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Article

The Effect of Acid Rain and Understory Vegetation Removal on the Biological Activity of the Soils of the Cinnamomum camphora (Linn) Presl Plantation

by
Zaihua He
1,†,
Yini Liu
1,†,
Yonghui Lin
1,
Xiangshi Kong
2,
Hong Lin
3 and
Xingbing He
1,*
1
College of Biology and Environmental Sciences, Jishou University, Jishou 416000, China
2
Key Laboratory for Ecotourism of Hunan Province, School of Tourism, Jishou University, Jishou 416000, China
3
Institute of Applied Ecology, School of Food Science, Nanjing Xiaozhuang University, 3601 Hongjing Avenue, Nanjing 211171, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2025, 16(3), 525; https://doi.org/10.3390/f16030525
Submission received: 19 February 2025 / Revised: 12 March 2025 / Accepted: 14 March 2025 / Published: 16 March 2025
(This article belongs to the Special Issue How Does Forest Management Affect Soil Dynamics?)
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Abstract

:
Acid rain and understory vegetation removal are critical drivers altering soil ecosystem alterations. However, the mechanisms by which these factors influence soil moisture dynamics, nutrient availability, and microbially mediated enzyme activities remain insufficiently elucidated. This study investigated the impacts of simulated acid rain and understory vegetation removal on soil properties, enzyme activities, and microbial community in a subtropical Cinnamomum camphor (Linn) Presl plantation. The results indicated that acid rain and understory vegetation removal significantly decreased the soil organic carbon (SOC) while concurrently elevating the C-acquiring enzyme activities and microbial C limitation. Understory vegetation removal markedly reduced the soil moisture, nutrient availability, and N- and P-acquiring enzyme activities. Additionally, acid rain increased the bacterial diversity, but the understory vegetation removal increased the fungal diversity. Moreover, both acid rain and understory vegetation removal enhanced the bacterial community deterministic processes and destabilized the community by shifting generalists toward specialists, but had no significant effect on the fungal community structure. Partial least squares path modeling revealed that the bacterial stability loss intensified the C limitation, while the fungal stability regulated the P limitation. Collectively, the findings highlighted the critical role of understory vegetation in buffering the soil microclimate and nutrient cycling, and demonstrated that bacterial communities are more responsive to acid rain and understory vegetation removal than fungal communities. This study provides insights into the mechanisms by which anthropogenic disturbances alter soil ecological functions in subtropical plantations, emphasizing the need for integrated forest management strategies to conserve and manage soil ecosystems in subtropical plantations.

1. Introduction

Acid rain, one of the most pervasive anthropogenic environmental stressors, has attracted widespread concerns due to its negative impact on ecosystems [1,2,3]. Soil, as an essential component of forest ecosystems, is highly susceptible to external environmental disturbances [4,5]. Numerous studies have shown that acid rain can accelerate soil acidification, reduce nutrients availability, and elevate toxic aluminum (Al3+) levels, thus destabilizing soil structure and functions [1,3,6]. Soil microorganisms and enzymes play central roles in soil ecological processes, such as carbon (C) sequestration and nutrient cycling, and their activities are sensitive to environmental changes, such as acid rain [7,8]. In general, soil acidification caused by acid rain can inhibit microbial activity, reduce microbial biomass, and, subsequently, alter microbial communities and enzyme activities [9,10]. Existing studies have also suggested that soil microbial activity and enzyme activities can be directly suppressed by acid rain, thus influencing soil ecological functions [6,10,11]. For instance, Wang et al. reported that acid rain decreased soil hydrolytic enzyme activities and then slowed the rate of litter decomposition [6]. A meta-analysis also indicated that acid rain inhibited the growth of soil microbes and reduced soil enzyme activities [10,12]. In addition, acid rain can indirectly influence soil microorganisms and enzyme activities by influencing plant diversity, as well as the aboveground and underground biomass [1,13,14]. Meanwhile, for plants, as the main interceptors and receptors of acid rain, the understory vegetation modulates acid rain’s impacts on soil microorganisms and enzymatic processes to some extent [14,15].
Understory vegetation, as an essential components of forest ecosystem, is crucial to the processes and functions of forest ecosystems, like ecosystem productivity, nutrient cycling, and water conservation [14,16]. To decrease competition between canopy trees and understory vegetation, removing the latter has been a traditional forest management practice, particularly in plantations [17,18]. However, the removal of understory vegetation can alter the soil microenvironment and nutrient availability, which greatly impact the soil microbial community and enzyme activities [14,19]. For instance, several studies have reported that root exudates and litter from understory vegetation exert substantial impacts on soil nutrient dynamics (the content and bioavailability) [20,21], and, subsequently, result in significantly negative, positive, or no effects on soil enzymatic activities and microbial biomass [19,22,23,24]. These studies indicated that the effects of understory vegetation removal on microorganisms and soil enzyme activities exhibit significant variability across ecosystem types and environmental conditions, and needs further study.
Soil microorganisms, particularly bacteria and fungi, are recognized as important regulators of nutrient cycling and sensitive to external environmental disturbances. However, bacterial and fungal communities responded differently to acid rain or understory vegetation removal in forest ecosystems [25,26]. Microbial communities typically derive their energy from soil organic matter (e.g., organic matter mineralization) [27,28]; yet, bacteria and fungi have different metabolic preferences, where bacteria are normally characterized by using labile C resources, while fungi exhibit greater metabolic efficiency in processing recalcitrant C compounds [29,30]. Moreover, fungi generally have a considerably higher osmotic stress tolerance capabilities than bacteria [30,31]. Consequently, bacterial communities may be more responsive to acid rain or understory vegetation removal than fungal communities, due to the fact that bacteria are more susceptible to the soil microenvironment and nutrient availability [25,32]. Extracellular enzymes, secreted by soil microorganisms, are protein catalysts that drive essential ecological processes, including the decomposition of soil organic matter and the biogeochemical cycling of nutrients, such as C, nitrogen (N), and phosphorus (P) [7,33]. Consequently, the changes in the soil microbial community, combined with modifications to the nutrient availability, can alter extracellular enzyme secretion and their stoichiometry (e.g., the vector length and vector angle). Furthermore, we speculate that soil enzyme stoichiometry is closely related to the microbial community. However, in subtropical Cinnamomum camphor (Linn) Presl (C. camphor) plantations, the underlying mechanism by which the alterations in the soil moisture and nutrient availability, driven by distinct environmental stressors like acid rain and understory vegetation removal, regulate microbial community dynamics and enzyme activities remain poorly understood, necessitating further investigation.
In this study, we carried out a simulated acid rain experiment combined with understory vegetation removal in a subtropical C. camphor plantation forest located in Jishou, a region within southwestern China severely impacted by acid rain [34], to assess the effects of acid rain and understory vegetation removal on the soil microbial community and enzyme activities and stoichiometry, while elucidating their relevance. Specifically, we hypothesized that (1) the soil bacterial community would be more responsive to acid rain and understory vegetation removal than the soil fungal community; (2) soil enzyme activities and their stoichiometry would be significantly affected by the soil microbial community. By elucidating these mechanisms, this work aims to provide sustainable management practices that conserve soil ecological functions in subtropical plantations.

