2472 [Google Scholar]

[pone.0246263.ref065] 65.Dai Z.; Su W.; Chen H.; Barberán A.; Zhao H.; Yu M.; et al. Long-term nitrogen fertilization decreases bacterial diversity and favors the growth of Actinobacteria and Proteobacteria in agro-ecosystems across the globe. Glob. Chang. Biol. 2018; 24 (8): 3452–3461. 10.1111/gcb.14163 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref066] 66.Zechmeister-Boltenstern S.; Keiblinger K.M.; Mooshammer M.; PENuelas J.; Richter A.; Sardans J.; et al. The application of ecological stoichiometry to plant–-microbial–soil organic matter transformations. Ecol. Monogr. 2015; 85: 133–155. [Google Scholar]

[pone.0246263.ref067] 67.Zhang C.; Liu G.B.; Xue S.; Wang G.L. Soil bacterial community dynamics reflect changes in plant community and soil properties during the secondary succession of abandoned farmland in the Loess Plateau. Soil Biol. Biochem. 2016; 97: 40–49. [Google Scholar]

[pone.0246263.ref068] 68.Zhang X.; Liu W.; Zhang G.; Jiang L.; Han X. Mechanisms of soil acidification reducing bacterial diversity. Soil Biol. Biochem. 2015; 81: 275–281. [Google Scholar]

[pone.0246263.ref069] 69.Zhang H.; Wang L.; Liu H.; Zhao J.; Li G.; Wang H.; et al. Nitrogen deposition combined with elevated precipitation is conducive to maintaining the stability of the soil fungal diversity on the Stipa baicalensis steppe. Soil Biol. Biochem. 2018; 117: 135–138. [Google Scholar]

[pone.0246263.ref070] 70.Zhou J.; Guan D. W.; Zhou B. K.; Zhao B. S.; Ma M. C.; Qin J.; et al. Influence of 34-years of fertilization on bacterial communities in an intensively cultivated black soil in northeast China. Soil Biol. Biochem. 2015; 90: 42–51. [Google Scholar]

[pone.0246263.ref071] 71.Wang Q. F.; Jiang X.; Guan D.; Wei D.; Zhao B.; Ma M.; et al. Long-term fertilization changes bacterial diversity and bacterial communities in the maize rhizosphere of Chinese Mollisols. Appl. Soil Ecol. 2018; 125: 88–96. [Google Scholar]

[pone.0246263.ref072] 72.Cui J.; Wang J.; Xu J.; Xu C.; Xu X. Changes in soil bacterial communities in an evergreen broad-leaved forest in east China following 4 years of nitrogen addition. J. Soils Sediments, 2017; 17: 2156–2164. [Google Scholar]

[pone.0246263.ref073] 73.Högberg M.N.; Högberg P.; Myrold D.D. Is microbial community composition in boreal forest soils determined by pH, C-to-N ratio, the trees, or all three? Oecologia 2007; 150: 590–601. 10.1007/s00442-006-0562-5 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref074] 74.Rousk J.; Brookes P.C.; Bååth E. Fungal and bacterial growth responses to N fertilization and pH in the 150-year ‘Park Grass’ UK grassland experiment. Fems Microbiol.Ecol. 2011; 76: 89–99. 10.1111/j.1574-6941.2010.01032.x [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref075] 75.Chen D. M.; Lan Z. C.; Bai X.; Grace J.B.; Bai Y. F. Evidence that acidification-induced declines in plant diversity and productivity are mediated by changes in belowground communities and soil properties in a semi-arid steppe. J. Ecol. 2013; 101: 1322–1334. [Google Scholar]

[pone.0246263.ref076] 76.Ai C.; Liang G.; Sun J.; Wang X.; He P.; Zhou W.; et al. Reduced dependence of rhizosphere microbiome on plant-derived carbon in 32-year long-term inorganic and organic fertilized soils. Soil Biol. Biochem. 2015; 80: 70–78. [Google Scholar]

