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Variations of soil nitrogen-fixing microorganism communities and nitrogen fractions in a Robinia pseudoacacia chronosequence on the Loess Plateau of China
Catena 174 (2019) 316–323
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Variations of soil nitrogen-fixing microorganism communities and nitrogen fractions in a Robinia pseudoacacia chronosequence on the Loess Plateau of China
T
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Yadong Xua,b, Tao Wanga,b, Hui Lib,c, Chengjie Rena,b, Jianwei Chenb,c, Gaihe Yanga,b, , ⁎ Xinhui Hana,b, , Yongzhong Fenga,b, Guangxin Rena,b, Xiaojiao Wanga,b a b c
College of Agronomy, Northwest A&F University, Yangling, 712100, Shaanxi, China The Research Center of Recycle Agricultural Engineering and Technology of Shaanxi Province, Yangling, 712100, Shaanxi, China College of Forestry, Northwest A&F University, Yangling, 712100, Shaanxi, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Robinia pseudoacacia L Chronosequence N fractions nifH gene Loess Plateau
Afforestation greatly alters soil nitrogen (N) dynamics and soil microbial community, but the response of soil N dynamics to microbial activity, particularly to that of N-fixing microbes, remains unclear. In the present study, soil samples were collected from areas with a chronosequence of 17, 27 and 42 years of Robinia pseudoacacia L (RP17a, RP27a, and RP42a) and a sloped farmland (FL) as a control. The nifH gene was sequenced with the Illumina MiSeq platform to analyze soil microbial diversity and composition, and soil N fractions (TN, AN, NN, MBN, DON) were estimated. The soil N stocks, N fractions, and diversity of N-fixing microorganisms increased with the chronosequence of R. pseudoacacia. Beta diversity, analyzed using nonmetric multidimensional scaling, revealed that afforestation effectively improved soil microbial communities. The profiles of the N-fixing microorganism communities at sites 17a, 27a and 42a were clearly separated from the farmland, and soil N-fixing microbial community compositions generally changed in the farmland from Actinobacteria-dominated to Proteobacteria- dominated with stand ages. Redundancy analysis revealed that soil N fractions were positively correlated with the main species, particularly for AN, which had a 34.7% explanatory space in axis one and was significantly correlated with the Proteobacteria at the phylum level. Results implied the evolutionary tendency of the dominant microbial groups, and demonstrated the variation of the N fractions and some other physicalchemistry properties along the chronosequence of R. pseudoacacia, and that AN was the most sensitive N fraction to the N-fixing microorganism composition. Thus, our results provide evidence that the variation of soil compositions of N-fixing microbial communities is linked to the level of N fractions, especially the content of AN.
1. Introduction The nitrogen cycle is one of the most important basic material cycles in the biosphere which describes the process of conversion between nitrogen elements and nitrogen-containing compounds in nature. The nitrogen cycle includes four microbial processes: N-fixation, mineralization, nitrification and denitrification. The nitrogen transformation processes are related to each other, and together determine the balance and fate of nitrogen in the soil ecosystem. Nitrogen fixation is a process of reducing molecular nitrogen to ammonia and other nitrogen-containing compounds. It is one of the important methods of nitrogen input
and plays a key role in the nitrogen cycle. Without human influence, there are two processes fixing atmospheric N2 into biologically available forms: lighting and biological nitrogen fixation (BNF). Approximately 97% of the natural N input comes from BNF (Galloway et al., 2008) and is formed by Bacteria and Archaea. Most of these Nfixation microorganisms (diazotrophs) exist in a free-living condition and provide an average of 110 million tons of nitrogen a year to terrestrial ecosystems (Liu and Liu, 2018). The process of microorganism N-fixation in the soil can be influenced by multiple factors. Hai et al. (2009) used real-time PCR found that after applying organic fertilizer (organic fertilizer and straw), the nitrogen-fixing population occupied
Abbreviations: FL, Farmland; RP, Robinia pseudoacacia; TN, Total nitrogen; AN, ammonium nitrogen; NN, Nitrate nitrogen; MBN, Microbial biomass nitrogen; SOC, Soil organic carbon; DON, Dissolved organic nitrogen; SWC, Soil water content; SBD, 9. Soil bulk density ⁎ Corresponding authors at: College of Agronomy, Northwest A&F University, Yangling, 712100, Shaanxi, China. E-mail addresses: [email protected] (Y. Xu), [email protected] (T. Wang), [email protected] (C. Ren), [email protected] (G. Yang), [email protected] (X. Han), [email protected] (Y. Feng), [email protected] (G. Ren), [email protected] (X. Wang). https://doi.org/10.1016/j.catena.2018.11.009 Received 16 March 2018; Received in revised form 13 October 2018; Accepted 8 November 2018 0341-8162/ © 2018 Elsevier B.V. All rights reserved.
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2. Materials and methods
the dominant position in the investigated community, but the size of the nitrogen-fixing population decreased in the plot treated with urea. Land management methods like crop rotation have been shown to have a significant effect on total bacterial diversity (and that of free-living N fixers) (P < 0.001) (Orr et al., 2011). Low pH level and high concentrations of inorganic N in soil may reduce the population’s size and activity of free-living N-fixing bacteria (Deluca et al., 1996). Roper and Smith (1991) also found that different pH values and clay content caused the soils with similar histories of straw retention to differ in their potential for straw-associated N2 fixation. Furthermore, the number of nitrogen-fixing bacteria was generally greater in the surface layer of soil (5 cm) and decreased with the increase in soil depth. With seasonal fluctuations, the greatest total number of nitrogen-fixing bacteria appeared in autumn, winter, and early spring, and was low in summer (Mergel et al., 2001). Despite the importance of nitrogen-fixing microorganism communities for ecosystem processes, our understanding of the species composition, community diversity and abundance involved in the process is still very limited, especially for a certain time chronosequence. The Loess Plateau is one of the most vulnerable areas in China because of its complex topography and high intensity of artificial disturbance (Fu et al., 2011). Afforestation has often been proposed as one of the most commonly used technique for preventing soil degradation and restoring landscapes (Nunez-Mir et al., 2015). Herrera and Garcia (2010) found that the woody leguminous plants are the most useful plants for vegetation restoration in the barren and water-deficient wastelands. Black locust (Robinia pseudoacacia, RP) was considered a promising tree species for reforestation programs because of its rapid growth rate, and ability to fix nitrogen in the atmosphere. The first time that RP was planted in the Loess Plateau was in the 1950s (Jiao et al., 2012). After several years, the RP had been widely restored in the hilly Loess Plateau (Ren et al., 2017). In the Loess Plateau, RP, a non-native species, was considered a pioneer species for restoration programs. Since the 1990s, because of water, nutrient, and density restricted conditions and site mismatches, forest degradation, low ecological benefit, dry shoots, sparse undergrowth, and single tree species, which is commonly called “small old tree,” have appeared (Zhu et al., 2018). However, the climate has improved with the vegetation restoration in the ecosystem. Because of its strong adaptability, rapid growth rate, high resistance to stress, drought resistance, and the ability to fix nitrogen, the distribution of RP in China has gradually expanded. It has now evolved into a native tree species in China (Han, 2018). It has been used in several studies, especially in N fractions. Rice et al. (2004) found that R. pseudoacacia could replenish the soil N pool by decomposing litter or root following afforestation. Moreover, soil structure (Quanhou et al., 2008), soil chemical character [e.g., soil organic carbon (C), total nitrogen (N), dissolved organic nitrogen (DON) (Kou et al., 2016; Niu et al., 2017; Qiu et al., 2010; Ren et al., 2016; Wang et al., 2012a; Zheng et al., 2011), and soil C:N:phosphorous (P) stoichiometry (Cao and Chen, 2017)], soil respiration (Sha et al., 2007), soil enzyme activities (e.g., urease and catalase) (Wang et al., 2012b), soil microbial biomass (Bolat et al., 2016), and soil rhizosphere microbial community diversity and composition were improved in relation to the control soils (Xiaogang et al., 2008). R. pseudoacacia is an N-fixing leguminous tree; however, how soil N fractions respond to N-fixing microbes remain unclear. In this study, soil samples were collected from areas with a chronosequence of 17, 27, and 42 years of Robinia pseudoacacia L and farmland. We hypothesized that the changes in chronosequence would make a difference in soil diazotrophs, including changes in abundance and community diversity, and increase soil N stocks and fractions. Therefore, the main purposes of our study were to (i) study the response of soil diazotrophs to soil N fractions and (ii) describe the evolution of N-fixing bacteria communities along the chronosequence.
