Impact of Robinia pseudoacacia stand conversion on soil bacterial communities and soil properties

Background: Robinia pseudoacacia is a widely planted pioneer tree species in reforestations on barren mountains in northern China. Because of its nitrogen-xing ability, it can play a positive role in soil and forest restoration. After clear-cutting of planted stands, R. pseudoacacia stands become coppice plantations. The impacts of shifting from seedling to coppice plantations on soil bacterial community and soil properties have not been well described. This study aims to quantify how soil properties and bacterial community composition vary between planted seedling versus coppice stands. Methods: Three 20 × 20 m plots were randomly selected in each seedling and coppice stand. The bulk soil and rhizosphere soil were sampled in the nine above-mentioned sample plots in the summer of 2017. Bulk soil was sampled at 10 cm from the soil surface using a soil auger. Rhizosphere soil samples were collected by brush. The soil samples were transported to the laboratory for chemical analysis and bacterial community composition and diversity was obtanied through DNA extraction, 16S rRNA gene amplication and high throughput sequencing. Results: The results showed that, compared to seedling plantations, soil quality decreased signicantly in coppice stands, but without affecting soil exchangeable Mg 2+ and K 2+ . Total carbon (C) and nitrogen (N) were lower in the rhizosphere than in bulk soil, whereas nutrient availability showed an opposite trend. The conversion from seedling to coppice plantations was also related to signicant differences in soil bacterial community structure and to the reduction of soil bacterial α-diversity. Principal component analysis (PCA) showed that, bacterial community composition was similar in both bulk and rhizosphere soils in second generation coppice plantations. Specially, the conversion from seedling to coppice increased the relative abundance of Proteobacteria and Rhizobium, but reduced that of Actinobacteria, which may result in a decline of soil nutrient availability. Mantel tests revealed that C, N, Soil organic matter (SOM), nitrate nitrogen (NO 3− -N) and available phosphorus positively correlated with bacterial community composition, while a variation partition analysis (VPA) showed that NO 3− -N explained a relatively greater proportion of bacterial distribution ± SE (n = 5) are shown. FR, SR and TR represent the rhizosphere of seedling plantations, rst generation coppice plantations and second generation coppice plantations, respectively; FNR, SNR and TNR represent bulk soil of seedling plantations, rst generation coppice plantations and second generation coppice plantations, respectively. Different lowercase letters indicate signicant differences in soil properties among the bulk soil or rhizosphere in different R. pseudoacacia plantations (p < 0.05).

Rhizosphere is a critical interface supporting the exchange of resources between plants and the surrounding soil environment, which provides microhabitats and niches for diverse microorganisms and microbial species (Philippot et al. 2013; Mendes et al. 2013). Rhizosphere microorganisms play a key role in plant growth and soil properties, especially in the rhizosphere niche (Philippot et al. 2013; Zhang et al. 2018a), which in uences several plant physiological processes such as growth and energy metabolism affecting overall plant health (Fonseca et al. 2018). Generally, there are signi cant differences between rhizosphere and bulk soil microenvironments, the most obvious of which is that the higher nutrient content and root exudates in the rhizosphere contribute to improving soil carbon and nitrogen concentrations (Yin et al. 2018). Such differences may affect the composition of the rhizosphere microbial community (Neumann et al. 2014). Soil properties and their ecological processes provide a scienti c basis for understanding the interaction between root physiological activity and soil physical and biological environments. At the same time, rhizosphere micro-ecology may be a key driver for predicting tree growth mechanisms.
Previous research has reported the high capacity of R. pseudoacacia for nitrogen xation (Buzhdygan et al. 2016), and higher N mineralization and nitri cation rates in black locust plantations compared to surrounding soils (Williard et al. 2005). Moreover, the excess of N can accumulate in the soil (Berthold et al. 2009) by means of root exudates, contributing to increasing soil fertility (Joëlle et al. 2010). The main nitrogen form uptaken by plants is inorganic nitrogen including nitrate and ammonium. R. pseudoacacia bene ts from nitrogen xation associated with symbiotic rhizobia in root nodules (Cierjacks et al. 2013).
The reduction of soil N availability induces nodulation and biological nitrogen xing of R. pseudoacacia in order to sustain the required nitrogen amounts for plant growth (Mantovani et al. 2015). Therefore, both bacteria and N play an important role in the growth and development of R. pseudoacacia plantations.
With the development of R. pseudoacacia coppice plantations, unexpected problems have arisen in Mount Tai (China) forest ecosystems, including the decline of landscape quality, soil erosion and plant dwar ng, in line with previous research suggesting tree growth decline and trunk shape worsening (Geng et al. 2013). However, to date, most studies have attempted to investigate the effects of conversion from natural forests to plantations on soil properties, soil microbes and their community structure (Zhang et al. 2017;Yang et al. 2018). But there is a gap in knowledge concerning the effects of the transtion from seedling plantations to coppice stands. Radtke et al. (2013) showed that repeated clear cuttings every 20-30 years favored the spread of R. pseudoacacia. Yet, the effects of shift from seedling to coppice plantations on soil properties and soil microbes are not yet well understood, and information is scarce.
The aim of this study was to (1) shed light on the effects of shifting from seedling to coppice stands in black locust plantations on soil properties and soil bacterial community composition, especially Rhizobium, and (2) investigate the relationships beween soil properties and bacterial community composition in seedling and coppice plantations, respectively. The study was performed in rst generation seedling plantation stands (F), rst generation coppice plantations (S) and second generation coppice plantations (T) in Mount Tai, China. We hypothesized that (1) the changes caused by the conversion of seedling to coppice stands lead to decline of soil quality, and to alterations in soil bacterial community composition, (2) nutrient availability plays an important role in shaping the bacterial community, and (3) the relative abundance of Rhizobium decreases in coppice plantations.