2. Materials and Methods

2.1. Study Site Description

The study was implemented in a subtropical C. camphor plantation located at 28°17′ N, 109°43′ E, with an elevation of 258 m above sea level. The study site spanned 1000 m2 adjacent to Jishou University in Jishou City, China. The mean annual temperature is about 16.5 °C, the mean annual precipitation is about 1400 mm–1800 mm, primarily falling during the rainy reason from April to June, and the mean annual humidity is about 82%. The soil is commonly classified as Ultisol, which is primarily developed from limestone [35]. The soil moisture content, pH, total organic C, and ammonium nitrogen is about 25.81%, 6.26, 40.93 g/kg, and 35.05 mg/kg, respectively.
The C. camphor plantation, established 40 years ago on land cleared from a Pinus massoniana forest, has an understory vegetation coverage of approximately 85%. The current understory plants are dominated by five fern species (Cyclosorus acuminatus, Arachniodes rhomboidea, Polystichum tsus-simense, Cyrtomium fortunei, and Microlepia marginata), a palm (Trachycarpus fortunei), and a grass (Veronicastrum stenostachyum). These species play critical roles in maintaining ecosystem functions, including nutrient cycling, soil and water conservation, and so on [14,35]. Some other plantation characteristics were recorded in prior studies [35,36].

2.2. Experimental Design and Soil Sampling

In January 2023, four 10 m × 10 m plots, characterized by similar site conditions, were established within the C. camphor plantation, with each plot separated by >20 m. Each plot has four 1 m × 1 m subplots, separated by more than 2 m buffer zones to minimize cross-treatment interference. Briefly, we conducted a complete factorial combination experiment of simulated acid rain and understory vegetation removal to give a total of four treatments. The treatments included (1) the control (CK, no acid rain and understory vegetation removal), (2) acid rain only (AR), (3) understory vegetation removal only (UR), and combined acid rain and understory vegetation removal (UA), with four replicates for each treatment in the C. camphor plantation.
The mother solution (0.1 mol/L) of acid rain was formed using H2SO4 (98, wt%) to avoid nitrogen fertilizer effects. The mother solution was then diluted with deionized water to obtain a pH = 4.0 for the simulated acid rain. For the AR and UA, 2 L simulated acid rain was sprayed on each subplot every 15 days [26], while the UR and CK received an equivalent volume of deionized water. For the UR and UA, the shoots and visible roots on the surface of all understory vegetation were manually excised using machetes. Each month, germinating understory vegetation were removed manually during the experiment. The experiment lasted for 18 months (from January 2023 to July 2024).
In July 2024, the topsoil (0–10 cm) was sampled via a five-point method [2], put into plastic bags, mixed thoroughly, and then the 16 soil samples were immediately transported back to the laboratory in an icebox. After taking out visible stones and plant residues, the soil samples were sieved (2 mm mesh) and then split into three parts. The first part was air-dried for soil physicochemical properties analysis, the second part was kept fresh for soil enzymatic activity determination, and the third part was stored at −80 °C to examine the soil microbial community.