2.1. Site description and sampling These study sites were conducted in the Wuliwan catchment, located in Ansai District, North Shaanxi Province, China (36°51′41.23″–36°52′50.87″N, 109°19′49.20″–109°21′46.46″E), where the annual mean temperature is 8.8 °C. The area has a semi-arid temperate climate and receives approximately 510 mm of annual precipitation (mainly from July to September). According to the soil distribution map of the world (FAO, 1974), the soils are classified as Calcic Cambisols and are highly erodible. The research area has been used as a test area for water and soil erosion control by the Institute of Soil and Water Conservation, Chinese Academy of Science (CAS) since 1973. The main crops grown in these areas were millet (Setaria italica) and pea (Pisum sativum L.), and irrigation was not available throughout the growing season. The method of substitution of space for time is commonly used in ecosystem research and a successful practice to analyze the changes in soil characteristics and vegetation communities in the period of natural succession (Ligi et al., 2014; Williams et al., 2013). We used this method to research the influence on soil nifH community structure and the development of N fractions in soil based on the chronosequence (17a, 27a, 42a) of Robinia pseudoacacia L, and a sloped farmland (FL) was used as a reference. Soil sampling occurred in August 2016 during the periods of vegetation growth. Three 20 × 20 m plots were set up at each site which were considered to be true replicates in our study. The distance between any two plots did not exceed 13.5 m to ensure that they had similar environmental conditions (Martinez-Alcala et al., 2010). After the litter layer was completely removed, the 0–10 cm soil samples were collected using a stainless-steel sampler with a diameter of 5 cm. Along an S-shaped pattern, 10 soil cores were collected from each plot and then mixed together. Visible plant roots, stones, litters and debris were removed, and the soil sample was divided into two sub-samples. One of the sub-samples was stored at −80 °C for DNA sequencing analysis. The other sample was air-dried for physical and chemical analysis. Characteristics of the sampling sites are presented in Table 1. 2.2. Analysis of plant characteristics and soil physicochemical properties All plant leaves, litters and biomass were dried at 105 °C for 15 min, and then put them under 80 °C until a constant weight. Then the dry materials were crushed and passed through a 100 mesh sieve after grinding finely. The N content in plants (biomass N, foliar N, and litter N) was determined according to the H2SO4-H2O2 digestion and Kjeldahl method (Lin et al., 2011). Soil water content (SWC) was measured by oven drying to constant mass (when weight remains constant) at 105 °C. Soil bulk density (SBD) was expressed as a ratio of the mass of dry soil (after oven dried for 24 h at 105 °C) to the volume of the core (Vos et al., 2005). The soil pH was determined with a pH meter (PHS-3C, Shanghai, China) in a 1:2.5 soil-water mixture. We used the percentage of clay (< 2 μm), silt (50–2 μm) and sand (2000–50 μm) to describe the soil particle size distribution, which was measured by a Malvern MS 2000 (Malvern Instruments, Malvern, England) (Jin et al., 2013). Soil organic carbon (SOC) was measured with dichromate oxidation method. Total nitrogen (TN) was extracted by Kjeldahl digestion previously described (Zhang et al., 2011). The N stocks (Ren et al., 2016) on behalf of the soil total nitrogen storage (STNS) at each sampling depth. Ammonium nitrogen (AN) and nitrate nitrogen (NN) in soil were measured using KCl digestion (Jones and Willett, 2006) and analyzed with CleverChem, whereas dissolved organic nitrogen (DON) was measured using potassium persulfate oxidation before the treated liquids were assessed by an Automated Chemistry Analyzer (Ren et al., 2016). The soil microbial biomass nitrogen (MBN) was measured using the chloroform fumigation extraction method (Brookes et al., 1985). 317
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Table 1 The geographical information, soil physical-chemistry properties and vegetation N contents of the stands. Sample characteristics
Farmland (FL)
Robinia pseudoacacia L 17a (RP 17a)
Robinia pseudoacacia L 27a (RP 27a)
Robinia pseudoacacia L 42a (RP 42a)
Location Elevation (m) Slope aspect (°) SBD (g cm−3) SWC (%) pH Clay (%) Silt (%) Sand (%) SOC (g kg−1) Biomass N (g kg−1) Foliar N (g kg−1) Litter N (g kg−1)
36.865 N, 109.351 E 1205 N by E 75 1.24 ± 0.06A 10.95 ± 0.71B 8.51 ± 0.10A 20.03 ± 0.10A 40.57 ± 0.25A 39.41 ± 0.16AB 2.98 ± 0.05D
36.869 N, 109.352 E 1298.4 S by W 75 1.24 ± 0.06A 13.69 ± 0.69A 8.35 ± 0.07A 18.43 ± 0.66AB 42.75 ± 1.52A 38.82 ± 0.85B 4.67 ± 0.35C 12.73 ± 1.14B 24.16 ± 0.55C 11.65 ± 0.48C
36.860 N, 109.349 E 1303.9 N by W 10 1.16 ± 0.08A 14.70 ± 0.64A 8.49 ± 0.11A 18.60 ± 0.36AB 41.23 ± 0.80A 40.17 ± 0.48AB 5.91 ± 0.48B 14.24 ± 0.67AB 28.21 ± 1.01B 14.19 ± 0.62B
36.871 N, 109.348 E 1293.8 S by W 80 1.11 ± 0.11A 13.52 ± 1.61A 8.62 ± 0.15A 18.15 ± 0.68B 40.64 ± 0.28A 41.20 ± 0.52A 8.79 ± 0.25A 16.89 ± 0.66A 33.82 ± 0.81A 17.71 ± 0.64A
Notes: SBD = soil bulk density; SWC = soil water content; Capital letters indicate significant difference among different stands by the LSD test (P < 0.05). Error bars: SE (n = 3). The same below.
2.3. DNA extraction
2.6. Sequence accession numbers
Soil genomic DNA was extracted from 1.5 g of fresh soil (3 × 0.5 g) using a FastDNA spin kit for soil (MP Biomedical, Carlsbad, CA, USA) as described by the manufacturer's instructions. The DNA concentration and purity were detected by using 2% agarose gel electrophoresis and a spectrophotometer (NanoDrop ND-2000, NanoDrop Technologies, Wilmington, USA).
We used the NCBI (BLAST, v1.2.0) to compare and confirm the nifH gene sequences and the nifH gene sequences obtained in our study have been deposited in the NCBI Sequence Read Archive database with the accession number SRP142236. 2.7. Statistical analyses The changes in soil and vegetation properties, such as pH, soil texture, SOC, N fractions and litter N, were tested for difference among sites with a one-way ANOVA. The microbial community diversity index (Shannon) was compared using one-way ANOVA. The least significant difference (LSD) was calculated to examine differences between mean values at a value of P < 0.05. Data analysis and graphing were performed with SPSS version 20.0 statistical software (SPSS, Chicago, USA) and OriginLab Origin Pro software (version 9.2) (OriginLab, Northampton, MA, U.S.). To illustrate the clustering of different samples and to further determine of the microbial community structure, non-metric multi-dimensional scaling (NMDS) was performed using the UniFrac method and R, whereas changes in microbial structure along the chronosequence were referred to as microbial beta diversity. To determine the relationship between the soil nifH gene compositions and soil N fractions, a redundancy analysis (RDA) was conducted using CANOCO (version 5.0) for Windows.
2.4. PCR amplification and the Illumina MiSeq platform The design of primers: PolyF (TGCGAYCCSAARGCBGACTC) and PolyR (ATSGCCATCATYTCRCCGGA) (Mirza et al., 2014) were used to amplify the nifH gene from the bacteria. The purified PCR products were mixed in equimolar ratios for sequencing. The reaction volume of 25 μl contained 5 μl reaction buffer, 5 μl GC buffer, 2 μl dNTP (100 mM), 1 μl each primer, 0.25 μl Q5® High-Fidelity DNA Polymerase (NEB, #M0491 L), 2 μl DNA template and 8.75 μl ddH2O. The PCR conditions were denatured for 2 min, followed by 30 rounds of temperature cycling (98 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s), and a final extension at 72 °C for 5 min. Each sample was amplified in triplicate, and the triplicate amplicons were pooled and purified by gel extraction and quantified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P7589). After quantitation, the purified amplicons from each reaction mixture were mixed at equimolar ratios based on concentration and subjected to emulsion PCR to generate amplicon libraries. The PCR products were submitted to Shanghai Personal Biotechnology Corporation, Ltd. for sequencing analysis using the Illumina MiSeq platform. Each sample produced about 60,000 high-quality sequences with an average length of about 360 bp.
3. Results 3.1. Sample characteristics analysis Since the crops on the field were harvested, the plant properties were not assessed for this site. Plant N properties differed significantly among the three ages as is shown in Table 1. From 17a to 42a, biomass N, foliar N and litter N increased to 32.7%, 40.0%, and 52.0%, respectively.
2.5. Processing of sequencing data After trimming of the barcodes and primers, sequences that were shorter than 150 bp in length and reads containing ambiguous bases or any unresolved nucleotides were removed. Quality filtering and chimerism detection of the sequence were conducted using the QIIME (Quantitative Insights Into Microbial Ecology, v1.8.0) workflow. Sequences with the same barcode were defined as the same sample (Caporaso et al., 2010). Using the UCHIME (v5.2.236) algorithm to filter raw flowgrams and remove noise and chimeras (Edgar et al., 2011). Sequences with similarities of > 97% were assigned to one operational taxonomic unit (OTU) using the UCLUST method (Edgar, 2010). Community Alpha diversity indices (Shannon index) and rarefaction curves were obtained using the MOTHUR (v1.31.2) program.