Study area
This study was conducted in Mount tai region of Shandong Province, which is located in eastern China. The region is characterized by a typical temperature climate. The mean annual temperature is 12.8 ℃, and the mean annual precipitation is 1124.6 mm. In the 1920s, R. pseudoacacia was introduced to Mount Tai because of its potential for soil and forest restoration. Afforestation was mainly conducted between 1956 and 1958 by seedling direct planting. However, with increasing timber demand for use in construction, seedling plantations were gradually harvested leading to naturally-regenerated coppice plantations. Nowadays, most R. pseudoacacia stands are coppice plantations, mainly distributed along an elevational gradient from 500 to 1000 meters above sea level, and southern aspects.

Sampling
Three 20 × 20 m plots were randomly selected in each seedling and coppice stand (i.e., a total of nine plots). The bulk soil and rhizosphere soil were sampled in the nine above-mentioned sample plots in the summer of 2017. Bulk soil was sampled at 10 cm from the soil surface by using a soil auger (length 50 cm, diameter 5 cm, volume 100 cm 3 ). Rhizosphere soil samples were collected by brush (5 samples per plot). The soil samples were transported on ice to the laboratory, where they were sieved (mesh size 2 mm) and divided into two parts, one was air-dried and stored at room temperature prior to chemical analysis and the other was stored at -80℃ for further analysis. Hereafter in this manuscript, FR, SR and TR refer to the rhizosphere of F, S and T, respectively; and FNR, SNR and TNR refer to bulk soil of F, S and T, respectively.
Analysis of soil physicochemical properties DNA extraction, 16S rRNA gene ampli cation, and high throughput sequencing Total genomic DNA from samples was extracted using CTAB method. 16S rRNA genes of distinct regions (16SV4-V5) were ampli ed using a speci c primer with the barcode. All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs). The 16S rRNA genes were analyzed to evaluate bacterial diversity using IlluminaHiSeq (Novogene Bioinformatics Technology Co., Ltd., Beijing, China).
Sequences were analyzed using QIIME software package (Quantitative Insights Into Microbial Ecology), and in-house Perl scripts were used to analyze alpha-(within samples) and beta-(among samples) diversities. We picked a representative sequence for each OTU and used the RDP classi er to annotate taxonomic information for each representative sequence (Wang et al. 2007).

Statistical analysis
Duncanʼs one-way ANOVA was conducted to examine differences in soil characteristics, SQI and relative abundance of Rhizobioum between bulk and rhizosphere soils. A T-test was conducted to examine differences in Shannon and Simpson indices between bulk and rhizosphere soils. These analyses were performed using SPSS 24.0 (IBM, USA). Principal component analysis (PCA) was conducted to test for differences in the OUT-based community composition using Bray-Curtis distance. The relationships between soil properties and dominant bacterial community composition (TOP 10) were determined using Spearman correlation analysis. Mantel-tests and variation partition analysis (VPA) were used to determine the relative importance of the measured soil properties in shaping soil bacterial community, which were calculated using the Bray-Curtis distance. These analysis were carried out using the "vegan" package of R software (Version 2.15.3). The graphics were drawn using Origin 2019.

