1. Introduction
Carbon (C) input from plants into soil is an important flux in the terrestrial C cycle, and it is crucial not only for C accumulation, but also for the maintenance of soil fertility and ecosystem stability and function [
1,
2]. Soil organic carbon (SOC) is thought to derive mostly from the decomposition of litter [
3]. However, recent studies have found that SOC is mainly derived from roots and from the microbial community around them [
4]. Plant-microbe interactions in soil play a central role in terrestrial ecosystem functions and may control up to half of the total carbon dioxide (CO
2) released from terrestrial ecosystems at the global scale [
5]. Roots can strongly change the turnover of soil organic matter and greatly affect soil C accumulation [
6]. While mycorrhizae supply nitrogen and phosphorus to plants, the mycelium transfers a large amount of photosynthetic organic C to the underground [
7]. Additionally, the root-microbial system can transfer organic C directly from the plant to the soil carbon pool [
8], and the biomass and secretions of microbes are also large sources of SOC [
9]. Therefore, studies on root-microbial systems are essential for revealing the mechanisms regulating SOC accumulation.
Forests are the largest C sinks worldwide, accounting for about two-thirds of the total C accumulation in terrestrial ecosystems [
10]. Tree roots have the potential to shift underground microbial compositions [
11,
12,
13], and, according to the ecological stoichiometry theory, this change can affect organic matter decomposition and biogeochemical cycling [
6,
14,
15]. Tree species and microbial diversity also affect soil C accumulation in forests [
11,
16,
17]. Thus, changes in the physical, chemical, and biological properties of the soil affected by tree roots have been evaluated in recent studies [
17,
18,
19,
20]. However, limitations due to the use of traditional techniques hinder an accurate evaluation of soil microbial taxa, which has greatly delayed our understanding of several core ecosystem processes, such as variations in microbial communities, soil C accumulation, ecosystem succession, and ecosystem functions [
20,
21]. Identifying and quantifying the relative influence of root-microbial systems in forests remains a challenge for researchers [
22]. Thus, there is still a limited understanding of the relationship between soil microbial taxa and soil C accumulation in a changing environment, especially in restored forest ecosystems.
Plant succession is intrinsically linked to the succession of microbes, as these utilize all forms of plant-derived C and are important drivers of plant community productivity and diversity [
23]. Root-associated microbes have evolved close links to living plants, albeit being a highly heterogeneous group [
24]. Roots are colonized by obligate or facultative microbes, but also by many fungi commonly termed root endophytes, which have a variable and only partially known trophic and functional relationship with plants [
25]. Similar to the aboveground biomass of plants, rhizosphere microbes in forests also present a succession from the degraded ecosystem to climax community during the restoration of forest ecosystems [
26]. However, microbial succession has received less attention than the successional dynamics of plants [
24].
Afforestation, as a type of ecosystem degradation counter measure, can add terrestrial C sink capacity and alleviate atmospheric CO
2 accumulation [
27]. In the last century, the “Grain for Green” Project conducted nationwide aimed to rehabilitate and recover degraded ecosystems in China. The conversion of cropland to forest offers opportunities to conserve soil and water and improve the microclimate, among other benefits [
28].
Pinus tabuliformis Carr. and
Quercus variabilis Blume. are two primary conifer and broad leave afforestation species due to their capacity to limit soil erosion, their well-developed root system, and their role in C restoration [
29,
30]. Although the effects of afforestation on C restoration have been studied in China, the microbial mechanisms of C accumulation following afforestation have not been explored [
28]. Therefore, a better understanding of the effects of soil microbial communities on soil C dynamics is fundamental for providing further insight into the microbial mechanisms of C accumulation following afforestation.
Because root-associated microbes are important for SOC accumulation in forest ecosystems, disclosing key microbial taxa for SOC accumulation driven by tree roots is not only helpful for a better understanding of the microbial mechanisms of C accumulation, but it can also improve forest management, degraded ecosystem restoration, and afforestation. In the present study, we estimated variations in microbial communities and SOC accumulation driven by tree roots during the succession from a degraded to climax forest ecosystem using high-throughput DNA sequencing. The current study aimed to reveal the variations occurring in rhizosphere microbial species in restored forest ecosystems and which species benefit rhizosphere SOC accumulation. These results might contribute to future research and the management of forest C accumulation.
3. Results
3.1. Changes in Soil Bacterial and Fungal Community Compositions
High-throughput sequencing and subsequent quality filtering allowed 476,667 bacterial and 496,873 fungal clean reads (tags) to be obtained with the 338F/806R (bacterial 16S rRNA) and ITS1F/ITS2 (fungal ITS) primer sets, respectively, across all soil samples. The number of bacterial clean tags found in each soil sample was 77, 617 in TPR, 73,809 in MPR, 73,704 in CS, 73,154 in NFS, 57,130 in DPR, 48,660 in TQR, 37,756 in MQR, and 34,837 in FPR; fungal clean tags reached 111,512 in TPR, 75,803 in MPR, 68,202 in DPR, 56,802 in MQR, 55,589 in NFS, 49,117 in TQR, 41,797 in CS, and 38,051 in FPR.