3.2. Variations in the soil N stocks and fractions The soil N stocks and total nitrogen (TN) content in 17a, 27a, and 42a were significantly higher by 49.2%–127.3% and 50.3%–155.2% than those in FL (Fig. 1a, b), respectively. The contents of AN and NN in 42a were > 28.8% and 193.5% greater than that of FL, respectively; and with the increase in age, the difference decreased (Fig. 1c, d). MBN content in 17a, 27a, and 42a increased by 239.2%, 385.4%, and 761.6%, respectively, compared with that of FL (Fig. 1e). However, the DON content was the highest in 27a (Fig. 1f). 318
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Fig. 1. Changes of soil nitrogen (N) stocks (a), soil nitrogen (N) fractions (TN-b, AN-c, NN-d, MBN-e, DON-f). Different capital letters indicate significant differences among different stands by the LSD test (P < 0.05). Error bars: SE (n = 3). The same below.
presented an increasing trend (Fig. 2). To contrast with the similarity and dissimilarity between all sample sites, nonmetric multidimensional scaling analysis (NMDS) was performed. The NMDS plot (Fig. 3) indicated that the weighted UniFrac distance for soil nitrogen-fixing microorganism communities found in the different ages were well separated, and the relative abundance of each population varies with time. The distribution of the nitrogenfixing microorganism communities at the 17a, 27a, and 42a sites were clearly separated from FL. However, the 42a and 27a sites tended to group together; in other words, the difference between 42a and 27a was
3.3. Microbial diversity and community composition Overall, 537,686 quality sequences and a mean of 59,743 sequences per sample were obtained across all soil samples. In total 4642 OTUs were identified, after leveling, the average OTU, for subsequent analysis was 1160.5 per region. Fig. S1 shows that 285 OTUs are shared in all four groups. There were 887, 497, 429, and 834 unique OTUs in 42a, 27a, 17a and FL, respectively. A depth of 32,600 sequences was chosen as a threshold for comparing the α-diversity among samples. The Shannon index was from 5.98 to 7.54. In general, alpha diversity 319
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microorganism communities at the phyla level, redundancy analysis (RDA) was performed (Fig. 5a). The first two canonical axes explain 57.84% and 20.49% (P = 0.006) of the total variance within all data, respectively. The vectors on the RDA plot suggested that AN, TN, MBN, and NN were the most important variables related to the changes in the composition and diversity of the communities. In particular, AN was the most variables associated with the first axis. In addition, the relative abundance of Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria, and Deltaproteobacteria at the class level (Fig. 5b) and Myxococcales, Rhizobiales, and Sphingomonadales at the order level were sensitive to the changes in N fractions (Fig. 5c). 4. Discussion 4.1. Changes in soil N fractions in a Robinia pseudoacacia chronosequence Changes from farmland to afforestation mean that the annual cycle of cultivation and harvesting crops were substituted for a long-term forest process. Therefore, the biogeochemical characteristics of soil can change because of the influence of the afforestation process, and then affect the N dynamics (Li et al., 2012; Liu et al., 2013). In the present study, the N fractions including TN, AN, NN, DON, and MBN in soil were increased synchronously following afforestation, and such changes were evidenced by increased plant inputs (Table 1). It is possible that the litter and biomass in afforestation sites were higher than that of farmland and that the decomposition of organic matter (Schimel and Bennett, 2004; Xin et al., 2015) further increases the N stocks and cycles (Li et al., 2012). In the hilly regions of the Loess Plateau, 50 years might have been a turning point for the leaf properties of black locust (Niu et al., 2017), and in the present study, the N content of leaves of 42-year-old Robinia pseudoacacia was the highest in the forest chronosequence (Table 1). Along with the growth of RP, more rRNA is needed to increase the protein synthesis, which leads to the improvement of the N content of leaves. Moreover, as the plant grows, leaves fall and are converted into litter. Accompanied by a reduction in bulk density, it was mainly caused by the recycling of annual litter fall and nutrient elements to the trees for nutrient supply. Microbial decomposition forms humus from litter, thereby increasing soil organic matter content. In addition, with plant growth, the root systems would release a large number of ions and organic matter into the soil. Therefore, soil N stocks and fractions were higher in the soil of R. pseudoacacia forests than in the farmland soil, suggesting that afforestation has potential effects on soil N fractions.
Fig. 2. Changes of microbial (nitrogen-fixing microorganism) alpha diversity (Shannon index) along the Robinia pseudoacacia L chronosequence.
Fig. 3. Changes of soil nitrogen-fixing microorganism beta diversity along the RP chronosequence.
4.2. Correlation of nifH-based soil community diversity with soil properties In the present study, we confirmed that soil community alpha diversity (Shannon index) assessed from nifH diversity, was higher along with the chronosequence, suggesting that the changes in ages drove the differences in microbial diversity. Newly exposed areas are usually invaded by some opportunistic species, followed by an increase in species diversity related to increased accumulation of resources (Zhang et al., 2016). Accordingly, we found that the highest value of Shannon index, found at the 42a site, was significantly higher than the value of the Shannon index at the 27a, 17a, and farmland sites. These findings were similar to those of Schutte et al. (2010), where in the diversity of soil Nfixation microorganisms in the leading edge of the Arctic glacier was generally high and increased remarkably along with the succession gradient. Zeng et al. (2016) also found that a higher diversity of diazotrophs along a successional gradient of deglaciated forelands of the Tianshan Mountain in China. Afforestation on farmland, in addition to affecting the composition of aboveground plants, will also lead to changes in the properties of underground soil (e.g., pH and SWC), N stocks, N fractions (i.e., TN, AN, NN, DON, MBN), and soil microbial community structure. We found that changes in the diversity of N-fixing microorganisms were
decreased. The composition of soil nitrogen-fixing microbial communities and their differences among different sites were determined by sequence analysis. Different community structure patterns were observed at the phyla, class and order levels across all sites. The dominant phyla (relative abundance > 0.5%) were Proteobacteria, Actinobacteria, Firmicutes and Nitrospirae, with contributions of 34.44%, 13.15%, 1.99%, and 1.68%, respectively (Fig. 4I). Among them, the relative abundance of Actinobacteria decreased and Proteobacteria significantly increased with the increase in chronosequence. Alphaproteobacteria and Gammaproteobacteria were the most dominant class with a range of 7.25–13.59% and 2.70–9.42%, respectively (Fig. 4II). Rhizobiales, one of the most dominant order, was significantly decreased in 42a and synchronized with the changes in Gammaproteobacteria (Fig. 4III). 3.4. Relationships between soil N fractions and soil nitrogen-fixing microorganisms To explore the effect of the soil N fractions on the nitrogen-fixing 320
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Fig. 4. Relative abundance of the soil nitrogen-fixing microorganism communities at the phylum level (I), class level (II) and order level (III). The data for the average relative abundances from three replicates were calculated as the ratio between the abundance of the sequence type and the total number of sequences. All calculations used normalized data. The abbreviation were as follow: (I) Proteobacteria (Prot), Actinobacteria (Acti), Nitrospirae (Nitr), Firmicutes (Firm). (II) Alphaproteobacteria (Alph), Gammaproteobacteria (Gamm), Betaproteobacteria (Beta), Deltaproteobacteria (Delt), Nitrospira (Nitr), Bacilli (Baci). (III) Myxococcales (Myxo), Rhizobiales (Rhiz), Xanthomonadales (Xant), Frankiales(Fran), Burkholderiales (Burk), Sphingomonadales (Sphi), Rhodocyclales (Rhodo), Micrococcales (Micr), Nitrospirales (Nitr), Streptomycetales (Stre), Bacillales (Baci), Pseudomonadales (Pseudom), Rhodospirillales (Rhodos), Planctomycetales (Plan), Pseudonocardiales (Pseudon). The same below.