Results
Impact of the conversion to coppice stands on soil quality Soil nutrient contents diminished mostly from seedling to coppice plantations (Table 1). Soil characteristics varied considerably in both rhizosphere and bulk soild from F stands to T stands. Total C, N and NO 3 − -N concentration and SOM content in both the rhizosphere and bulk soil was signi cantly higher in seedling stands compared to rst and second generation coppice stands. There were signi cant differences in P concentration in the rhizosphere and bulk soil. There were no statistically signi cant difference in available phosphorous (A.P) concentrations between FNR and SNR, but A.P concentration was signi cantly greater in FNR and SNR compared to TNR. No differences were found regarding exchangeable ions in bulk soil between seedling and coppice plantation, while signi cantly higher contentrations appeared in the rhizosphere of coppice plantations compared to seedling stands. The SQI of both bulk soil and rhizosphere was higher in seedling plantations than in coppice stands, i.e., the highest SQI value (29.14) was found in the rhizosphere of seedling stands whereas the lowest SQI (24.33) was found in the bulk soil of second generation coppice stands.  1A) were Proteobacteria (30.54%), Actinobacteria (25.30%), Acidobacteria (13.94%), Firmicutes (7.19%), Verrucomicrobia (6.86%), Planctomycetes (5.22%), Chloro exi (3.87%), Gemmatimonadetes (2.37%), Bacteroidetes (1.14%), and Cyanobacteria (0.40%), and these groups accounted for more than 96.43% of the bacterial sequences. Moreover, the Shannon and Simpson indices for alpha bacterial diversity declined from seedling to coppice plantations and from rst-rotation to second-rotation coppice plantations by 2% and 0.2%, respectively (Table 2). At the genus level (Fig. 1B), the six most abundant bacteria (≥ 1%) were Bacillus (4.22%), Bradyrhizobium (2.82%), Acidothermus (1.88%), Bryobacter (1.44%), Burkholderia-Paraburkholderia (2.00%) and Streptomyces (1.41%). The relative abundance of Bacillus and Burkholderia-Paraburkholderia in the rhizosphere were lower than that of bulk soil in seedling plantations, but the opposite trend was found in coppice plantations. In addition, the relative abundance of other bacteria in the rhizosphere was higher than that of bulk soil in seedling and coppice plantations.
Relative abundance of Rhizobium in seedling and coppice plantations The relative abundance of Rhizobium in both bulk soil and rhizosphere in second generation coppice stands was signi cantly higher than in seedling and rst generation coopice stands. The relative abundance of Rhizobium was the highest in the rhizosphere of T stands (0.32%), while the lowest was foudn in the bulk soil of seedling stands (0.11%). Moreover, the difference in Rhizobium abundance between rhizosphere soil and bulk soil was signi cant in seedling plantations (p = 0.002), while there was no difference in coppice plantations (Fig. 2).