2.3. Soil Physicochemical Property, CO2 Release, and Enzyme Analysis

The soil moisture content (MC) was assessed by drying a fresh soil sample at 105 °C for 48 h. The soil pH was determined using a pH meter after shaking soil/deionized water (w/v) suspensions at a ratio of 1:2.5 for 30 min. The soil ammonium nitrogen (AN) was measured using a continuous flow autoanalyzer (AA3, iFIA, Beijing, China) following extraction with 2 mol/L KCl. Dissolved organic carbon (DOC) was extracted with a 0.5 mol/L K2SO4, passed through a 0.45 μm filter, and then measured via a TOC analyzer (TOC-L, Shimadzu, Kyoto, Japan). The soil organic carbon (SOC) was analyzed using dichromate–sulfuric acid oxidation method [37]. The soil total nitrogen (STN) was determined through the Kjeldahl method [37]. The soil total phosphorus (STP) was analyzed colorimetrically after the soil was digested with H2SO4-HClO4 solution [37].
Approximately 0.5 g of fresh soil was placed in a sealed flask and then incubated in 25 °C darkness for 2 days. The released CO2 was absorbed using a 0.5 mol/L NaOH solution and titrated by using 0.05 mol/L HCl. The released CO2 was expressed as μmol·g−1dry soil·d−1.
The potential activity of the extracellular enzymes was quantified in soil suspensions using a spectrophotometry protocol with appropriate substrates [38]. Briefly, we homogenized 20 g of soil in 100 mL of acetic acid buffer (pH = 5.5). The resulting slurry was used to perform spectrophotometry for the potential enzyme activity. These included β-1,4-glucosidase (BG) and cellobiohydrolase (CBH) using a 4-nitrophenyl-β-D-linked substrates (cellobioside and glucopyranoside); leucine aminopeptidase (LAP) using L-leucine-p-nitroanilide; β-1,4-N-acetyl-glucosaminnidase (NAG) using 4-nitrophenyl N-acetyl-β-D-glucosaminide; acid phosphatase (AP) using p-nitrophenyl disodium phosphate substrates. The units of enzyme activity were calculated as units of substrate hydrolyzed product per gram per hour (nmol·g−1 dry soil·h−1).

2.4. Soil Microbial Community Analysis

Total genomic DNA was extracted from well-homogenized soil sample using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer’s instructions. The soil bacterial V4–V5 regions of the 16S rRNA were amplified with the primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′). Fungal communities were targeted through the amplification of the ITS1 region using the primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′), with adapter and barcode sequences appended. PCR reactions were performed in triplicate with a 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions. Purified PCR products were quantified by Qubit®3.0 (Life Invitrogen, Waltham, MA, USA) and the pooled DNA product was used to construct the Illumina pair-end library following Illumina’s genomic DNA library preparation procedure. Then, the amplicon library was paired-end sequenced (2 × 300) on an NGS platform (Shanghai BIOZERON Biotech. Co., Ltd., Shanghai, China) following the standard protocols. Raw sequences were deposited in the NCBI SRA database under accession number PRJNA1224727.
Raw sequences were first demultiplexed–filtered using Trimmomatic [39] complemented by custom perl scripts, implementing the following stringent criteria: (i) the 300 bp reads were truncated at positions with an average quality score < 20 within a 10 bp sliding window, with the subsequent removal of any truncated reads shorter than 50 bp; (ii) strict sequence-matching requirements were enforced—exact barcode matching with ≤2 nucleotide mismatches allowed in the primer regions, coupled with the elimination of reads containing ambiguous base calls; (iii) overlap-based assembly required minimum 10-bp overlaps between paired reads, discarding unassembled fragments. The curated sequences were then analyzed in QIIME2 (2020.11) [40] using the DADA2 algorithm to obtain the biological reads (i.e., amplicon sequence variants, ASVs) [41].
The bacterial and fungal sequences were classified with the SILVA (v.138) and UNITE databases, respectively. After the sequence classification, we retained only the classified bacterial and fungal ASVs. We then rarefied the 16S and ITS dataset to 11,579 and 3774 ASVs per sample, respectively.

