Natural restoration of degraded karst vegetation shifts the acquisition strategy of soil microbial phosphorus by enhancing organic phosphorus and decreasing inorganic phosphorus cycling potentials

doi:10.21203/rs.3.rs-2465720/v1

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Research Article

Natural restoration of degraded karst vegetation shifts the acquisition strategy of soil microbial phosphorus by enhancing organic phosphorus and decreasing inorganic phosphorus cycling potentials

https://doi.org/10.21203/rs.3.rs-2465720/v1

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published 12 May, 2023

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Aims Soil phosphorus (P) cycling in karst regions is mainly regulated by microbial activities. Natural restoration has been widely adopted in the degraded karst regions of southwestern China. However, the responses of functional genes and microbial communities involved in soil P cycling to revegetation have not been well characterized.

Methods We used metagenomic sequencing to investigate the genes and microorganisms related to soil P cycling derived from natural restoration stages (shrubbery, TG; secondary forest, SG; old-growth forest, OG) in the southeast of Guizhou Province, China.

Results Natural restoration affected the composition of soil P cycling genes. When TG returned to OG, the relative abundance of organic P (OP) mineralization genes increased from 45.78% to 48.38%, while the genes related to inorganic P (IP) solubilization decreased from 27.19% to 25.03%. Compared to aboveground plant diversity, soil nutrients more affected the relative abundances of OP and IP genes. Structural equation model (SEM) further indicated that soil nutrients directly drove the increase in the relative abundance of OP genes and indirectly impacted the relative abundance of IP genes. We also found that Proteobacteria (38.97%–52.72%) and Actinobacteria (13.44%–29.34%) were the main contributors to soil OP and IP cycling genes but their contributions varied among the restoration stages.

Conclusions Natural restoration of the degraded karst vegetation shifted the acquisition strategy of soil microbial P by enhancing OP but decreasing IP cycling potentials. This study provides a novel insight into the regulation of P cycling in the ecological restoration of degraded karst regions from microbial perspective.

Metagenomics

Functional gene

Soil phosphorus cycling

Karst region

Natural restoration

Phosphorus (P) is an essential element for all biotas and participates in many metabolic processes, such as energy transfer, respiration, signal transduction, and macromolecular biosynthesis (Huang et al., 2005). Based on the data of 258 different soils from 41 publications, P reserves in soils are sufficient to sustain maximum agricultural production worldwide for approximately 350 years (Menezes-Blackburn et al., 2013). Although P is abundant in soils, it exists mainly in unavailable inorganic and organic forms and cannot be used directly by plants and microorganisms. Therefore, the deficiency of bioavailable P is widespread in terrestrial ecosystems (Elser et al., 2007). The cycling of soil nutrients depends on microbial activities; this is especially true of P cycling since it cannot be introduced into soils by atmospheric deposition like C and N (Chadwick et al., 1999; Walker and Syers, 1976). Most soil P is sequestered in recalcitrant minerals and organic compounds, and only a small part of soil P is utilized by microorganisms and plants as only inorganic orthophosphate forms (H₂PO₄²⁻ or H₂PO₄⁻) (Schneider et al., 2019). Therefore, effective strategies, including secreting organic acids to solubilize inorganic P (IP) and synthesizing hydrolytic enzymes to mineralize organic P (OP), have been developed by soil microorganisms to fulfill their own growth needs and enhance the abundance of plant available P in soils.