[pone.0246263.ref077] 77.Cavaletti L.; Monciardini P.; Bamonte R.; Schumann P.; Rohde M.; Sosio M.; et al. New lineage of filamentous, spore-forming, gram-positive bacteria from soil. Appl. Environ. Microbiol. 2006; 72: 4360–4369. 10.1128/AEM.00132-06 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref078] 78.Yabe S.; Aiba Y.; Sakai Y.; Hazaka M.; and Yokota A. Thermosporothrix hazakensis gen. nov., sp. nov., isolated from compost, description of Thermosporotrichaceae fam. nov. within the class Ktedonobacteria Cavaletti et al. 2007 and emended description of the class Ktedonobacteria. Int. J. Syst. Evol. Microbiol. 2010; 60: 1794–1801. 10.1099/ijs.0.018069-0 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref079] 79.Albertsen M.; Hansen L.B.S.; Saunders A.M.; Nielsen P.H.; Nielsen K.L. A metagenome of a full-scale microbial community carrying out enhanced biological phosphorus removal. ISME J. 2012; 6: 1094 10.1038/ismej.2011.176 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref080] 80.Sellstedt A.; Richau K.H. Aspects of nitrogen-fixing Actinobacteria, in particular free-living and symbiotic Frankia. FEMS Microbiol. Lett. 2013; 342: 179–186. 10.1111/1574-6968.12116 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref081] 81.Vetrovsky T.; Steffen K.T.; Baldrian P. Potential of cometabolic transformation of polysaccharides and lignin in lignocellulose by soil Actinobacteria. PLoS One 2014; 9: e0089108 10.1371/journal.pone.0089108 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref082] 82.Anderson I.; Abt B.; Lykidis A.; Klenk H.P.; Kyrpides N.; Ivanova N. Genomics of aerobic cellulose utilization systems in actinobacteria. PLoS One 2012; 7: e0039331 10.1371/journal.pone.0039331 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref083] 83.Pujic P.; Bolotin A.; Fournier P.; Sorokin A.; Lapidus A.; Richau K.H.; et al. Genome sequence of the atypical symbiotic Frankia R43 strain, a nitrogen-fixing and hydrogen-producing actinobacterium. Genome Announcements 2015; 3: 01387–01315. 10.1128/genomeA.01387-15 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref084] 84.Nouioui I.; Ghodhbane-Gtari F.; del Carmen Monterro-Calasanz M.; Rohde M.; Tisa L.S.; Gtari M.; et al. Frankia inefficax sp. nov., an actinobacterial endophyte inducing ineffective, non nitrogen-fixing, root nodules on its actinorhizal host plants. Antonie van Leeuwenhoek 2017; 110: 313–320. 10.1007/s10482-016-0801-7 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref085] 85.Paungfoo-Lonhienne C.; Yeoh Y.K.; Kasinadhuni N.R.P.; Lonhienne T.G.A.; Robinson N.; Hugenholtz P.; et al. Nitrogen fertilizer dose alters fungal communities in sugarcane soil and rhizosphere. Scientific Reports 2015; 5: 8678 10.1038/srep08678 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref086] 86.Klaubauf S.; Inselsbacher E.; Zechmeister-Boltenstern S.; Wanek W.; Gottsberger R.; Strauss J.; et al. Molecular diversity of fungal communities in agricultural soils from Lower Austria. Fungal Diversity 2010; 44: 65–75. 10.1007/s13225-010-0053-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref087] 87.Chen W.; Xu R.; Wu Y.; Chen J.; Zhang Y.; Hu T.; et al. Plant diversity is coupled with beta not alpha diversity of soil fungal communities following N enrichment in a semi-arid grassland. Soil Biol. Biochem. 2018; 116: 388–398. [Google Scholar]

[pone.0246263.ref088] 88.She W.; Bai Y.; Zhang Y.; Qin S.; Feng W.; Sun Y.; et al. Resource availability drives responses of soil microbial communities to short-term precipitation and nitrogen addition in a desert shrubland. Front. Microbiol. 2018; 9: 186 10.3389/fmicb.2018.00186 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref089] 89.Yao F.; Yang S.; Wang Z.; Wang X.; Ye J.; Wang X.; et al. Microbial taxa distribution is associated with ecological trophic cascades along an elevation gradient. Front. Microbiol. 2017; 8: 2071 10.3389/fmicb.2017.02071 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246263.ref090] 90.Niu S.; Classen AT.; Dukes JS.; Kardol P.; Liu L.; Luo Y.; et al. Global patterns and substrate based mechanisms of the terrestrial nitrogen cycle. Ecol. Letters 2016; 19(6): 697–709. 10.1111/ele.12591 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref091] 91.DALPÉ Y., Ericoid mycorrhizal fungi in the Myxotrichaceae and Gymnoascaceae. New Phytologist 1989; 113(4): 523–527. [Google Scholar]

[pone.0246263.ref092] 92.Serna-Chavez H.M.; Fierer N.; van Bodegom P.M. Global drivers and patterns of microbial abundance in soil. Global Ecol. and Biogeogr. 2013; 22: 1162–1172. [Google Scholar]

[pone.0246263.ref093] 93.Magill A.H.; Aber J.D. Long-term effects of experimental nitrogen additions on foliar litter decay and humus formation in forest ecosystems. Plant Soil 1998; 203: 301–311. [Google Scholar]