Fig. 5. RDA analysis to identify the relationships between soil N fractions (red arrows) and soil nitrogen-fixing microorganism composition (blue arrows) (a-phyla level; b-class level; c-order level). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
percentages of sand and organic carbon increased, whereas the SBD decreased. These effects improved the permeability of the soil significantly, and subsequently promoted microbial growth, leading to changes in species compositions. In our study, the N-fixing microorganism communities commonly varied from Actinobacteria-dominated in cropland to Proteobacteria-dominated in forest soil during the changes of chronosequence, showing that the underground community transitioned from a slow-growing oligotrophic group to a fast-growing symbiotic group. Therefore, soil properties, such as organic carbon level and the percentage of sand may affect biological N fixation of specific diazotrophs taxonomic groups (Calderoli et al., 2017). Our results provide a plausible explanation for the N fractions and the composition of soil nifH communities in the RP chronosequence. Moreover, some uncertainties lead to changes in the relative abundance of species, such as temporal variability, landform, and perturbed environments. Future studies need to consider these factors at the same time and study the changes in nifH communities and N fractions over a longer chronosequence. In addition, we need to study the process considering ammoxidation, nitrification, and denitrification.
significantly associated with the changes in N fractions of the soil (Fig. 5), particularly for AN. Therefore, it could be inferred that AN could be easily affected during the changes of chronosequence because of the process of biological N fixation (Fowler et al., 2013; Vitousek et al., 2013). The nifH communities consisted of Proteobacteria, Actinobacteria, Nitrospirae, and Firmicutes (Fig. 4). The phyla level classification was similar to that of other soils and environments (Mirza et al., 2014). Regardless of the age of succession, Proteobacteria and Actinobacteria were the richest phylum which was commonly consistent with previous findings that soils usually including two common and ubiquitous bacterial groups (Kim et al., 2014; Kolton et al., 2011; Shen et al., 2013). Jangid et al. (2013) have recently reported an increasing number of Proteobacteria following the plant succession of the Franz Josef in New Zealand. Li et al. (2014) showed the similar findings in a 20 years chronosequence of soil restoration. Many soil Proteobacteria are symbiotic, and when active substrates are present, they become dominant (Goldfarb et al., 2011). However, Actinobacteria are oligotrophic groups and like poor nutrition environment. Most of the N-fixing microorganisms belong to the Proteobacteria and are aerobic organisms. In the present study, with changes in chronosequence, the 321
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5. Conclusions
Biochem. 38, 991–999. Kim, H.M., et al., 2014. Bacterial community structure and soil properties of a subarctic tundra soil in council, Alaska. FEMS Microbiol. Ecol. 89, 465–475. Kolton, M., et al., 2011. Impact of biochar application to soil on the root-associated bacterial community structure of fully developed greenhouse pepper plants. Appl. Environ. Microbiol. 77, 4924–4930. Kou, M., Garcia-Fayos, P., Hu, S., Jiao, J., 2016. The effect of Robinia pseudoacacia afforestation on soil and vegetation properties in the Loess Plateau (China): a chronosequence approach. For. Ecol. Manag. 375, 146–158. Li, D.J., Niu, S.L., Luo, Y.Q., 2012. Global patterns of the dynamics of soil carbon and nitrogen stocks following afforestation: a meta-analysis. New Phytol. 195, 172–181. Li, Y.Y., Wen, H.Y., Chen, L.Q., Yin, T.T., 2014. Succession of bacterial community structure and diversity in soil along a chronosequence of reclamation and re-vegetation on coal mine spoils in China. PLoS One 9. Ligi, T., et al., 2014. Effects of soil chemical characteristics and water regime on denitrification genes (nirS, nirK, and nosZ) abundances in a created riverine wetland complex. Ecol. Eng. 72, 47–55. Lin, C., Yang, Y., Guo, J., Chen, G., Xie, J., 2011. Fine root decomposition of evergreen broadleaved and coniferous tree species in mid-subtropical China: dynamics of dry mass, nutrient and organic fractions. Plant Soil 338, 311–327. Liu, J.G., Liu, W.G., 2018. Advances in microbial-mediated nitrogen cycling. Acta Agrestia Sin. 26, 277–283. Liu, Z.P., Shao, M.A., Wang, Y.Q., 2013. Spatial patterns of soil total nitrogen and soil total phosphorus across the entire Loess Plateau region of China. Geoderma 197, 67–78. Martinez-Alcala, I., Walker, D.J., Bernal, M.P., 2010. Chemical and biological properties in the rhizosphere of Lupinus albus alter soil heavy metal fractionation. Ecotoxicol. Environ. Saf. 73, 595–602. Mergel, A., Kloos, K., Bothe, H., 2001. Seasonal fluctuations in the population of denitrifying and N-2-fixing bacteria in an acid soil of a Norway spruce forest. Plant Soil 230, 145–160. Mirza, B.S., Potisap, C., Nusslein, K., Bohannan, B.J.M., Rodrigues, J.L.M., 2014. Response of free-living nitrogen-fixing microorganisms to land use change in the Amazon rainforest. Appl. Environ. Microbiol. 80, 281–288. Niu, S., et al., 2017. Plant stoichiometry characteristics and relationships with soil nutrients in Robinia pseudoacacia communities of different planting ages. Acta Ecol. Sin. 37, 355–362. Nunez-Mir, G.C., Iannone, B.V., Curtis, K., Fei, S.L., 2015. Evaluating the evolution of forest restoration research in a changing world: a “big literature” review. New For. 46, 669–682. Orr, C.H., James, A., Leifert, C., Cooper, J.M., Cummings, S.P., 2011. Diversity and activity of free-living nitrogen-fixing bacteria and total bacteria in organic and conventionally managed soils. Appl. Environ. Microbiol. 77, 911–919. Qiu, L.P., Zhang, X.C., Cheng, J.M., Yin, X.Q., 2010. Effects of black locust (Robinia pseudoacacia) on soil properties in the loessial gully region of the Loess Plateau, China. Plant Soil 332, 207–217. Quanhou, D., Guobin, L., Xue, S., Yu, N., Zhang, C., 2008. Dynamics of soil water-stable aggregates in the restoration process of artificial Robinia pseudoacacia under erosion environment. Bull. Soil Water Conserv. 28, 56–59. Ren, C.J., et al., 2016. Responsiveness of soil nitrogen fractions and bacterial communities to afforestation in the loess hilly region (LHR) of China. Sci. Rep. 6. Ren, C., et al., 2017. Response of microbial diversity to C:N:P stoichiometry in fine root and microbial biomass following afforestation. Biol. Fertil. Soils 1–12. Rice, S.K., Westerman, B., Federici, R., 2004. Impacts of the exotic, nitrogen-fixing black locust (Robinia pseudoacacia) on nitrogen-cycling in a pine-oak ecosystem. Plant Ecol. 174, 97–107. Roper, M.M., Smith, N.A., 1991. Straw decomposition and nitrogenase activity (C2H2 reduction) by free-living microorganisms from soi: effects of pH and clay content. Soil Biol. Biochem. 23, 275–283. Schimel, J.P., Bennett, J., 2004. Nitrogen mineralization: challenges of a changing paradigm. Ecology 85, 591–602. Schutte, U.M.E., et al., 2010. Bacterial diversity in a glacier foreland of the high Arctic. Mol. Ecol. 19, 54–66. Sha, X., Liu, G.B., Dai, Q.H., Wei, W., 2007. Evolution of soil microbial biomass in the restoration process of artificial Robinia pseudoacacia under erosion environment. Acta Ecol. Sin. 27, 909–917. Shen, C.C., et al., 2013. Soil pH drives the spatial distribution of bacterial communities along elevation on Changbai Mountain. Soil Biol. Biochem. 57, 204–211. Vitousek, P.M., Menge, D.N.L., Reed, S.C., Cleveland, C.C., 2013. Biological nitrogen fixation: rates, patterns and ecological controls in terrestrial ecosystems. Philos. Trans. R. Soc. B 368. Vos, B.D., Meirvenne, M.V., Quataert, P., Deckers, J., Muys, B., 2005. Predictive quality of pedotransfer functions for estimating bulk density of forest soils. Soil Sci. Soc. Am. J. 69, 500–510. Wang, B., Liu, G., Xue, S., 2012a. Effect of black locust (Robinia pseudoacacia) on soil chemical and microbiological properties in the eroded hilly area of China's Loess Plateau. Environ. Earth Sci. 65, 597–607. Wang, B., Liu, G.B., Xue, S., 2012b. Effect of black locust (Robinia pseudoacacia) on soil chemical and microbiological properties in the eroded hilly area of China's Loess Plateau. Environ. Earth Sci. 65, 597–607. Williams, M.A., Jangid, K., Shanmugam, S.G., Whitman, W.B., 2013. Bacterial communities in soil mimic patterns of vegetative succession and ecosystem climax but are resilient to change between seasons. Soil Biol. Biochem. 57, 749–757. Xiaogang, D., Ming, T., Hui, C., Haihan, Z., Yongan, Z., 2008. Mycorrhizae and diversity of microbial community in rhizosphere soils of Robinia pseudoacacia at different ages on the Loess Plateau. Sci. Silvae Sin. 78–82.