The transformation processes of microbial P are mainly mediated by several functional gene groups and include OP mineralization, IP solubilization, P uptake and transport, and P-starvation response regulation (Dai et al., 2019). When facing the scarcity of bioavailable P, microorganisms upregulate the expression of the genes coding for organic anions to solubilize IP or enzymes to mineralize OP. Organic anions play their roles via acidification, chelation, and exchange reactions to desorb sparingly available IP forms (Jones, 1998; Oburger et al., 2011; Wang et al., 2016), including gluconic acid, acetic acid, fumaric acid, formic acid, and malic acid (Pradhan et al., 2017; Rawat et al., 2021). Among them, gluconic acid is usually regarded as the most common agent directly participating in the oxidation pathway of glucose and the acidification of the periplasmic space. The gcd genes coding for quinoprotein glucose dehydrogenase (PQQ-GDH) that govern the production of gluconic acid have been widely studied and reported (Santos-Torres et al., 2021). In addition, the microbial conversion of soil OP compounds into bioaccessible forms is achieved through the action of organophosphorus hydrolases, such as alkaline phosphatases (phoD, phoA, and phoX), acid phosphatases (olpA), phosphodiesterases (ugp), phosphonatases (ptxD), phytases (appA), and C-P lyases (phn) (Pradhan et al., 2017). Among them, alkaline phosphatases (EC: 3.1.3.1) are deemed to be principally derived from soil microbes and are non-specific enzymes that catalyze the hydrolysis of ester-phosphate bonds of many orthophosphate monoesters (Spohn and Kuzyakov, 2013; Wasaki et al., 2018; Wei et al., 2019), thereby contributing to P bioavailability in soils. Furthermore, the changes in the availability conditions of soil P trigger the expression of genes related to the uptake and transport of microbial P and the system of P-starvation response regulation. The genes coding for the system of P uptake and transport allow microbes to assimilate IP under P-low and P-rich conditions through the high-affinity (pst) and low-affinity (pit) IP transporters, respectively (Dai et al., 2019; Rawat et al., 2021). A set of genes, including phoU, phoR, and phoB, involved in the system of P-starvation response regulation enable soil microorganisms to utilize external or alternative P sources during phosphate limitation (Bergkemper et al., 2016; Liang et al., 2020). Recent studies indicated that these genes were closely connected to the genes involved in P uptake and transport and alkaline phosphatase production, especially under P-low conditions (Dai et al., 2019).

Karst ecosystems account for around 6% of the area of China’s land surface and are widely located in the southwest region (Jiang et al., 2014). However, they are fragile due to particularities, such as soluble carbonate rock, calcium abundance, soil scarcity and water leakage. (Yuan, 2001). In the last century, under the pressure of increasing population, large areas of forests in karst regions were cut down and destroyed for agriculture. Consequently, ecological disasters, such as soil erosion, vegetation loss, and rocky desertification, were formed (Jiang et al., 2014). After that, the vegetation restoration project named “Grain for Green Project” has been widely implemented in karst regions to overcome ecological degradation (Qi et al., 2013). Natural restoration in degraded karst regions is accompanied by changes in aboveground plants and belowground soils. Soil microbial communities are assumed to shift to adapt to the changes in these environmental factors (Salvioli Di Fossalunga and Novero, 2019; Wang et al., 2018). Numerous studies revealed that soil microorganisms mediated P cycling across various functions, and the change in microbial communities may affect the transformation and availability of soil P (Huang et al., 2017; Li et al., 2013; Wang et al., 2018). Therefore, the natural restoration of the degraded karst vegetation may greatly impact functional genes and microorganisms involved in the transformation and cycling of soil P.

The study mainly aimed (i) to investigate the composition of the functional genes involved in soil P cycling affected by the natural restoration of the degraded karst vegetation; (ii) to detect the relationships between soil nutrients, plant diversity indexes, and the abundance of the shifted P cycling genes; (iii) to identify the key factors driving these shifts; (iv) to recognize the main microbial phyla contributing to soil P transformation. Our findings may provide considerable knowledge to promote ecological restoration in karst regions by regulating soil P cycling.

Site description

The study area is located in Maolan National Nature Reserve in the southeast of Guizhou Province, China (25°09′–25°20′ N, 107°52′–108°05′ E, 550–850 m above sea level). The Reserve has a humid subtropical monsoon climate with an average annual temperature of 15.3℃, an annual relative humidity of 83%, and an annual precipitation of 1 752 mm. The precipitation between April and October accounts for 80% of the annual precipitation. The soil in the studied karst region is mainly black lime soil, and the vegetation is mainly subtropical evergreen deciduous broad-leaved mixed forest. We selected the shrubbery (TG), secondary forest (SG), and old-growth forest (OG) stages as the representatives of three typical natural restoration stages in the studied karst region. Then, 10 TG plots (10 m × 10 m), 10 SG plots (30 m × 30 m), and 10 OG plots (30 m × 30 m) were randomly selected and constructed based on the standard raised by Condit in 1998 (Condit, 1998). The distance between every two adjacent plots was more than 50 m to eliminate mutual interference.