[pone.0246263.ref094] 94.Zhang T.; Luo Y.; Chen H. Y. H.; Ruan H. Responses of litter decomposition and nutrient release to N addition: A meta-analysis of terrestrial ecosystems. Appl. Soil Ecol. 2018; 128: 35–42. [Google Scholar]

[pone.0246263.ref095] 95.Allison SD.; Weintraub MN.; Gartner TB.; Waldrop MP. Evolutionary-economic principles as regulators of soil enzyme production and ecosystem function. In: Shukla G,Varma A(eds) Soil enzymology, 1st edn Springer, Berlin, 2011; pp 229–243. [Google Scholar]

[pone.0246263.ref096] 96.Carreiro MM.; Sinsabaugh RL.; Repert DA.; Parkhurst DF. Microbial enzyme shifts explain litter decay responses to simulated nitrogen deposition. Ecology 2000; 81, 2359–2365. [Google Scholar]

[pone.0246263.ref097] 97.Aber J.D.; Nadelhoffer K.J.; Steudler P.; Melillo J.M. Nitrogen saturation in northern forest ecosystems. Bioscience 1989; 39: 378–386. [Google Scholar]

[pone.0246263.ref098] 98.Gress S.E.; Nichols T.D.; Northcraft C.C.; Peterjohn W.T. Nutrient limitation in soils exhibiting differing nitrogen availabilities: what lies beyond nitrogen saturation? Ecology 2007; 88: 119 10.1890/0012-9658(2007)88[119:nlised]2.0.co;2 [DOI] [PubMed] [Google Scholar]

[pone.0246263.ref099] 99.Kopáček J.; Cosby B.; Evans C.; Hruška J.; Moldan F.; Oulehle F.; et al. Nitrogen, organic carbon and sulphur cycling in terrestrial ecosystems: linking nitrogen saturation to carbon limitation of soil microbial processes. Biogeochemistry 2013; 115: 33–51. [Google Scholar]

[pone.0246263.ref100] 100.Rousk J.; Frey S.D. Revisiting the hypothesis that fungal-to-bacterial dominance characterizes turnover of soil organic matter and nutrients. Ecol. Monogr. 2015; 85: 457–472. [Google Scholar]

[pone.0246263.ref101] 101.Zhou J.; Jiang X.; Zhou B.; Zhao B.; Ma M.; Guan D.; et al. Thirty-four years of nitrogen fertilization decreases fungal diversity and alters fungal community composition in black soil in northeast China. Soil Biol. Biochem. 2016; 95: 135–143. [Google Scholar]

[pone.0246263.ref102] 102.Farrer E.C.; Suding K.N. Teasing apart plant community responses to N enrichment: the roles of resource limitation, competition and soil microbes. Ecol. Letters 2016; 19: 1287–1296. [DOI] [PubMed] [Google Scholar]

PERMALINK

Nutrient availability is a dominant predictor of soil bacterial and fungal community composition after nitrogen addition in subtropical acidic forests

Juyan Cui

Xiaochun Yuan

Qiufang Zhang

Jiacong Zhou

Kaimiao Lin

Jianguo Xu

Yaozhong Zeng

Yue Wu

Lei Cheng

Quanxin Zeng

Kongcan Mei

Yuehmin Chen

Roles

Abstract

Introduction

Materials and methods

Site description and experimental design

Fig 1. Study site location.

Soil sampling and analysis

Quantitative PCR (qPCR) analysis of soil microbial abundance

Illumina Miseq sequencing and bioinformatics analysis

Statistical analysis

Results

Responses of soil properties and microbial biomass to nitrogen additions

Table 1. Responses of physicochemical properties of soil at different depths to nitrogen addition in the Daiyun Mountain Nature Reserve in southeastern China.

Effects of N addition on bacterial and fungal diversity

Table 2. Effects of nitrogen deposition, soil layer, and their interactions on soil bacterial (B) and fungal (F) α diversity analyzed by two-way ANOVA.

Fig 2. Pearson’s correlation coefficients between soil properties and soil bacterial (A) and fungal (B) α-diversity.

Relative abundance of dominant microbial taxa and species variation

Fig 3. Relative abundance of dominant phyla of bacteria and fungi in the topsoil and subsoil under different N addition treatments.

Bacterial and fungal community structure and correlations with environmental parameters

Fig 4. Redundancy discriminate analysis (RDA) plots illustrating the relationships between the dominant bacterial (A) and fungal (B) phyla and soil physicochemical properties and microbial biomass.

Table 3. Mantel test analysis of soil physicochemical properties and soil microbial biomass and community composition in topsoil and subsoil under different nitrogen addition treatments.

Discussion

Effect of nitrogen addition on bacterial and fungal gene abundance and diversity

Influence of nitrogen addition on bacterial and fungal community structure

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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