2.5. Statistical Analysis

The microbial observed ASVs, Chao1, and phylogenetic diversity (PD) were calculated using the “microeco” package [42]. The microbial community niche breadth index was determined according to Levins [43], and the generalist species and specialist species were classified by calculating the occurrences of ASVs from 1000 permutations simulated via the “EcolUtils” package [44], and an ASV was classified generalist or specialist depending on whether its observed occurrence exceeded the upper 95% confidence interval or was below the lower 95% confidence interval [45,46]. The average variation degree (AVD) was calculated to estimate the soil microbial community stability among different treatments, where a lower AVD indicates higher microbiome stability [47]. Phylogenetic normalized stochasticity ratio (pNST) calculations were performed with the “NST” package to characterize the bacterial and fungal community assembly processes [48,49]. The pNST index had a cutoff at 0.5 to delineate the assembly processes that were more deterministic (<0.5) and more stochastic (>0.5).
To quantify soil microbial metabolic limitations in C, N, and P, we employed vector analysis based on extracellular enzyme stoichiometry. This method interprets enzyme activity ratios as proxies for microbial resource allocation. The vector length reflects the relative investment of the microbial metabolism in C versus nutrient acquisition, while the vector angle indicates the preferential limitation between P and N [50]. Specifically, vector length quantifies the degree of C limitation, that is, a longer vector length corresponds to a stronger microbial C restriction. The vector angle distinguishes N and P limitation, that is, an angle below 45° indicates N limitation; otherwise, it is the P limitation. These metrics were calculated using the following equations:
V e c t o r   l e n g t h = sqrt ( x 2 + y 2 )
V e c t o r   a n g l e = degrees ( atan 2 ( x , y ) )
where x represents ln(CBH + BG)/ln(AP) and y represents ln(CBH + BG)/ln(NAG + LAP).
Additionally, the effects of acid rain and the understory vegetation removal on soil physicochemical properties, enzyme activities, and the microbial community indices were quantified and compared as follows:
R = l n ( T C )
where T and C represent the specific values of physicochemical properties, enzyme activities, or microbial community indices for the treatments and control, respectively. Values of R > 0 indicate a positive effect (i.e., acid rain increased the variable value relative to the control), while R < 0 indicates a negative effect. The vector length, vector angle, and pNST was analyzed using the ANOVA test, and a post hoc Tukey test was performed to detect differences between individual treatments, with Holm correction applied to adjust the p-values for multiple comparisons, ensuring a robust statistical analysis. Statistical significance was determined at p < 0.05.
Redundancy analysis (RDA) was performed to examine the relationships between enzyme activities or their stoichiometry, soil physicochemical properties, and biotic factors using the “vegan” package. Key discriminating variables were identified through a ‘stepwise selection’ process. Variable importance quantification was implemented using the “rdacca.hp” package [51], which calculates the relative contribution of each explanatory variable to dependent variables. Finally, the “plspm” package was employed to develop a partial least squares path modeling (PLS-PM) to study the primary mechanisms on which acid rain and understory vegetation removal mediate the microbial metabolism limitation through soil physicochemical properties and the soil microbial community. The PLS-PM model fitness was assessed using the goodness-of-fit (GoF) index [52], with interpretation thresholds defined as weak (GoF > 0.1), moderate (GoF > 0.25), and strong (GoF > 0.36) [53]. All of the analyses were performed in R 4.4.0.

3. Results

3.1. Soil Physicochemical Properties and CO2 Release

Regardless of the acid rain treatment, understory vegetation removal significantly decreased the soil MC compared to the control (Figure 1), and the interaction of the understory vegetation and acid rain also had significant negative effect on the MC (p < 0.001; Figure 1). The soil pH value in the CK, UR, AR, and UA treatments was 6.28, 6.28, 6.19, and 6.22, respectively, indicating that acid rain slightly reduced the soil pH, but there were no significant difference among all of the treatments in the C. camphor plantation (Figure 1; Table S1). However, the DOC in the UR, AR, and UA treatments decreased by 26.32%, 3.83%, and 16.18%, respectively. The AN in UR, AR, and UA treatments decreased by 16.83%, 15.32%, and 25.99%, respectively. Moreover, understory vegetation removal and the interaction of understory removal with acid rain had an obviously negative effect on the DOC and AN (Figure 1). In addition, understory vegetation removal and acid rain had significantly negative main and interactive effects on the SOC (Figure 1). Compared with the CK, the UR, AR, and UA treatments decreased the SOC by 23.38%, 16.24%, and 18.34%, respectively. Only the understory vegetation had a significantly negative effect on the STN, and neither the understory vegetation removal nor acid rain had a marked effect on the STP in the C. camphor plantation (Figure 1). For the CO2 release, compared to the CK, there were significant positive effects in the AR and UA treatments (Figure 1).

3.2. Soil Enzyme Activities

In general, soil potential enzyme activities were significantly affected by either acid rain or understory vegetation removal in the C. camphor plantation (Figure 2). Specifically, acid rain had a significantly positive effect on the CBH and BG activities, understory vegetation removal had a markedly positive effect on the BG and had a significantly negative effect on the NAG and AP activities, while the understory vegetation and acid rain had a significantly positive interactive effect on the BG activity and a negative effect on the LAP activity in the C. camphor plantation (Figure 2). Compared to the CK, the enzyme vector lengths of the UR, AR, and UA increased by 9.08%, 10.35%, and 12.43%, respectively, and understory vegetation removal and acid rain had significantly positive main and interactive effects on the vector length (Figure 2). However, understory vegetation removal and acid rain did not have any marked effects on the vector angle (Figure 2). Additionally, the enzyme vector lengths were > 1 and the vector angles were > 45° in all of the treatments, indicating that the microbial metabolism was likely limited by both C and P, but P restriction was more obvious in the C. camphor plantation (Figure 2).