Plant survey

A plant survey was then conducted on each plot. Plants with a diameter at breast height greater than 1 cm were investigated and recorded. In the TG restoration stage, Mahonia fortunei (Lindl.) Fedde, Lindera communs Hemsl., and Swida parviflora (Chien) Holub are the main vegetation species. Clausena dunniana Levl., Platycarya strobilacea Sieb. et Zucc., and Viburnum henryi Hemsl were the dominant vegetation species in the SG restoration stage. Acer wangchii Fang, Clausena dunniana Levl., and Boniodendron minus (Hemsl.) T. Chen were the dominant species in the OG stage. For plant diversity indexes, we defined Plant Species as the total number of species in each plot. We then calculated the plant Shannon-Wiener index (Shannon), Pielou Evenness index (Pielou), and Rarefied Species Richness index (Rarefied SR) for each plot based on the abundance information on Plant Species:

$$Plant Shannon=-{\sum }_{i=1}^{s}{p}_{i}ln{p}_{i}$$

Plant Pielou = Plant Shannon / log(S) (2)

$Plant Rarefied SR={\sum }_{i=1}^{s}(1-{q}_{i}$ ) (3)

where ${q}_{i}=\frac{\left(\frac{N-{x}_{i}}{n}\right)}{\left(\frac{N}{n}\right)}$S represents the total number of species. p_i represents the proportion of the i-th species to the total. x_i is the count of species i. $\left(\frac{N}{n}\right)$ is the binomial coefficient, or the number of ways we can choose n from N. q_i gives the probability that species i does not occur in a sample of size n. In this study, we randomly selected 20 species of plants from each plot (n = 20) and then calculated Rarefied SR.

Soil sampling and analyses

Five soil samples (0–20 cm) were taken from each plot and mixed homogenously in early October 2021. We obtained 10 soil samples from each restoration stage, and 30 soil samples were used for chemical and microbial analyses. Each sample was divided into two parts. One was air-dried to determine soil environmental factors, and the other was frozen in liquid N₂ immediately for microbial analysis. To determine soil nutrients, we measured soil total carbon (TC), total nitrogen (TN), and total phosphorus (TP) by the dichromate oxidation method (Pribyl, 2010), the Kjeldahl method (Bremner and Mulvaney, 1982), and the molybdate colorimetry method (Pansu and Gautheyrou, 2007), respectively. Soil total calcium (TCa) was measured using an atomic absorption spectrophotometer (ICE 3500, Thermo Scientific, MA, USA) after HNO₃-HClO₄ digestion (Bao, 2008).

DNA extraction, library construction, and metagenomics sequencing

According to the manufacturer’s instruction, we extracted the total genomic DNA from 0.5 g of each soil sample using the FastDNA® Spin Kit for Soil (MP Biomedicals, CA, USA). The concentration and purity of the extracted DNA were determined with a TBS-380 micro-fluorometer (TurnerBioSystems, Sunnyvale, CA, USA) and a NanoDrop 2000 ultra micro-spectrophotometer (Thermo Scientific, Waltham, MA, USA), respectively. After that, the quality of the extracted DNA was checked on 1% agarose gel. We fragmented the extracted DNA to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for the construction of the paired-end library. The paired-end library was constructed using NEXTflex™ Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). The adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt end of fragments. We performed paired-end sequencing on Illumina NovaSeq (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits according to the manufacturer’s instruction (www.illumina.com).

Sequence quality control and genome assembly

The raw reads from metagenome sequencing were used to generate clean reads by removing adaptor sequences and trimming and removing low-quality reads (reads with N bases, with a minimum length threshold of 50bp and a minimum quality threshold of 20) using the fastp (Chen et al., 2018) (https://github.com/OpenGene/fastp, version 0.20.0) on the free online Majorbio Cloud Platform (cloud.majorbio.com). Then, we assembled these high-quality reads to contigs using MEGAHIT (Li et al., 2015) (parameters: kmer_min = 47, kmer_max = 97, and step = 10) (https://github.com/voutcn/megahit, version 1.1.2), which makes use of succinct de Bruijn graphs. Contigs with a length being or over 300 bp were selected as the final assembling result.