Our data show the effects of changes in the chronosequence in sites afforested with RP on nifH microorganism communities, N fractions, and some other properties, e.g., SWC, and SBD. We found that with the changes with forest age, the tendency of dominant species varied and AN was the most sensitive N fraction to N-fixing microorganisms. Understanding the effects of nitrogen fractions and soil physical-chemical properties on nitrogen-fixing microbes plays an important role in the later study of nitrogen fixation capacity in this area. However, the measured species composition only reflects the microbial potential for nitrogen fixing and do not describe actual turnover rates in soils. Therefore, investigations of gene expression and enzyme activity remain to be performed with further study. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.catena.2018.11.009. Acknowledgements The project of National Natural Science Foundation of China (No. 41571501) and the Key Project of the National Key Research and Development Program of China (No. 2017YFC0504601) sustained our research. We are grateful to An'sai Ecological Experimental Station of Soil and Water Conservation for the assistance. We thank our colleagues Wei Zhang and Zekun Zhong for their help of field experiments. References Bolat, I., Kara, O., Sensoy, H., Yuksel, K., 2016. Influences of black locust (Robinia pseudoacacia L.) afforestation on soil microbial biomass and activity. IforestBiogeosciences and Forestry 9, 171–177. Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985. Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. Biochem. 17, 837–842. Calderoli, P.A., Collavino, M.M., Kraemer, F.B., Morrás, H.J.M., Aguilar, O.M., 2017. Analysis of nifH-RNA reveals phylotypes related to geobacter and cyanobacteria as important functional components of the N2-fixing community depending on depth and agriculturaluse of soil. Microbiology 6, e00502. Cao, Y., Chen, Y.M., 2017. Coupling of plant and soil C:N:P stoichiometry in black locust (Robinia pseudoacacia) plantations on the Loess Plateau, China. In: Trees-Structure and Function. 31. pp. 1559–1570. Caporaso, J.G., et al., 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336. Deluca, T.H., Drinkwater, L.E., Wiefling, B.A., Denicola, D.M., 1996. Free-living nitrogenfixing bacteria in temperature cropping systems: influence of nitrogen source. Biol. Fertil. Soils 23, 140–144. Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461. Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200. Food and Agriculture Organization (FAO), 1974. Soil Map of the World. volume 1 Legend, Paris. Fowler, D., et al., 2013. The global nitrogen cycle in the twenty-first century. Philos. Trans. R. Soc. B 368 (1621), 20130164. Fu, B.J., et al., 2011. Assessing the soil erosion control service of ecosystems change in the Loess Plateau of China. Ecol. Complex. 8, 284–293. Galloway, J.N., et al., 2008. Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320, 889–892. Goldfarb, K.C., et al., 2011. Differential growth responses of soil bacterial taxa to carbon substrates of varying chemical recalcitrance. Front. Microbiol. 2. Hai, B., et al., 2009. Quantification of key genes steering the microbial nitrogen cycle in the rhizosphere of sorghum cultivars in tropical agroecosystems. Appl. Environ. Microbiol. 75, 4993–5000. Han, X.H., 2018. Evaluation and Ecological Effects of Returning Farmland to Forest in Loess Hilly and Gully Region. Science Press, Beijing. Herrera, J.M., Garcia, D., 2010. Effects of Forest fragmentation on seed dispersal and seedling establishment in ornithochorous trees. Conserv. Biol. 24, 1089–1098. Jangid, K., Whitman, W.B., Condron, L.M., Turner, B.L., Williams, M.A., 2013. Soil bacterial community succession during long-term ecosystem development. Mol. Ecol. 22, 3415–3424. Jiao, J., Zhang, Z., Bai, W., Jia, Y., Wang, N., 2012. Assessing the ecological success of restoration by afforestation on the Chinese Loess Plateau. Restor. Ecol. 20, 240–249. Jin, Z., Dong, Y.S., Qi, Y.C., Liu, W.G., An, Z.S., 2013. Characterizing variations in soil particle-size distribution along a grass–desert shrub transition in the Ordos plateau of inner Mongolia, China. Land Degrad. Dev. 24, 141–146. Jones, D.L., Willett, V.B., 2006. Experimental evaluation of methods to quantify dissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soil. Soil Biol.
322
Catena 174 (2019) 316–323
Y. Xu et al.
changes in plant community and soil properties during the secondary succession of abandoned farmland in the Loess Plateau. Soil Biol. Biochem. 97, 40–49. Zheng, Y.A., et al., 2011. The importance of slope aspect and stand age on the photosynthetic carbon fixation capacity of forest: a case study with black locust (Robinia pseudoacacia) plantations on the Loess Plateau. Acta Physiol. Plant. 33, 419–429. Zhu, D.J., et al., 2018. Effects of alien species Robinia pseudoacacia on plant community functional structure in hilly-gully region of Loess Plateau, China. Chin. J. Appl. Ecol. 29, 459–466.
Xin, L.I., Rui-Ping, M.A., Shao-Shan, A.N., Zeng, Q.C., Ya-Yun, L.I., 2015. Characteristics of soil organic carbon and enzyme activities in soil aggregates under different vegetation zones on the Loess Plateau. Chin. J. Appl. Ecol. 26, 2282. Zeng, J., et al., 2016. Primary succession of nitrogen cycling microbial communities along the deglaciated forelands of Tianshan mountain, China. Front. Microbiol. 7 (e42149). Zhang, C., Xue, S., Liu, G.B., Song, Z.L., 2011. A comparison of soil qualities of different revegetation types in the Loess Plateau, China. Plant Soil 347, 163–178. Zhang, C., Liu, G.B., Xue, S., Wang, G.L., 2016. Soil bacterial community dynamics reflect
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Variations of soil nitrogen-fixing microorganism communities and nitrogen fractions in a Robinia pseudoacacia chronosequence on the Loess Plateau of China
T
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Yadong Xua,b, Tao Wanga,b, Hui Lib,c, Chengjie Rena,b, Jianwei Chenb,c, Gaihe Yanga,b, , ⁎ Xinhui Hana,b, , Yongzhong Fenga,b, Guangxin Rena,b, Xiaojiao Wanga,b a b c
College of Agronomy, Northwest A&F University, Yangling, 712100, Shaanxi, China The Research Center of Recycle Agricultural Engineering and Technology of Shaanxi Province, Yangling, 712100, Shaanxi, China College of Forestry, Northwest A&F University, Yangling, 712100, Shaanxi, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Robinia pseudoacacia L Chronosequence N fractions nifH gene Loess Plateau
Afforestation greatly alters soil nitrogen (N) dynamics and soil microbial community, but the response of soil N dynamics to microbial activity, particularly to that of N-fixing microbes, remains unclear. In the present study, soil samples were collected from areas with a chronosequence of 17, 27 and 42 years of Robinia pseudoacacia L (RP17a, RP27a, and RP42a) and a sloped farmland (FL) as a control. The nifH gene was sequenced with the Illumina MiSeq platform to analyze soil microbial diversity and composition, and soil N fractions (TN, AN, NN, MBN, DON) were estimated. The soil N stocks, N fractions, and diversity of N-fixing microorganisms increased with the chronosequence of R. pseudoacacia. Beta diversity, analyzed using nonmetric multidimensional scaling, revealed that afforestation effectively improved soil microbial communities. The profiles of the N-fixing microorganism communities at sites 17a, 27a and 42a were clearly separated from the farmland, and soil N-fixing microbial community compositions generally changed in the farmland from Actinobacteria-dominated to Proteobacteria- dominated with stand ages. Redundancy analysis revealed that soil N fractions were positively correlated with the main species, particularly for AN, which had a 34.7% explanatory space in axis one and was significantly correlated with the Proteobacteria at the phylum level. Results implied the evolutionary tendency of the dominant microbial groups, and demonstrated the variation of the N fractions and some other physicalchemistry properties along the chronosequence of R. pseudoacacia, and that AN was the most sensitive N fraction to the N-fixing microorganism composition. Thus, our results provide evidence that the variation of soil compositions of N-fixing microbial communities is linked to the level of N fractions, especially the content of AN.
1. Introduction The nitrogen cycle is one of the most important basic material cycles in the biosphere which describes the process of conversion between nitrogen elements and nitrogen-containing compounds in nature. The nitrogen cycle includes four microbial processes: N-fixation, mineralization, nitrification and denitrification. The nitrogen transformation processes are related to each other, and together determine the balance and fate of nitrogen in the soil ecosystem. Nitrogen fixation is a process of reducing molecular nitrogen to ammonia and other nitrogen-containing compounds. It is one of the important methods of nitrogen input
and plays a key role in the nitrogen cycle. Without human influence, there are two processes fixing atmospheric N2 into biologically available forms: lighting and biological nitrogen fixation (BNF). Approximately 97% of the natural N input comes from BNF (Galloway et al., 2008) and is formed by Bacteria and Archaea. Most of these Nfixation microorganisms (diazotrophs) exist in a free-living condition and provide an average of 110 million tons of nitrogen a year to terrestrial ecosystems (Liu and Liu, 2018). The process of microorganism N-fixation in the soil can be influenced by multiple factors. Hai et al. (2009) used real-time PCR found that after applying organic fertilizer (organic fertilizer and straw), the nitrogen-fixing population occupied
Abbreviations: FL, Farmland; RP, Robinia pseudoacacia; TN, Total nitrogen; AN, ammonium nitrogen; NN, Nitrate nitrogen; MBN, Microbial biomass nitrogen; SOC, Soil organic carbon; DON, Dissolved organic nitrogen; SWC, Soil water content; SBD, 9. Soil bulk density ⁎ Corresponding authors at: College of Agronomy, Northwest A&F University, Yangling, 712100, Shaanxi, China. E-mail addresses: [email protected] (Y. Xu), [email protected] (T. Wang), [email protected] (C. Ren), [email protected] (G. Yang), [email protected] (X. Han), [email protected] (Y. Feng), [email protected] (G. Ren), [email protected] (X. Wang). https://doi.org/10.1016/j.catena.2018.11.009 Received 16 March 2018; Received in revised form 13 October 2018; Accepted 8 November 2018 0341-8162/ © 2018 Elsevier B.V. All rights reserved.