Regarding bacterial communities, Cyanobacteria were dominant in CS (45.30%) and DPR (38.36%); Proteobacteria and Actinobacteria in MQR (35.18% and 28.41%, respectively), TQR (33.05% and 25.35%, respectively), and FPR (31.43% and 44.92%, respectively); and Proteobacteria in TPR (44.01%), MPR (38.57%), and NFS (46.39%) (
Figure 1). Notably, Proteobacteria and Actinobacteria were more abundant in MQR, TQR, TPR, MPR, and NFS groups (containing living roots), whereas Cyanobacteria were more abundant in CS and DPR groups (not containing living roots) (
Figure 1). Fungal communities of all groups except NFS were dominated by Ascomycota: 91.15% in CS, 87.25% in DPR, 79.58% in MPR, 66.63% in MQR, 62.98% in TPR, and 60.99% in FPR (
Figure 2). Basidiomycota accounted for about 59.82% of the fungal community in NFS (
Figure 2).
Soil bacterial and fungal community similarities are shown in
Figure 3 and
Figure 4, respectively. Based on hierarchical clustering, bacterial CS communities were most similar to DPR communities, TPR communities were most similar to MPR communities, TQR communities were most similar to MQR communities, and NFS communities were most similar to FPR communities. Fungal TPR and MPR communities, and TQR and MQR communities, were also clustered, but CS and DPR were in different branches of the dendrogram. Bacterial and fungal FPR and NFS were the least similar (
Figure 3 and
Figure 4).
3.2. Changes in SOC Accumulation and Microbial Taxa
Soil organic C content (%) was lower in CS than in all other groups, except FPR, and decreased in the following order: NFS > MQR > TPR > TQR > DPR > MPR > CS > FPR (
Figure 5a). After one year, SOC accumulation was 2.49, 1.81, 1.20, 0.91, 0.62, and −0.19 g/cm
3 in MQR, TPR, TQR, DPR, MPR, and FPR, respectively (
Figure 5b). Thus, SOC contents were greatly affected by tree roots.
The abundances of Proteobsacteria, Acidobacteria, and Gemmatimonadetes were positively correlated with SOC accumulation in groups comprising
P. tabuliformis living roots (TPR and MPR;
Table 1), and Proteobacteria, Actinobacteria, Chloroflexi, and Gemmatimonadetes abundances were positively correlated with SOC accumulation in groups comprising
Q. variabilis living roots (TQR and MQR;
Table 1). The abundance of Cyanobacteria was negatively correlated with SOC accumulation in all groups comprising living tree roots (TPR, MPR, TQR, and MQR;
Table 1). The abundances of Verrucomicrobia and Gemmatimonadetes were positively correlated with SOC accumulation in DPR and Proteobacteria and Acidobacteria abundances were positively correlated with SOC accumulation in FPR (
Table 1).
The abundances of Basidiomycota and Zygomycota were positively correlated with SOC accumulation in all groups containing living tree roots (TPR, MPR, TQR, and MQR;
Table 2), while Ascomycota abundance was negatively correlated with SOC in these groups. Zygomycota abundance was positively correlated and Ascomycota abundance negatively correlated with SOC accumulation in FPR (
Table 2). No significant correlation was found between SOC accumulation and fungi abundance in DPR (
Table 2).
5. Conclusions
In this study, we demonstrated that microbial community composition changed according to root-associated SOC accumulation along the primary succession of forest ecosystems. Soil organic C contents were greatly affected by tree roots and rhizomicrobes in the primary stage and climax of succession. Tree roots can shift underground microbial compositions and benefit SOC restoration in degraded ecosystems. Proteobacteria were keystone organisms for root-driven C accumulation in primary succession and Basidiomycota were the keystone for root-driven C accumulation in climax communities. This work has therefore enabled a deeper insight into root-associated microbial communities, increased awareness on root-associated SOC accumulation mechanisms, and contributes to a better understanding of microbial ecology in forest succession.
Based on our results, we suggest the forest management practices listed below. The establishment of P. tabuliformis and Q. variabilis plantations in the study area promoted SOC accumulation and the recovery of soil ecological environment; nevertheless, different species should be used in different circumstances during afforestation. Forest fire was harmful for both microbial community and SOC restoration, and, therefore, should be avoided in forest management. Microbial biofilms should be filtered from the apical region of P. tabuliformis roots or middle region of Q. variabilis roots and applied to soil to facilitate C accumulation in degraded forests and to improve the abundance of beneficial rhizomicrobes in afforestation.
In order to achieve the consistency of the study, we only experimented in the site mentioned. We are not sure whether these results would occur in other locations since the geographic factors were ignored. For this reason, the microbial phyla cannot be fully correlated with root-associated SOC in other forests. Whether the phyla were determined by the rhizosphere environment or the locations is still an important question and needs to be clarified. More replicates and geographic factors should be taken into account in future studies.