Gene prediction, taxonomy, and functional annotation

We used MetaGene (Noguchi et al., 2006) (http://metagene.cb.k.u-tokyo.ac.jp/) to identify open reading frames (ORFs) in contigs. The predicted ORFs with a length being or over 100 bp were retrieved and translated into amino acid sequences using the NCBI translation table (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/index.cgi?chapter=tgencodes#SG1). We constructed a non-redundant gene catalog using CD-HIT (Fu et al., 2012) (http://www.bioinformatics.org/cd-hit/, version 4.6.1) with 90% sequence identity and 90% coverage. The reads after quality control were mapped to the non-redundant gene catalog with 95% identity using SOAPaligner (Li et al., 2009) (http://soap.genomics.org.cn/, version 2.21), and the gene abundance in each sample was evaluated. The representative sequences of the non-redundant gene catalog were annotated based on the NCBI NR database using blastp as implemented in DIAMOND v0.9.19 with an e-value cutoff of 1e^− 5 using Diamond (Buchfink et al., 2015) (http://www.diamondsearch.org/index.php, version 0.8.35) for taxonomic annotations. We annotated the cluster of orthologous groups of proteins (COG) for the representative sequences using Diamond (http://www.diamondsearch.org/index.php, version 0.8.35) against the eggNOG database (version 4.5.1) with an e-value cutoff of 1e^− 5. We conducted the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation using Diamond (http://www.diamondsearch.org/index.php, version 0.8.35) against the KEGG database (http://www.genome.jp/keeg/, version 94.2) with an e-value cutoff of 1e^− 5. The observed relative functional contributions of microbial groups to the functional pathway were to assign the total abundance of all groups involved in soil P cycling by summing the abundances of the groups involved in the function.

Statistical analysis

Data organizing was performed by Excel 2016. The similarities and differences in the gene composition for P cycling were explored through the analysis of similarities (Clarke, 1993) using the “vegan” R package (Oksanen et al., 2022) and presented by nonmetric multidimensional scaling (NMDS) plots using the Bray-Curtis dissimilarity matrix. Then, we conducted the one-way analysis of variance to investigate the significant differences in the soil environmental factors and the genes related to P cycling between the three restoration stages using the SPSS 21.0 statistical software package (IBM, Armonk, NY, USA). The relationships between the relative abundances of genes, including genes involved in soil OP cycling and cycling, and soil environmental factors (e.g., pH and soil TC) were tested using Spearman’s rank correlations. We further constructed a structural equation model (SEM) using SPSS Amos version 26.0 (IBM, Armonk, NY, USA) to investigate the direct and indirect effects of soil nutrients and plant diversity on the relative abundances of the shifted P cycling genes.

The composition of genes involved in soil P cycling

The NMDS plots showed a clear separation of the three scatter groups for the restoration soils in the TG, SG, and OG stages, indicating significant differences in gene composition between the three restoration stages (NMDS stress, 0.080; p < 0.05) (Fig. 1). When the genes were assigned to four P cycling categories, the genes involved in soil OP mineralization had the highest relative abundance in all restoration soils and followed the sequence of OG (48.38%) ≥ SG (47.81%) > TG (45.78%) (there was no significant difference between OG and SG soils) (Fig. 2). The genes responsible for soil IP solubilization presented a higher relative abundance in the TG (27.19%) than in the SG (25.47%) and OG (25.03%) soils. The ranges of the relative abundances of genes participating in the system of soil P uptake and transport and P-starvation response regulation were 20.21–20.81% and 6.22–6.38%, respectively, and no significant difference was observed between the three restoration soils (Fig. 2).