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2. Materials and methods
the dominant position in the investigated community, but the size of the nitrogen-fixing population decreased in the plot treated with urea. Land management methods like crop rotation have been shown to have a significant effect on total bacterial diversity (and that of free-living N fixers) (P < 0.001) (Orr et al., 2011). Low pH level and high concentrations of inorganic N in soil may reduce the population’s size and activity of free-living N-fixing bacteria (Deluca et al., 1996). Roper and Smith (1991) also found that different pH values and clay content caused the soils with similar histories of straw retention to differ in their potential for straw-associated N2 fixation. Furthermore, the number of nitrogen-fixing bacteria was generally greater in the surface layer of soil (5 cm) and decreased with the increase in soil depth. With seasonal fluctuations, the greatest total number of nitrogen-fixing bacteria appeared in autumn, winter, and early spring, and was low in summer (Mergel et al., 2001). Despite the importance of nitrogen-fixing microorganism communities for ecosystem processes, our understanding of the species composition, community diversity and abundance involved in the process is still very limited, especially for a certain time chronosequence. The Loess Plateau is one of the most vulnerable areas in China because of its complex topography and high intensity of artificial disturbance (Fu et al., 2011). Afforestation has often been proposed as one of the most commonly used technique for preventing soil degradation and restoring landscapes (Nunez-Mir et al., 2015). Herrera and Garcia (2010) found that the woody leguminous plants are the most useful plants for vegetation restoration in the barren and water-deficient wastelands. Black locust (Robinia pseudoacacia, RP) was considered a promising tree species for reforestation programs because of its rapid growth rate, and ability to fix nitrogen in the atmosphere. The first time that RP was planted in the Loess Plateau was in the 1950s (Jiao et al., 2012). After several years, the RP had been widely restored in the hilly Loess Plateau (Ren et al., 2017). In the Loess Plateau, RP, a non-native species, was considered a pioneer species for restoration programs. Since the 1990s, because of water, nutrient, and density restricted conditions and site mismatches, forest degradation, low ecological benefit, dry shoots, sparse undergrowth, and single tree species, which is commonly called “small old tree,” have appeared (Zhu et al., 2018). However, the climate has improved with the vegetation restoration in the ecosystem. Because of its strong adaptability, rapid growth rate, high resistance to stress, drought resistance, and the ability to fix nitrogen, the distribution of RP in China has gradually expanded. It has now evolved into a native tree species in China (Han, 2018). It has been used in several studies, especially in N fractions. Rice et al. (2004) found that R. pseudoacacia could replenish the soil N pool by decomposing litter or root following afforestation. Moreover, soil structure (Quanhou et al., 2008), soil chemical character [e.g., soil organic carbon (C), total nitrogen (N), dissolved organic nitrogen (DON) (Kou et al., 2016; Niu et al., 2017; Qiu et al., 2010; Ren et al., 2016; Wang et al., 2012a; Zheng et al., 2011), and soil C:N:phosphorous (P) stoichiometry (Cao and Chen, 2017)], soil respiration (Sha et al., 2007), soil enzyme activities (e.g., urease and catalase) (Wang et al., 2012b), soil microbial biomass (Bolat et al., 2016), and soil rhizosphere microbial community diversity and composition were improved in relation to the control soils (Xiaogang et al., 2008). R. pseudoacacia is an N-fixing leguminous tree; however, how soil N fractions respond to N-fixing microbes remain unclear. In this study, soil samples were collected from areas with a chronosequence of 17, 27, and 42 years of Robinia pseudoacacia L and farmland. We hypothesized that the changes in chronosequence would make a difference in soil diazotrophs, including changes in abundance and community diversity, and increase soil N stocks and fractions. Therefore, the main purposes of our study were to (i) study the response of soil diazotrophs to soil N fractions and (ii) describe the evolution of N-fixing bacteria communities along the chronosequence.
2.1. Site description and sampling These study sites were conducted in the Wuliwan catchment, located in Ansai District, North Shaanxi Province, China (36°51′41.23″–36°52′50.87″N, 109°19′49.20″–109°21′46.46″E), where the annual mean temperature is 8.8 °C. The area has a semi-arid temperate climate and receives approximately 510 mm of annual precipitation (mainly from July to September). According to the soil distribution map of the world (FAO, 1974), the soils are classified as Calcic Cambisols and are highly erodible. The research area has been used as a test area for water and soil erosion control by the Institute of Soil and Water Conservation, Chinese Academy of Science (CAS) since 1973. The main crops grown in these areas were millet (Setaria italica) and pea (Pisum sativum L.), and irrigation was not available throughout the growing season. The method of substitution of space for time is commonly used in ecosystem research and a successful practice to analyze the changes in soil characteristics and vegetation communities in the period of natural succession (Ligi et al., 2014; Williams et al., 2013). We used this method to research the influence on soil nifH community structure and the development of N fractions in soil based on the chronosequence (17a, 27a, 42a) of Robinia pseudoacacia L, and a sloped farmland (FL) was used as a reference. Soil sampling occurred in August 2016 during the periods of vegetation growth. Three 20 × 20 m plots were set up at each site which were considered to be true replicates in our study. The distance between any two plots did not exceed 13.5 m to ensure that they had similar environmental conditions (Martinez-Alcala et al., 2010). After the litter layer was completely removed, the 0–10 cm soil samples were collected using a stainless-steel sampler with a diameter of 5 cm. Along an S-shaped pattern, 10 soil cores were collected from each plot and then mixed together. Visible plant roots, stones, litters and debris were removed, and the soil sample was divided into two sub-samples. One of the sub-samples was stored at −80 °C for DNA sequencing analysis. The other sample was air-dried for physical and chemical analysis. Characteristics of the sampling sites are presented in Table 1. 2.2. Analysis of plant characteristics and soil physicochemical properties All plant leaves, litters and biomass were dried at 105 °C for 15 min, and then put them under 80 °C until a constant weight. Then the dry materials were crushed and passed through a 100 mesh sieve after grinding finely. The N content in plants (biomass N, foliar N, and litter N) was determined according to the H2SO4-H2O2 digestion and Kjeldahl method (Lin et al., 2011). Soil water content (SWC) was measured by oven drying to constant mass (when weight remains constant) at 105 °C. Soil bulk density (SBD) was expressed as a ratio of the mass of dry soil (after oven dried for 24 h at 105 °C) to the volume of the core (Vos et al., 2005). The soil pH was determined with a pH meter (PHS-3C, Shanghai, China) in a 1:2.5 soil-water mixture. We used the percentage of clay (< 2 μm), silt (50–2 μm) and sand (2000–50 μm) to describe the soil particle size distribution, which was measured by a Malvern MS 2000 (Malvern Instruments, Malvern, England) (Jin et al., 2013). Soil organic carbon (SOC) was measured with dichromate oxidation method. Total nitrogen (TN) was extracted by Kjeldahl digestion previously described (Zhang et al., 2011). The N stocks (Ren et al., 2016) on behalf of the soil total nitrogen storage (STNS) at each sampling depth. Ammonium nitrogen (AN) and nitrate nitrogen (NN) in soil were measured using KCl digestion (Jones and Willett, 2006) and analyzed with CleverChem, whereas dissolved organic nitrogen (DON) was measured using potassium persulfate oxidation before the treated liquids were assessed by an Automated Chemistry Analyzer (Ren et al., 2016). The soil microbial biomass nitrogen (MBN) was measured using the chloroform fumigation extraction method (Brookes et al., 1985). 317
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Table 1 The geographical information, soil physical-chemistry properties and vegetation N contents of the stands. Sample characteristics
Farmland (FL)
Robinia pseudoacacia L 17a (RP 17a)
Robinia pseudoacacia L 27a (RP 27a)
Robinia pseudoacacia L 42a (RP 42a)
Location Elevation (m) Slope aspect (°) SBD (g cm−3) SWC (%) pH Clay (%) Silt (%) Sand (%) SOC (g kg−1) Biomass N (g kg−1) Foliar N (g kg−1) Litter N (g kg−1)
36.865 N, 109.351 E 1205 N by E 75 1.24 ± 0.06A 10.95 ± 0.71B 8.51 ± 0.10A 20.03 ± 0.10A 40.57 ± 0.25A 39.41 ± 0.16AB 2.98 ± 0.05D
36.869 N, 109.352 E 1298.4 S by W 75 1.24 ± 0.06A 13.69 ± 0.69A 8.35 ± 0.07A 18.43 ± 0.66AB 42.75 ± 1.52A 38.82 ± 0.85B 4.67 ± 0.35C 12.73 ± 1.14B 24.16 ± 0.55C 11.65 ± 0.48C
36.860 N, 109.349 E 1303.9 N by W 10 1.16 ± 0.08A 14.70 ± 0.64A 8.49 ± 0.11A 18.60 ± 0.36AB 41.23 ± 0.80A 40.17 ± 0.48AB 5.91 ± 0.48B 14.24 ± 0.67AB 28.21 ± 1.01B 14.19 ± 0.62B
36.871 N, 109.348 E 1293.8 S by W 80 1.11 ± 0.11A 13.52 ± 1.61A 8.62 ± 0.15A 18.15 ± 0.68B 40.64 ± 0.28A 41.20 ± 0.52A 8.79 ± 0.25A 16.89 ± 0.66A 33.82 ± 0.81A 17.71 ± 0.64A
Notes: SBD = soil bulk density; SWC = soil water content; Capital letters indicate significant difference among different stands by the LSD test (P < 0.05). Error bars: SE (n = 3). The same below.