3.3. Soil Microbial Community

Acid rain and the interaction of understory vegetation removal and acid rain had significantly positive effects on the bacterial-observed ASVs, Chao1, and PD; however, only the understory vegetation removal had markedly positive effects on the fungal-observed species, Chao1, and PD in the C. camphor plantation (Figure 3).
To study the differences in the microbial community structure, we calculated the community-level habitat niche breadth indices, the AVD (average variation degree), and the phylogenetic normalized stochasticity ratio (pNST) in all of the treatments. For the bacterial community structure, compared to the CK, the niche breadth indices of the UR, AR, and UA decreased by 7.41%, 18.92%, and 15.19%, respectively, but the understory vegetation and acid rain had no significant interactive effect on niche breadth (Figure 3). Simultaneously, the UR had a significantly positive and negative effect on the specialist ratio and generalist ratio, respectively. Acid rain only had a markedly negative effect on the generalist ratio. Conversely, the AVD values for the bacterial community were 8.64%, 27.40%, and 19.50% higher in the UR, AR, and UA treatments than in the CK treatment. Moreover, the increase in the AR treatment was significant (Figure 3), and the lower AVD value indicates higher microbiome stability. The results indicated that the bacterial community in the CK was more stable than in the other three treatments, that is, understory vegetation removal and acid rain would make the bacterial community more vulnerable in the C. camphor plantation. However, for the fungal community, the understory vegetation removal had an obviously positive effect on the generalist ratio, and the understory vegetation and acid rain had significantly positive effect on the niche breadth indices and generalist ratio (Figure 3). Moreover, there was no significant difference in the fungal community AVD values across all of the treatments in the C. camphor plantation (Figure 3).
The pNST based on the null model was used to quantitatively assess the microbial community assembly process of the four treatments in the C. camphor plantation. For the bacterial community, the pNST values for all of the treatments were lower than 50%, indicating that the bacterial community assembly process was more deterministic in all of the treatments. Despite that, the pNST values were distinctly higher in the CK treatment, at 30.43%, 22.54%, and 24.01%, than in the UR, AR, and UA treatments (Figure 4), thus demonstrating that understory vegetation removal, acid rain, and its interaction strengthened the deterministic assembly process. Different from the bacterial community, an opposite trend was observed for the fungal community assembly. Specifically, the pNST values were 0.48, 0.60, 0.62, and 0.58 for the CK, UR, AR, and UA treatments, respectively, indicating that the fungal community assembly process changed from more deterministic to more stochastic (Figure 4). However, there was no significant difference between the treatments.

3.4. The Relationship of Soil Physicochemical Properties, Enzyme Activities, and Microbial Community

The RDA results indicated that the soil enzyme activities were significantly affected by the MC, bacterial AVD values, and fungal diversity (Figure 5), and their explained variations were 22.57%, 13.93%, and 4.19%, respectively, in this study. The NAG, LAP, and AP activities were strongly correlated with the MC and fungal diversity, while the BG and CBH activities were strongly related to the bacterial AVD values and microbial activities in the C. camphor plantation. However, the enzyme stoichiometries were significantly affected by the AN, MC, AVD_fungi, and pH, and their explained variations were 17.04%, 15.18%, 12.49%, and 11.08%, respectively. Moreover, the vector length was closely related to the AN and pH, while the vector angle was strongly correlated with the fungal AVD values in this study.
The PLS-PM showed the effects of the soil physicochemical properties, bacterial diversity, and bacterial stability on the microbial C limitation in the C. camphor plantation. The soil properties (−0.684), pH (−0.300), soil elements (−0.218), bacterial niche (−0.361), and bacterial stability (−0.364) had a negative total effect on the microbial C restriction, while acid rain (0.311), understory vegetation removal (0.554), and C-acquiring enzyme activities (0.701) showed a positive total effect on it (Figure 6). Meanwhile, the soil properties (−0.477) had a negative effect on the microbial P restriction, but understory vegetation removal (0.391), soil elements (0.111), fungal generalists ratio (0.338), fungal niche breadth (0.366), and fungal stability (0.537) had a positive effect on the microbial P limitation in the C. camphor plantation.

4. Discussion

4.1. Responses of Soil Physicochemical Properties, CO2 Release, and Enzyme Activities

In the present study, simulated acid rain had a noticeable negative effect on the SOC, which was similar to the results published by previous studies [3,5]. There are several reasons to explain the findings. One possible explanation is that the decreased SOC could be ascribed to the solubility of the soil organic C promoted by acid rain and leached out [3,15,25], particularly in subtropical plantations with a high rainfall intensity. Another contributing factor involved microbial metabolic trade-offs [54,55], in which, under acid rain stress, soil microorganisms might reallocate C and energy from growth and maintenance toward stress tolerance and resource acquisition, thereby reducing the SOC stabilization [30,56]. This interpretation aligns with the elevated CO2 release and extracellular enzyme stoichiometry patterns observed in our study, which reflect intensified microbial C limitation and organic matter mineralization. It is commonly anticipated that, once organic matter decomposition increases, the microbial-derived CO2 release from soil will also increase [57]. Moreover, the increased C-acquiring enzyme CBH and BG activities under acid rain also support this interpretation. These results showed that soil microorganisms secreted more extracellular enzymes to metabolize organic matter in order to resist adverse environmental conditions. Furthermore, acid rain might indirectly reduce SOC inputs by impairing the plant photosynthetic efficiency and root exudate production, thereby reducing the amount and quality of C entering the soil [3,12,15]. Collectively, these mechanisms—leaching, microbial metabolic shifts, and reduced carbon inputs—synergistically drive SOC depletion in acid rain-affected subtropical plantations.
Similar with acid rain, there was a strong negative effect of understory vegetation removal on the SOC, but its mechanism was different. It is generally accepted that understory vegetation has a substantial impact on the soil microclimate and nutrient cycling in forest ecosystems [14,16]. Understory vegetation removal can change the amount and quality of C input to the soil [14,23,24]. Therefore, on the one hand, the decreased SOC could be ascribed to directly reducing the aboveground and belowground C quantity inputs [23,24]. On the other hand, understory vegetation removal reduced the soil nutrient availability, such as soil DOC and AN, which could intensify the microbial C restriction and subsequently accelerate soil organic matter mineralization, ultimately reducing the SOC [19,24,58]. The latter interpretation is also supported by the results of the increased C-acquiring enzyme CBH and BG activities in the present study. Additionally, the presence of understory vegetation can alleviate soil surface runoff and nutrient loss by modifying the amount of throughfall interception [14,59], which was supported by the reduced soil MC, DOC, and AN in understory vegetation removal in this study. Consequently, considering the significant influences from understory vegetation on soil properties, the above explanations probably account for the patterns observed here. Collectively, our results underscored the crucial role of understory vegetation in regulating soil C cycling in the C. camphor plantations.