Changes in the relative abundances of the genes related to soil OP and IP cycling

The genes involved in the formation of organic acid (19.88–21.84%) contributed the most to the IP solubilization, and the changes in the relative abundance followed the same pattern as changes in the total IP solubilization genes (TG > SG ≥ OG) (Fig. 3a). Specifically, the relative abundances of acetic acid and fumaric acid genes were higher in the TG soils than in the SG and OG soils (Fig. 3c). However, there was no significant difference in the relative abundances of inorganic pyrophosphatases (1.19–1.24%) and PQQ-GDH (3.90–4.17%) between the three restoration soils (Fig. 3a). Six sub-categories were included in OP mineralization (Fig. 3a). The relative abundances of the genes responsible for the synthesis of phosphomonoesterase (18.11–20.49%), phosphodiesterase (17.86–18.80%), and organic pyrophosphatase (4.75–5.33%) contributed top three to OP mineralization, and all of them increased with the restoration process (Fig. 3a). The relative abundances of the genes involved in C-P lyase and phosphonatase were higher in the TG soils than in the SG and OG soils (Fig. 3a). For the genes involved in the production of alkaline phosphatase, the OG and SG soils had higher relative abundances of phoD and phoX genes but a low relative abundance of phoA genes than the TG soils (Fig. 3b).

Taxonomic composition and contributions of genes involved in soil OP and IP cycling

In this study, 82 microbial phyla were assigned to be involved in soil P cycling. The NMDS plots indicated significant differences in the composition of microbial communities between TG, SG, and OG soils (Fig. S1). For microbes related to soil OP mineralization and IP solubilization, the top 10 identified predominant microbial phyla were the same. From the TG stage to the OG stage, the relative abundances of Actinobacteria (13.44–29.34%), Unclassified_d_Bacteria (1.78–3.22%), Chloroflexi (1.54–2.07%), Nitrospirae (0.51–1.79%), and Thaumarchaeota (0.40–2.05%) increased, whereas those of Proteobacteria (38.97–52.72%) and Verrucomicrobia (2.62–5.98%) decreased (Fig. 4). In addition, compared to the microbes involved in soil IP solubilization, some of the microbes involved in soil OP mineralization, such as Actinobacteria (OP, 25.13%; IP, 20.57%), Candidatus_Rokubacteria (OP, 8.32%; IP, 7.08%), and Nitrospirae (OP, 1.51%; IP, 0.80%), had higher relative abundances, but some, such as Proteobacteria (OP, 43.48%; IP, 46.01%), Acidobacteria (OP, 7.70%; IP, 9.91%), and Verrucomicrobia (OP, 3.68%; IP, 4.44%), had lower relative abundances.

The relationships between soil nutrients, plant diversity indexes, and the relative abundances of OP and IP genes

Soil nutrients (including soil TC, TN, TP, and TCa) and plant diversity indexes (including Shannon, Rarefied SR, Pielou, and Species indexes) in the three restoration stages are presented in Table S1, indicating that plant restoration in the studied karst regions significantly enhanced underground soil nutrients and aboveground plant diversity.

During natural restoration, all four soil nutrient indexes showed significant positive correlations (R = 0.409^*–0.627^**, p < 0.05 or p < 0.01) with the relative abundance of the functional genes responsible for soil OP mineralization (Fig. 5). On the contrary, they were all negatively correlated with the relative abundance of IP genes (R = -0.570–-0.416, p < 0.05 or p < 0.01) (Fig. 6). Among the four plant diversity indexes, only Plant Species was significantly correlated with the relative abundances of OP (R = 0.502^**, p < 0.01) and IP genes (R = -0.508^**, p < 0.01) (Fig. 5, Fig. 6). Shannon, Rarefied SR and Pielou indexes were not correlated with OP genes and IP genes.

The driving modes of soil nutrients and plant diversity to the relative abundances of OP and IP genes

The SEM model (Fig. 7) was constructed based on the results in TG, SG, and OG restoration stages and had high goodness of fit. The SEM model showed that the natural restoration of degraded karst vegetation significantly and positively influenced soil nutrients and plant diversity. For the genes responsible for OP mineralization, soil nutrients had a positive and significant effect on their abundance, while plant diversity showed a weak effect. Only OP genes negatively affected the abundance of the genes responsible for IP solubilization, and other variables had no significant effect on them. In addition, a significant tandem pathway was formed by the natural restoration of degraded karst vegetation-soil nutrients-the abundance of OP genes-the abundance of IP genes.