2.3. DNA extraction
2.6. Sequence accession numbers
Soil genomic DNA was extracted from 1.5 g of fresh soil (3 × 0.5 g) using a FastDNA spin kit for soil (MP Biomedical, Carlsbad, CA, USA) as described by the manufacturer's instructions. The DNA concentration and purity were detected by using 2% agarose gel electrophoresis and a spectrophotometer (NanoDrop ND-2000, NanoDrop Technologies, Wilmington, USA).
We used the NCBI (BLAST, v1.2.0) to compare and confirm the nifH gene sequences and the nifH gene sequences obtained in our study have been deposited in the NCBI Sequence Read Archive database with the accession number SRP142236. 2.7. Statistical analyses The changes in soil and vegetation properties, such as pH, soil texture, SOC, N fractions and litter N, were tested for difference among sites with a one-way ANOVA. The microbial community diversity index (Shannon) was compared using one-way ANOVA. The least significant difference (LSD) was calculated to examine differences between mean values at a value of P < 0.05. Data analysis and graphing were performed with SPSS version 20.0 statistical software (SPSS, Chicago, USA) and OriginLab Origin Pro software (version 9.2) (OriginLab, Northampton, MA, U.S.). To illustrate the clustering of different samples and to further determine of the microbial community structure, non-metric multi-dimensional scaling (NMDS) was performed using the UniFrac method and R, whereas changes in microbial structure along the chronosequence were referred to as microbial beta diversity. To determine the relationship between the soil nifH gene compositions and soil N fractions, a redundancy analysis (RDA) was conducted using CANOCO (version 5.0) for Windows.
2.4. PCR amplification and the Illumina MiSeq platform The design of primers: PolyF (TGCGAYCCSAARGCBGACTC) and PolyR (ATSGCCATCATYTCRCCGGA) (Mirza et al., 2014) were used to amplify the nifH gene from the bacteria. The purified PCR products were mixed in equimolar ratios for sequencing. The reaction volume of 25 μl contained 5 μl reaction buffer, 5 μl GC buffer, 2 μl dNTP (100 mM), 1 μl each primer, 0.25 μl Q5® High-Fidelity DNA Polymerase (NEB, #M0491 L), 2 μl DNA template and 8.75 μl ddH2O. The PCR conditions were denatured for 2 min, followed by 30 rounds of temperature cycling (98 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s), and a final extension at 72 °C for 5 min. Each sample was amplified in triplicate, and the triplicate amplicons were pooled and purified by gel extraction and quantified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P7589). After quantitation, the purified amplicons from each reaction mixture were mixed at equimolar ratios based on concentration and subjected to emulsion PCR to generate amplicon libraries. The PCR products were submitted to Shanghai Personal Biotechnology Corporation, Ltd. for sequencing analysis using the Illumina MiSeq platform. Each sample produced about 60,000 high-quality sequences with an average length of about 360 bp.
3. Results 3.1. Sample characteristics analysis Since the crops on the field were harvested, the plant properties were not assessed for this site. Plant N properties differed significantly among the three ages as is shown in Table 1. From 17a to 42a, biomass N, foliar N and litter N increased to 32.7%, 40.0%, and 52.0%, respectively.
2.5. Processing of sequencing data After trimming of the barcodes and primers, sequences that were shorter than 150 bp in length and reads containing ambiguous bases or any unresolved nucleotides were removed. Quality filtering and chimerism detection of the sequence were conducted using the QIIME (Quantitative Insights Into Microbial Ecology, v1.8.0) workflow. Sequences with the same barcode were defined as the same sample (Caporaso et al., 2010). Using the UCHIME (v5.2.236) algorithm to filter raw flowgrams and remove noise and chimeras (Edgar et al., 2011). Sequences with similarities of > 97% were assigned to one operational taxonomic unit (OTU) using the UCLUST method (Edgar, 2010). Community Alpha diversity indices (Shannon index) and rarefaction curves were obtained using the MOTHUR (v1.31.2) program.
3.2. Variations in the soil N stocks and fractions The soil N stocks and total nitrogen (TN) content in 17a, 27a, and 42a were significantly higher by 49.2%–127.3% and 50.3%–155.2% than those in FL (Fig. 1a, b), respectively. The contents of AN and NN in 42a were > 28.8% and 193.5% greater than that of FL, respectively; and with the increase in age, the difference decreased (Fig. 1c, d). MBN content in 17a, 27a, and 42a increased by 239.2%, 385.4%, and 761.6%, respectively, compared with that of FL (Fig. 1e). However, the DON content was the highest in 27a (Fig. 1f). 318
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Fig. 1. Changes of soil nitrogen (N) stocks (a), soil nitrogen (N) fractions (TN-b, AN-c, NN-d, MBN-e, DON-f). Different capital letters indicate significant differences among different stands by the LSD test (P < 0.05). Error bars: SE (n = 3). The same below.
presented an increasing trend (Fig. 2). To contrast with the similarity and dissimilarity between all sample sites, nonmetric multidimensional scaling analysis (NMDS) was performed. The NMDS plot (Fig. 3) indicated that the weighted UniFrac distance for soil nitrogen-fixing microorganism communities found in the different ages were well separated, and the relative abundance of each population varies with time. The distribution of the nitrogenfixing microorganism communities at the 17a, 27a, and 42a sites were clearly separated from FL. However, the 42a and 27a sites tended to group together; in other words, the difference between 42a and 27a was
3.3. Microbial diversity and community composition Overall, 537,686 quality sequences and a mean of 59,743 sequences per sample were obtained across all soil samples. In total 4642 OTUs were identified, after leveling, the average OTU, for subsequent analysis was 1160.5 per region. Fig. S1 shows that 285 OTUs are shared in all four groups. There were 887, 497, 429, and 834 unique OTUs in 42a, 27a, 17a and FL, respectively. A depth of 32,600 sequences was chosen as a threshold for comparing the α-diversity among samples. The Shannon index was from 5.98 to 7.54. In general, alpha diversity 319
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microorganism communities at the phyla level, redundancy analysis (RDA) was performed (Fig. 5a). The first two canonical axes explain 57.84% and 20.49% (P = 0.006) of the total variance within all data, respectively. The vectors on the RDA plot suggested that AN, TN, MBN, and NN were the most important variables related to the changes in the composition and diversity of the communities. In particular, AN was the most variables associated with the first axis. In addition, the relative abundance of Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria, and Deltaproteobacteria at the class level (Fig. 5b) and Myxococcales, Rhizobiales, and Sphingomonadales at the order level were sensitive to the changes in N fractions (Fig. 5c). 4. Discussion 4.1. Changes in soil N fractions in a Robinia pseudoacacia chronosequence Changes from farmland to afforestation mean that the annual cycle of cultivation and harvesting crops were substituted for a long-term forest process. Therefore, the biogeochemical characteristics of soil can change because of the influence of the afforestation process, and then affect the N dynamics (Li et al., 2012; Liu et al., 2013). In the present study, the N fractions including TN, AN, NN, DON, and MBN in soil were increased synchronously following afforestation, and such changes were evidenced by increased plant inputs (Table 1). It is possible that the litter and biomass in afforestation sites were higher than that of farmland and that the decomposition of organic matter (Schimel and Bennett, 2004; Xin et al., 2015) further increases the N stocks and cycles (Li et al., 2012). In the hilly regions of the Loess Plateau, 50 years might have been a turning point for the leaf properties of black locust (Niu et al., 2017), and in the present study, the N content of leaves of 42-year-old Robinia pseudoacacia was the highest in the forest chronosequence (Table 1). Along with the growth of RP, more rRNA is needed to increase the protein synthesis, which leads to the improvement of the N content of leaves. Moreover, as the plant grows, leaves fall and are converted into litter. Accompanied by a reduction in bulk density, it was mainly caused by the recycling of annual litter fall and nutrient elements to the trees for nutrient supply. Microbial decomposition forms humus from litter, thereby increasing soil organic matter content. In addition, with plant growth, the root systems would release a large number of ions and organic matter into the soil. Therefore, soil N stocks and fractions were higher in the soil of R. pseudoacacia forests than in the farmland soil, suggesting that afforestation has potential effects on soil N fractions.