4.2. Responses of Microbial Community

In our study, the bacterial diversity was notably positively affected by acid rain and its interaction with understory vegetation removal, but the fungal diversity was only significantly positively affected by understory vegetation removal. In general, acid rain is expected to reduce soil bacterial diversity due to its toxic effects on bacterial communities [14,25]. However, we observed an increase in the bacterial diversity under the AR, which was consistent with prior studies reporting similar trends [3,60,61]. One possible reason for these findings is that soil acidification selectively suppresses acid-sensitive bacterial taxa while favoring acid-tolerant oligotrophic species through niche filtering [62,63]. Another possible reason is that different nutrient availability might drive shifts in the microbial diversity [26,64]; the soil low C availability induced by acid rain was likely to be mutually disadvantaged to bacteria with rather copiotrophic lifestyles, but was likely to be advantaged to oligotrophic bacteria through interspecific competition [25,65]. Previous studies also reported that the increase in C and P limitation significantly increased bacterial diversity [65], which was consistent with our study. In addition, these explanations were supported by the decrease in bacterial generalists and the increase in bacterial specialists in this study. Simultaneously, the change in bacterial generalists and specialists resulted in the notable reduction in the bacterial community niche breadth. The results indicated that the bacterial community was significantly affected by acid rain. In contrast, fungal diversity was not significantly affected by acid rain, but instead showed a pronounced response to understory vegetation removal, likely because fungi are more resistant to acidity [30,66]. The possible reasons for the significant increase in fungal diversity in the UR are that understory vegetation removal reduced the input labile organic compounds, such as the soil DOC, and fungi are often associated with recalcitrant organic compounds [30,32], which was supported by the increase in generalists.
In contrast to the microbial diversity, both acid rain and understory vegetation removal significantly destabilized the bacterial communities. This aligned with previous findings that canopy nitrogen addition and understory removal reduced bacterial and fungal community stability [67]. However, the cause of this phenomenon was not exactly the same. Although high microbial diversity is generally associated with enhanced community stability [47,67], in contrast to this, in the present study, bacterial communities with a high diversity had low community stability, which was presumably due to the high diversity of bacteria caused by the increase in specialists and the decrease in generalists. As a result, these specialists could lead to intense competition and less cooperation with each other [3], which could impede the exchange and efficient use of soil nutrients and resources, ultimately destabilizing the bacterial community structure [67]. Simultaneously, fewer generalists were present, leaving the community more vulnerable to environmental change [68], which could also destabilize the community. Alternatively, another possible reason was that acid rain and understory vegetation removal led to more deterministic processes of bacterial community assembly through niche partitioning and species interactions [67,69], which will eventually destabilize the community. However, the response of the fungal community to acid rain and understory vegetation removal was relatively stable. The fungal community stability and assembly processes were not notably influenced by acid rain and understory vegetation removal. The following reason might be used to explain the findings. On the one hand, fungi are known to be more adaptable to complex environmental variations, such as acid rain and drought [30]. Moreover, previous studies also reported that the fungal community did not respond sensitively to soil acidification and understory vegetation removal [25,70]. On the other hand, fungi can make more effective use of recalcitrant organic matter, whereas acid rain and understory vegetation removal might mainly reduce labile organic matter and eventually exacerbate the utilization of recalcitrant C [30,32], which was supported by the increase in the enzyme vector length. Therefore, the stability of fungal communities contributed to the absence of significant differences in fungal community assembly processes across the treatments.
However, the interactive effects of acid rain and understory vegetation removal did not significantly affect bacterial and fungal community assembly processes and stability, likely because the specialist and generalist ratios in the UA were not significantly affected, which consequently maintained the bacterial community niche breadth and stabilized the bacterial community. However, the stability of the fungal community structure might be attributed mainly to the fungal diversity stability. Overall, this work revealed that the response of the soil bacterial and fungal communities to acid rain and understory vegetation removal was different in the C. camphor plantations, that is, the soil bacterial community was generally more responsive to acid rain and understory vegetation removal in the C. camphor plantations, which was supported our first hypothesis.