The structures of soil microbial communities can be shaped by edaphic properties, such as nutrient content (Bakker et al., 2013; Chong et al., 2010; Chu et al., 2011; Lauber et al., 2009; Schlatter et al., 2015; Yergeau et al., 2007; Yu et al., 2012), and aboveground plant factors, such as vegetation types (Oh et al., 2012; Shi et al., 2015). In the present study, plant diversity and soil nutrients increased with revegetation time, indicating the positive effect of natural restoration on the recovery of plants and soils in degraded karst regions. As a result, the composition of the functional genes of soil P cycling varied across the chronosequence (Fig. 1). It should be noted that no significant difference existed in plant diversity indexes between the SG and OG soils (Table S1), indicating that the natural restoration of degraded karst vegetation led to the saturation of plant diversity at an early stage. This result was also confirmed by Liu et al. (2021), who found that the restoration of plant diversity was saturated during the restoration process of aerial seeding in the degraded Mu Us Desert. The increase in plant species means more diverse producers with a higher photosynthetic rate per unit area, promoting net primary productivity and providing soils with more nutrient sources such as plant litter and root exudates (Cardinale et al., 2011; Maestre et al., 2012). Therefore, soils can gradually restore and have positive linear relationships with plant diversity during the ecological restoration of degraded terrestrial ecosystems (Yan et al., 2020; Zhong et al., 2019). Thus, in the present study, the increases in soil TC, TN, TP, and TCa with natural restoration could be attributed to the diversified plant species. This was also confirmed by the constructed SEM model (Fig. 7), where plant diversity positively influenced soil nutrients during the vegetation restoration from the TG stage to the OG stage. Furthermore, in karst soils with sufficient calcium and carbonate ions, bacteria can induce calcium carbonate precipitation as part of their basic metabolic activities linked to the sequestration of atmospheric CO₂ (Buczynski and Chafetz, 1991). The high content of soil TCa in the SG and OG soils indicated that vegetation restoration activated soil bacterial communities rather than shaped microbial structures in karst regions. The present study revealed that natural restoration could restore degraded aboveground plant communities and improve soil nutrients in karst regions and could shift the composition of genes involved in soil P cycling.

Among these four P cycling categories presented in Fig. 2, the genes involved in soil OP mineralization had the highest relative abundance in all restoration soils, followed by the genes related to IP solubilization. Similar results were also obtained in grassland, cropland, and tropical forests (Dai et al., 2019; Siles et al., 2022), where the genes responsible for OP mineralization are dominant in soils. Previous studies demonstrated that OP mineralization was the main driver of microbial P turnover in P-depleted soils (Bergkemper et al., 2016; Dai et al., 2019). In addition, under P-low conditions, microorganisms require extra energy to obtain P by enhancing the release of phosphatases to mineralize soil OP (Dai et al., 2019). Therefore, the high proportion of the microbial mineralization processes of OP indicates a general shortage of bioavailable soil P in terrestrial ecosystems, including the studied karst regions. Soil functional genes related to the system of P uptake and transport and P-starvation response regulation are mainly controlled by soil P supply (Rawat et al., 2021; Santos-Torres et al., 2021). It should be noted that in the present study, the relative abundances of the genes related to the two P cycling categories did not show a significant difference between the TG, SG, and OG restoration soils (Fig. 2). The reason may be that the three restoration soils in our study all suffered the deficiency of bioavailable P (8.91–16.93 mg/kg, unpublished data), which was demonstrated by numerous previous studies (Hui et al., 2015; Ma et al., 2019; Shen et al., 2020).