Bacterial community composition in seedling and coppice plantations
The results showed ve replicates usually clustered closely (Fig. 3).. The rst and second PCA axes revealed that the rhizosphere-and bulk soil-associated bacterial microbiota were inhomogeneous at phylum (12.77% and 8.23%, respectively, Fig. 3A) and genus (17.21% and 13.16%, respectively, Fig. 3B) levels. The soil layer and plantation type rendered a signi cant effect on bacterial community composition. The similarities in bacterial community composition within rhizosphere and bulk soil were lower in seedling plantations than in coppice plantations (Fig. 3).
We found that C, N, SOM, NO 3 --N and A.P were positively correlated with bacterial community composition by Mantel tests at both the phylum and genus levels ( Table 3). Spearman correlation analysis of the relationships between soil properties and bacterial community at the phylum (Fig. 4A) and genus levels (  shown that R. pseudoacacia may induce signi cant changes on several physical and chemical properties of the soil (Khan et al. 2010). In R. pseudoacacia coppice plantations, intra-speci c competition increases because of the high stem density, which may result in differences in microclimatic and ecological conditions as compared to seedling stands. In this regard, our results provide incremental knowledge to previous research by further showing that the conversion from seedling to coppice stands reduced soil quality (Table 1), consistently with the ndings of Johnson (2001) and Luo (2006). Therefore, it supports hypothesis 1. that R. pseudoacacia is a N-xing species with a strong nitrogen xation ability. However, our results showed that soil N (N, NO 3 − -N and A.N) concentrations declined in coppice plantations. It possiblely indicates that the nitrogen xation ability of R. pseudoacacia coppice decreased to a certain extent, and the N mineralization rate was signi cantly lower than seedling plantation (Unpublished data).
The main reason may be that the conversion decreased the net primary production and aboveground biomass and productivity (Liao et al. 2012). Specially, the coppice plantation had a lower stand productivity than seedling plantation ( Figure S2), and which could modify soil structure and lead to less inputs and more losses of soil nutrients (Zheng et al. 2005), then nally affectthe absorption of N by trees (Zhang et al. 2018b). Additionally, we found that the greater moisture content occurred in coppice plantations (13.95%), which might reduce root and microbial activity (Banerjee et al. 2016), then reduce the soil total N concentration, N storage, N cycling and availability (Wang et al. 2010).
Due to root exudations, microbiota activity, and plant absorption, which may lead to the accumulation of nutrients in the rhizosphere, the microenvironments between the rhizosphere and bulk soil may differ markedly (Philippot et al. 2013). Our results showed that N and C contents in bulk soil were higher than those in the rhizosphere, but the concentrations of other nutrients (eg. SOM, NO 3 − -N and A.P) were lower in the bulk soil than in the rhizosphere (Table 1). These results are consistent with previous research (Chaudhary et al. 2015). One main possible reason is that plant roots directly take up lower available nutrients and reduce carbon loss in the rhizosphere (Jones et al. 2009), and they could also adapt to the change of soil nutrient availability through the elastic distribution of underground roots (Bardgett et al. 2014). The consumption of N for growth, the strong physiological metabolism function of root system and the activity of rhizosphere microorganisms drive the transformation of N to A.N, and this may be the reason why we found that rhizosphere soil had lower N content and higher A.N content (Table 1).
Conversion from seedling to coppice plantations altered the structure of bacterial communities Changes in forest community types can affect soil microbial structure (Cardenas et al. 2015) and αdiversity (Vitali et al. 2016). Our results showed that Shannon and Simpson indices declined from seedling to coppice plantations (Table 2). These shifts can be accompanied by changes in bacterial functional activity (Kaiser et al. 2014), contributing to one of reported changes of soil nutrients (Zhao et al. 2018). Previous research (Shi et al. 2016) found that rhizosphere microbes displayed higher levels of interactions than bulk soil microbes.However, we found that the bacterial community structures of bulk soil and rhizosphere were not signi cantly different in coppice plantations (Fig. 3), which supports the hypothesis that the bacterial community structures of rhizosphere soil and bulk soil tend to be consistent.
At the phylum level, the three most abundant bacteria in both rhizosphere and bulk soil samples were Proteobacteria, Actinobacteria and Acidobacteria, consistent with the ndings of Fonseca (2018). The relative abundance of Actinobacteria and Verrucomocrobia decreased from F to T, while Proteobacteria showed an opposite trend ( Figure. 1A). A possible explanation for this result is that the Proteobacteria is generally a fast-growing r-strategist with the ability to use a wide range of root-derived carbon substrates (Philippot et al. 2013). Furthermore, the decline in soil quality will drive Proteobacteria to acquire more abundant carbon sources to sustain growth, but the underlying mechanisms need to be further explored.
The main function of Actinobacteria is to absorb nutrients and excrete metabolic products, which results in the decline of soil quality (Wang et al. 2017a). At the genus level, the relative abundance of Bacillus and Bradyrhizobium increased from F to T, while Acidothermus and Bryobacter showed the opposite trend ( Figure. 1B). Therefore, the proportion of dominant species changed, which resulted in bacterial community composition homogeneity of bulk soil and rhizosphere in coppice plantations.
Conversion from seedling to coppice plantations increased the relative abundance of Rhizobium , which supports hypothesis 2, i.e., that nutrient availability plays an important role in shaping the bacterial community. C and N contents exhibited a strong signi cantly positive correlation with Bacteroidetes, and a negative correlation with Proteobacteria and Firmicutes, but no correlation with Actinobacteria and Acidobacteria (Fig. 4), which was consistent with the the results of Fierer (2007) and Zhao (2018 conversion from seedling to coppice stands altered the structure of the soil bacterial community and decreased soil resource availability (Zhang et al. 2017), which also partly supports the hypothesis that nutrient availability plays an important role in shaping the bacterial community. In this study, we found that bacterial communities in both the rhizosphere and bulk soil were strongly in uenced by soil NO 3 − -N ( Fig. 5). Nitrogen in soil can be decomposed by bacteria to promote N absorption by trees. All N transformation and uptake processes are correlated with soil carbon resources and regulated by soil microbes (Geisseler et al. 2010). Our results showed that C and NO 3 − -N contents in the coppice plantations were lower than those in the seedling stands, leading to inhibition of microbial activity.

Conclusions
This research revealed three important ndings for assessing the impacts of converting seedling to coppice plantations on soil habitat. First, we found that this conversion can negatively affect soil properties. Second, the conversion from seedling to coppice stands could alter soil bacterial community composition, resulting in higher homogeneity of the bacterial community composition in bulk soil and rhizosphere in coppice plantations. Furthermore, this can lead to the imbalance of soil microenvironment structure and the decline of soil functions. Additionally, stand conversion increased the relative abundance of Rhizobium, but the soil N and available N decreased, implying that the activity of Rhizobium was limited. Eventually, we found that NO 3 − -N is the most important factor in shaping soil bacterial structure in this ecosystem.
Although the impacts of the conversion from seedling to coppice plantations on soil properties and soil bacterial community were studied, we can not state that the contribution rate of N to bacterial community was zero (Fig. 5). Further research with N cycling and understory coverages conversion from seedling to coppice plantations would help to better assess this phenomenon, including mineralization, nitri cation, anammox, denitri cation and nitrogen xation.

Declarations
Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests. Differences in the relative abundance of Rhizobium between the rhizosphere and bulk soil seedling and coppice plantations. α=0.05.