4.3. Relationship Among Measured Variables

Prior studies have reported that shifts in microbial C source utilization resulted from changes in the microbial community structure [65,71], which was consistent with our study, showing that microbial community stability significantly affected the microbial metabolic nutrition restriction. Specifically, in the present study, the RDA and PLS-PM indicated that acid rain and understory vegetation removal affected soil enzyme activities and their stoichiometry by altering soil physicochemical properties and microbial community stability. These results supported our second hypothesis. C-acquiring enzymes and the vector length were mainly regulated by the soil bacterial community, while N- and P-acquiring enzymes and the vector angle were mainly affected by the soil fungal community. It is possible to attribute this to microbial resource usage preferences, with bacteria being able to use labile C more efficiently and fungi being able to use recalcitrant C more effectively [30,32], while N and P are more often contained in complex organic matter. Therefore, the shift in the nutrient availability and microclimate under acid rain and understory vegetation removal would amplify the impact of environmental filtering on the bacterial community and reduce its stability [67]. However, the unstable bacterial community structure was mainly due to a decrease in the community niche breadth, which led to increased competition and decreased cooperation, requiring more energy to maintain, and ultimately to stronger microbial C limitation, which is in agreement with a previous study [65]. However, probably because fungi are more resistant to external disturbances, the fungal community stability was not significantly affected by acid rain and understory vegetation removal, allowing the vector angle to remain relatively stable. These interpretations were supported by the results of our PLS-PM. Collectively, our results elucidated the linkages between microbial stability and microbial nutrient limitation under acid rain and understory vegetation removal in the C. camphor plantations. While the bacterial stability was closely tied to the C limitation, the fungal stability correlated with the P limitation. However, given that this study is only a short-term experiment and lacks continuous samples, the underlying mechanisms between microbial community stability and diversity and microbial metabolism require further investigation in the future.

5. Conclusions

This study explored the impacts of simulated acid rain and understory vegetation removal on the biological activity of the soils in a C. camphor plantation. The findings revealed the distinct impacts of these external disturbances. Acid rain significantly altered the SOC, CO2 release, enzyme activities, and their stoichiometry, while also restructuring the microbial community. Understory vegetation removal markedly reduced the MC and nutrient availability, affected the enzyme stoichiometry balances, and also shifted the microbial community. The elevated bacterial diversity led to reduced bacterial community stability, likely due to the increase in specialists. In contrast, the fungal community demonstrated greater resistance to external perturbations, attributed to their metabolic preference and tolerance to osmotic stress. Mechanistically, the bacterial community instability was firmly closely related to the microbial C limitation (i.e., the vector length), whereas the fungal community stability was more closely associated with the microbial P limitation (i.e., the vector angle). Overall, our study advanced the understanding of the mechanisms by which acid rain and understory vegetation removal destabilize microbial communities and increase microbial metabolic limitations. Critically, they highlight the indispensable role of understory vegetation in buffering soil microclimates, sustaining nutrient cycling, and maintaining community stability in plantations. Finally, these finding emphasized the need for integrated forest management strategies to conserve and manage soil ecosystems in subtropical plantations.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f16030525/s1, Table S1: Soil physicochemical properties in treatments in the Cinnamomum. camphor (Linn) Presl plantation.

Author Contributions

Conceptualization, X.H. and Z.H.; methodology, Z.H. and Y.L. (Yonghui Lin); software, Z.H. and Y.L. (Yini Liu); validation, Y.L. (Yonghui Lin); formal analysis, X.H. and Z.H.; investigation, Y.L. (Yini Liu), Z.H. and X.K.; data curation, Z.H. and X.K.; writing—original draft preparation, Z.H. and Y.L. (Yini Liu); writing—review and editing, X.H., Z.H. and H.L.; visualization, Z.H. and Y.L. (Yini Liu); projection administration, X.H. and Z.H. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the General Program of Scientific Research Foundation of Hunan Provincial Education Department (22C0278), the Jishou University Doctoral Talent Introduction Project (1122007), the National Natural Science Foundation of China (grant numbers 32060332, 31670624, and 32160356), the Youth Program of Scientific Research Foundation of Hunan Provincial Education Department (24B0500), and the Natural Science Foundation of Hunan Province (2025JJ60205 and 2025JJ50112).