The deficiency of bioavailable soil P has greatly negatively impacted agricultural production and ecological restoration in the karst regions of southwest China. Especially, for natural revegetation, the available plant P mainly comes from microbial transformation. In the present study, the relative abundance of the genes involved in soil OP mineralization increased from the TG stage to the OG stage, indicating that the natural restoration of degraded karst vegetation promoted the microbial potential in the transformation of soil OP compounds. This result was consistent with the changes in plant diversity and soil nutrients. However, correlation analysis revealed that the changes in soil OP cycling genes were more affected by soil nutrients than plant diversity. This result was also proven by the further SEM model, where the driving path of soil nutrients to the abundance of OP genes was significant, but that of plant diversity to the abundance of OP genes was not (Fig. 7). Therefore, during natural restoration from shrubs to the old-growth forest in karst regions, soil nutrients directly affected the soil microbial transformation of OP. As the main OP fractions in soils, phosphomonoesters and phosphodiesters are easily hydrolyzed and can account for up to 90% of the soil OP content (Dai et al., 2019; Ragot et al., 2017). Our results indicated that the genes responsible for their catalysis-related enzymes (phosphomonoesterase and phosphodiesterase) increased from the TG stage to the OG stage (Fig. 3a). This can be explained by the fact that the increased soil nutrients brought more OP compounds that can be easily used for microorganisms. In addition, the relative proportion of recalcitrant OP (e.g., phosphonates) decreased, resulting in a lower relative abundance of the genes responsible for the production of C-P lyases (the main enzyme group involved in the hydrolysis of phosphonates) in the SG and OG soils (Kamat et al., 2011; Siddhesh and Raushel, 2013). Alkaline phosphatases are monomeric enzymes and contribute greatly to soil OP mineralization. Soil alkaline phosphatases have been identified as predominant from the microbial origin and are encoded by phoD, phoX, and phoA genes (Lu et al., 2022). In our study, among the three phosphatase-encoding genes, phoD had the highest relative abundance in all studied soils (Fig. 3b). In line with our result, previous studies have reported that phoD is the most widespread gene in soils and has become the reference marker in studies on soil P cycling (Ragot et al., 2017). Therefore, in our study, the natural restoration of degraded karst vegetation enhanced the relative abundance of the genes coding for alkaline phosphatases, and other OP-mineralizing enzymes might increase the OP mineralization ability of soil microbes.

The relative abundance of IP genes decreased with natural restoration, showing an opposite trend with the changes in the abundance of OP genes, soil nutrients, and plant diversity. The correlation analysis revealed that soil nutrients were negatively correlated with the relative abundance of IP genes. However, the further SEM model showed that the direct driving path of soil nutrients to the abundance of IP genes was weak and insignificant. It should be noted that soil nutrients had a significant, indirect, and negative impact on the relative abundance of IP genes through OP genes (Fig. 7). This indicated that during natural revegetation in the studied karst regions, the changes in soil P cycling genes were caused by soil nutrients. These results may be explained by two reasons. One is that over time in the initial phase of ecosystem development, the number of mineral phosphates constantly decreases (Vitousek et al., 2010). Therefore, in the present study, although soil total nutrients increased with restoration, the content of IP decreased, leading to a lower abundance of IP genes in the SG and OG soils. The other is that the functional genes involved in soil OP mineralization are much more sensitive to the changes in edaphic properties than the genes related to IP solubilization. Since there was no significant change in the relative abundances of the other two P cycling categories, the increased abundance of OP genes caused a decrease in the relative abundance of IP genes. As a result, the natural restoration of degraded karst vegetation shifted the acquisition strategy of soil microbial P by enhancing OP mineralization and decreasing IP solubilization potentials. Specifically, several genes coding for the formation of organic acid decreased from the TG stage to the OG stage (Fig. 3c). The content of microbial organic acid in soils greatly affected IP solubilization (Cui et al., 2020; Turner and Newman, 2005). The low relative abundances of the genes coding for the formation of acetic acid and fumaric acid in the SG and OG soils may result in a decrease in the solubilization potential of soil microbial IP (Fig. 3c). However, no significant difference existed in the genes coding for PQQ-GDH, which is one of the main drivers for soil IP transformation, between the three studied restoration soils (Fig. 3a). Our findings are in line with the results obtained by Li et al. (2021), who found no significant difference in the relative abundance of the genes involved in PQQ-GDH when the marsh degraded into the meadow. This might indicate that the genetic determinants of QQ-GDH are less sensitive to vegetation restoration-mediated environmental changes than those of organic acid. Since the genes coding for PQQ-GDH were fewer than those coding for organic acid in the studied soils, PQQ-GDH probably had a limited contribution to soil IP cycling during vegetation restoration in karst regions.

In the present study, we found that 82 microbial phyla were responsible for soil P cycling, which may imply the widespread existence of microbes that participate in soil P cycling. The dominant microbial phyla related to soil OP and IP cycling did not change in all studied soils (Fig. 4), indicating that these microbial strains had a strong competitive ability and could adapt to various environmental conditions. However, their community composition was greatly affected by the natural restoration of degraded karst vegetation. For example, as the most abundant bacterial phyla in all studied soils, Proteobacteria had a relative abundance decreasing from 49.67–52.72% in the TG soils to 38.97–40.95% in the OG soils. Previous research revealed that most of the Gammaproteobacteria and Alphaproteobacteria belonged to IP-solubilizing or OP-mineralizing bacteria, and their abundances could be reduced by the increase in soil N (Dai et al., 2019). Therefore, the decreased relative abundance of Proteobacteria related to P cycling in the SG and OG soils can be explained by the increased soil TN. However, Actinobacteria, which belong to copiotrophic microorganisms, mainly adopt the R selection strategy to drive the rapid response to resource availability and are easy to multiply in soils with high organic C and N content (Gu et al., 2017; Wang et al., 2018). Thus, in the present study, the relative abundance of Actinobacteria increased with the improvement of soil nutrient conditions. It should be noted that Actinobacteria, Candidatus_Rokubacteria, and Nitrospirae had greater contributions to OP mineralization genes than to IP solubilization genes, while Proteobacteria, Acidobacteria, and Verrucomicrobia performed the opposite. This probably implies that diverse microbes may have tendentious functions for soil P cycling (Li et al., 2018).

The natural restoration of degraded karst vegetation significantly impacted the composition of the functional genes and microbial communities involved in soil P cycling. When shrubbery was restored into the old-growth forest, soil nutrients, plant diversity, and the relative abundance of the genes related to soil OP mineralization increased, whereas the relative abundance of the genes related to soil IP solubilization decreased. In this restoration situation, soil nutrients had a significant direct driving effect on the microbial transformation of OP but an indirect negative impact on the microbial IP cycling through OP mineralization genes. Microbial phyla, including Proteobacteria and Actinobacteria, were the main contributors to the genes related to soil OP and IP transformations in all studied soils, and their relative contributions changed with vegetation restoration. In conclusion, the natural restoration of degraded karst vegetation increased soil nutrients and further shifted the acquisition strategy of soil microbial P by enhancing OP mineralization and decreasing IP solubilization potentials. These results provide some fundamental information on the microbial mechanisms underlying soil P cycling in karst regions and may be helpful in promoting the management of degraded karst vegetation.

Funding

This research was supported by the Basic Research Program in Guizhou Province under grant number (2022)036, the Gui Da Te Gang He Zi Program under grant number (2021)06, the Cultivation Project of Guizhou University under grant number (2020)04, and the Joint Fund of the Natural Science Foundation of China and the Karst Science Research Center of Guizhou Province under grant number U1812401.

Author Contributions

Yu Dai: Conceptualization, Methodology, Writing - Original Draft, Visualization. Danmei Chen: Writing - Review & Editing, Funding acquisition, Supervision, Project administration, Methodology. Lipeng Zang: Project administration, Supervision. Guangqi Zhang: Project administration, Supervision. Qingfu Liu: Project administration, Supervision. Yuejun He: Supervision. Fangjun Ding: Resources. Shasha Wang: Investigation. Chunjie Zhou: Investigation. Yousu Yang: Investigation. Yujuan Li: Investigation.

Confict of interest

The authors declare that we have no known competing fnancial interests or personal relationships that could have appeared to infuence the work reported in this paper.

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Natural restoration of degraded karst vegetation shifts the acquisition strategy of soil microbial phosphorus by enhancing organic phosphorus and decreasing inorganic phosphorus cycling potentials

Status:

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Version 1

Abstract

Figures

Introduction

Materials And Methods

Site description

Plant survey

Soil sampling and analyses

DNA extraction, library construction, and metagenomics sequencing

Sequence quality control and genome assembly

Gene prediction, taxonomy, and functional annotation

Statistical analysis

Results

The composition of genes involved in soil P cycling

Changes in the relative abundances of the genes related to soil OP and IP cycling

Taxonomic composition and contributions of genes involved in soil OP and IP cycling

The relationships between soil nutrients, plant diversity indexes, and the relative abundances of OP and IP genes

The driving modes of soil nutrients and plant diversity to the relative abundances of OP and IP genes

Discussion

Conclusions

Declarations

References

Supplementary Files

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