Data Availability Statement

The datasets in this study are available in Supplementary Material Table S1.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The effect sizes of simulated acid rain and understory vegetation removal on the soil physicochemical properties and CO2 release in the C. camphor plantation. Effect sizes are presented as mean values ± 95% confidence intervals (n = 4). *** p < 0.001, ** p < 0.01, and * p < 0.05. UR stands for understory vegetation removal, AR stands for simulated acid rain, and UA stands for understory vegetation removal and simulated acid rain.
Figure 1. The effect sizes of simulated acid rain and understory vegetation removal on the soil physicochemical properties and CO2 release in the C. camphor plantation. Effect sizes are presented as mean values ± 95% confidence intervals (n = 4). *** p < 0.001, ** p < 0.01, and * p < 0.05. UR stands for understory vegetation removal, AR stands for simulated acid rain, and UA stands for understory vegetation removal and simulated acid rain.
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Figure 2. The effect sizes of simulated acid rain and understory vegetation removal on the soil enzyme activities (a), the variation in the vector lengths and angles across the treatments (b,c), potential C and nutrient limitations across the treatments, (d) and the enzyme vector model of the extracellular enzyme stoichiometry (e) in the C. camphor plantation. Effect sizes are presented as mean values ± 95% confidence intervals (n = 4). *** p < 0.001, ** p < 0.01, and * p < 0.05. Distinct lowercase letters indicate significant differences between treatments (p < 0.05). UR stands for understory vegetation removal, AR stands for simulated acid rain, and UA stands for understory vegetation removal and simulated acid rain.
Figure 2. The effect sizes of simulated acid rain and understory vegetation removal on the soil enzyme activities (a), the variation in the vector lengths and angles across the treatments (b,c), potential C and nutrient limitations across the treatments, (d) and the enzyme vector model of the extracellular enzyme stoichiometry (e) in the C. camphor plantation. Effect sizes are presented as mean values ± 95% confidence intervals (n = 4). *** p < 0.001, ** p < 0.01, and * p < 0.05. Distinct lowercase letters indicate significant differences between treatments (p < 0.05). UR stands for understory vegetation removal, AR stands for simulated acid rain, and UA stands for understory vegetation removal and simulated acid rain.
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Figure 3. The effect sizes of simulated acid rain and understory vegetation removal on the soil microbial alpha diversity, niche breadth, specialists, generalists, and AVD in the C. camphor plantation. Effect sizes are presented as mean values ± 95% confidence intervals (n = 4). *** p < 0.001, ** p < 0.01, and * p < 0.05. UR stands for understory vegetation removal, AR stands for simulated acid rain, and UA stands for understory vegetation removal and simulated acid rain.
Figure 3. The effect sizes of simulated acid rain and understory vegetation removal on the soil microbial alpha diversity, niche breadth, specialists, generalists, and AVD in the C. camphor plantation. Effect sizes are presented as mean values ± 95% confidence intervals (n = 4). *** p < 0.001, ** p < 0.01, and * p < 0.05. UR stands for understory vegetation removal, AR stands for simulated acid rain, and UA stands for understory vegetation removal and simulated acid rain.
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Figure 4. The effect of simulated acid rain and understory vegetation removal on the soil microbial community assembly process in the C. camphor plantation. Distinct lowercase letters indicate significant differences between the treatments (p < 0.05). UR stands for understory vegetation removal, AR stands for simulated acid rain, and UA stands for understory vegetation removal and simulated acid rain.
Figure 4. The effect of simulated acid rain and understory vegetation removal on the soil microbial community assembly process in the C. camphor plantation. Distinct lowercase letters indicate significant differences between the treatments (p < 0.05). UR stands for understory vegetation removal, AR stands for simulated acid rain, and UA stands for understory vegetation removal and simulated acid rain.
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Figure 5. Effects of soil physicochemical properties and microbial community on soil enzyme activities (a) and their stoichiometry (c), and individual explanation ratios of predictors (b,d) calculated via hierarchical variation partitioning in the C. camphor plantation. UR stands for understory vegetation removal, AR stands for simulated acid rain, and UA stands for understory vegetation removal and simulated acid rain.
Figure 5. Effects of soil physicochemical properties and microbial community on soil enzyme activities (a) and their stoichiometry (c), and individual explanation ratios of predictors (b,d) calculated via hierarchical variation partitioning in the C. camphor plantation. UR stands for understory vegetation removal, AR stands for simulated acid rain, and UA stands for understory vegetation removal and simulated acid rain.
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Figure 6. The primary mechanism of acid rain and understory vegetation removal mediating the microbial metabolism C limitation (a) and P limitation (b) through soil physicochemical properties and microbial community using partial least squares path modeling. Red and blue solid arrows indicate significant positive and negative paths, respectively. Soild and dash line indicate significant and non-significant paths. The thickness represents the strength of the path. *** p < 0.001, ** p < 0.01, and * p < 0.05. The microbial metabolism C limitation is represented by the vector length, while the microbial metabolism P limitation is represented by the vector angle.
Figure 6. The primary mechanism of acid rain and understory vegetation removal mediating the microbial metabolism C limitation (a) and P limitation (b) through soil physicochemical properties and microbial community using partial least squares path modeling. Red and blue solid arrows indicate significant positive and negative paths, respectively. Soild and dash line indicate significant and non-significant paths. The thickness represents the strength of the path. *** p < 0.001, ** p < 0.01, and * p < 0.05. The microbial metabolism C limitation is represented by the vector length, while the microbial metabolism P limitation is represented by the vector angle.
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MDPI and ACS Style

He, Z.; Liu, Y.; Lin, Y.; Kong, X.; Lin, H.; He, X. The Effect of Acid Rain and Understory Vegetation Removal on the Biological Activity of the Soils of the Cinnamomum camphora (Linn) Presl Plantation. Forests 2025, 16, 525. https://doi.org/10.3390/f16030525

AMA Style

He Z, Liu Y, Lin Y, Kong X, Lin H, He X. The Effect of Acid Rain and Understory Vegetation Removal on the Biological Activity of the Soils of the Cinnamomum camphora (Linn) Presl Plantation. Forests. 2025; 16(3):525. https://doi.org/10.3390/f16030525

Chicago/Turabian Style

He, Zaihua, Yini Liu, Yonghui Lin, Xiangshi Kong, Hong Lin, and Xingbing He. 2025. "The Effect of Acid Rain and Understory Vegetation Removal on the Biological Activity of the Soils of the Cinnamomum camphora (Linn) Presl Plantation" Forests 16, no. 3: 525. https://doi.org/10.3390/f16030525

APA Style

He, Z., Liu, Y., Lin, Y., Kong, X., Lin, H., & He, X. (2025). The Effect of Acid Rain and Understory Vegetation Removal on the Biological Activity of the Soils of the Cinnamomum camphora (Linn) Presl Plantation. Forests, 16(3), 525. https://doi.org/10.3390/f16030525

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