Fig. 2. Changes of microbial (nitrogen-fixing microorganism) alpha diversity (Shannon index) along the Robinia pseudoacacia L chronosequence.
Fig. 3. Changes of soil nitrogen-fixing microorganism beta diversity along the RP chronosequence.
4.2. Correlation of nifH-based soil community diversity with soil properties In the present study, we confirmed that soil community alpha diversity (Shannon index) assessed from nifH diversity, was higher along with the chronosequence, suggesting that the changes in ages drove the differences in microbial diversity. Newly exposed areas are usually invaded by some opportunistic species, followed by an increase in species diversity related to increased accumulation of resources (Zhang et al., 2016). Accordingly, we found that the highest value of Shannon index, found at the 42a site, was significantly higher than the value of the Shannon index at the 27a, 17a, and farmland sites. These findings were similar to those of Schutte et al. (2010), where in the diversity of soil Nfixation microorganisms in the leading edge of the Arctic glacier was generally high and increased remarkably along with the succession gradient. Zeng et al. (2016) also found that a higher diversity of diazotrophs along a successional gradient of deglaciated forelands of the Tianshan Mountain in China. Afforestation on farmland, in addition to affecting the composition of aboveground plants, will also lead to changes in the properties of underground soil (e.g., pH and SWC), N stocks, N fractions (i.e., TN, AN, NN, DON, MBN), and soil microbial community structure. We found that changes in the diversity of N-fixing microorganisms were
decreased. The composition of soil nitrogen-fixing microbial communities and their differences among different sites were determined by sequence analysis. Different community structure patterns were observed at the phyla, class and order levels across all sites. The dominant phyla (relative abundance > 0.5%) were Proteobacteria, Actinobacteria, Firmicutes and Nitrospirae, with contributions of 34.44%, 13.15%, 1.99%, and 1.68%, respectively (Fig. 4I). Among them, the relative abundance of Actinobacteria decreased and Proteobacteria significantly increased with the increase in chronosequence. Alphaproteobacteria and Gammaproteobacteria were the most dominant class with a range of 7.25–13.59% and 2.70–9.42%, respectively (Fig. 4II). Rhizobiales, one of the most dominant order, was significantly decreased in 42a and synchronized with the changes in Gammaproteobacteria (Fig. 4III). 3.4. Relationships between soil N fractions and soil nitrogen-fixing microorganisms To explore the effect of the soil N fractions on the nitrogen-fixing 320
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Fig. 4. Relative abundance of the soil nitrogen-fixing microorganism communities at the phylum level (I), class level (II) and order level (III). The data for the average relative abundances from three replicates were calculated as the ratio between the abundance of the sequence type and the total number of sequences. All calculations used normalized data. The abbreviation were as follow: (I) Proteobacteria (Prot), Actinobacteria (Acti), Nitrospirae (Nitr), Firmicutes (Firm). (II) Alphaproteobacteria (Alph), Gammaproteobacteria (Gamm), Betaproteobacteria (Beta), Deltaproteobacteria (Delt), Nitrospira (Nitr), Bacilli (Baci). (III) Myxococcales (Myxo), Rhizobiales (Rhiz), Xanthomonadales (Xant), Frankiales(Fran), Burkholderiales (Burk), Sphingomonadales (Sphi), Rhodocyclales (Rhodo), Micrococcales (Micr), Nitrospirales (Nitr), Streptomycetales (Stre), Bacillales (Baci), Pseudomonadales (Pseudom), Rhodospirillales (Rhodos), Planctomycetales (Plan), Pseudonocardiales (Pseudon). The same below.
Fig. 5. RDA analysis to identify the relationships between soil N fractions (red arrows) and soil nitrogen-fixing microorganism composition (blue arrows) (a-phyla level; b-class level; c-order level). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
percentages of sand and organic carbon increased, whereas the SBD decreased. These effects improved the permeability of the soil significantly, and subsequently promoted microbial growth, leading to changes in species compositions. In our study, the N-fixing microorganism communities commonly varied from Actinobacteria-dominated in cropland to Proteobacteria-dominated in forest soil during the changes of chronosequence, showing that the underground community transitioned from a slow-growing oligotrophic group to a fast-growing symbiotic group. Therefore, soil properties, such as organic carbon level and the percentage of sand may affect biological N fixation of specific diazotrophs taxonomic groups (Calderoli et al., 2017). Our results provide a plausible explanation for the N fractions and the composition of soil nifH communities in the RP chronosequence. Moreover, some uncertainties lead to changes in the relative abundance of species, such as temporal variability, landform, and perturbed environments. Future studies need to consider these factors at the same time and study the changes in nifH communities and N fractions over a longer chronosequence. In addition, we need to study the process considering ammoxidation, nitrification, and denitrification.
significantly associated with the changes in N fractions of the soil (Fig. 5), particularly for AN. Therefore, it could be inferred that AN could be easily affected during the changes of chronosequence because of the process of biological N fixation (Fowler et al., 2013; Vitousek et al., 2013). The nifH communities consisted of Proteobacteria, Actinobacteria, Nitrospirae, and Firmicutes (Fig. 4). The phyla level classification was similar to that of other soils and environments (Mirza et al., 2014). Regardless of the age of succession, Proteobacteria and Actinobacteria were the richest phylum which was commonly consistent with previous findings that soils usually including two common and ubiquitous bacterial groups (Kim et al., 2014; Kolton et al., 2011; Shen et al., 2013). Jangid et al. (2013) have recently reported an increasing number of Proteobacteria following the plant succession of the Franz Josef in New Zealand. Li et al. (2014) showed the similar findings in a 20 years chronosequence of soil restoration. Many soil Proteobacteria are symbiotic, and when active substrates are present, they become dominant (Goldfarb et al., 2011). However, Actinobacteria are oligotrophic groups and like poor nutrition environment. Most of the N-fixing microorganisms belong to the Proteobacteria and are aerobic organisms. In the present study, with changes in chronosequence, the 321
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5. Conclusions
Biochem. 38, 991–999. Kim, H.M., et al., 2014. Bacterial community structure and soil properties of a subarctic tundra soil in council, Alaska. FEMS Microbiol. Ecol. 89, 465–475. Kolton, M., et al., 2011. Impact of biochar application to soil on the root-associated bacterial community structure of fully developed greenhouse pepper plants. Appl. Environ. Microbiol. 77, 4924–4930. Kou, M., Garcia-Fayos, P., Hu, S., Jiao, J., 2016. The effect of Robinia pseudoacacia afforestation on soil and vegetation properties in the Loess Plateau (China): a chronosequence approach. For. Ecol. Manag. 375, 146–158. Li, D.J., Niu, S.L., Luo, Y.Q., 2012. Global patterns of the dynamics of soil carbon and nitrogen stocks following afforestation: a meta-analysis. New Phytol. 195, 172–181. Li, Y.Y., Wen, H.Y., Chen, L.Q., Yin, T.T., 2014. Succession of bacterial community structure and diversity in soil along a chronosequence of reclamation and re-vegetation on coal mine spoils in China. PLoS One 9. Ligi, T., et al., 2014. Effects of soil chemical characteristics and water regime on denitrification genes (nirS, nirK, and nosZ) abundances in a created riverine wetland complex. Ecol. Eng. 72, 47–55. Lin, C., Yang, Y., Guo, J., Chen, G., Xie, J., 2011. Fine root decomposition of evergreen broadleaved and coniferous tree species in mid-subtropical China: dynamics of dry mass, nutrient and organic fractions. Plant Soil 338, 311–327. Liu, J.G., Liu, W.G., 2018. Advances in microbial-mediated nitrogen cycling. Acta Agrestia Sin. 26, 277–283. Liu, Z.P., Shao, M.A., Wang, Y.Q., 2013. Spatial patterns of soil total nitrogen and soil total phosphorus across the entire Loess Plateau region of China. Geoderma 197, 67–78. Martinez-Alcala, I., Walker, D.J., Bernal, M.P., 2010. Chemical and biological properties in the rhizosphere of Lupinus albus alter soil heavy metal fractionation. Ecotoxicol. Environ. Saf. 73, 595–602. Mergel, A., Kloos, K., Bothe, H., 2001. Seasonal fluctuations in the population of denitrifying and N-2-fixing bacteria in an acid soil of a Norway spruce forest. Plant Soil 230, 145–160. Mirza, B.S., Potisap, C., Nusslein, K., Bohannan, B.J.M., Rodrigues, J.L.M., 2014. Response of free-living nitrogen-fixing microorganisms to land use change in the Amazon rainforest. Appl. Environ. Microbiol. 80, 281–288. Niu, S., et al., 2017. Plant stoichiometry characteristics and relationships with soil nutrients in Robinia pseudoacacia communities of different planting ages. Acta Ecol. Sin. 37, 355–362. Nunez-Mir, G.C., Iannone, B.V., Curtis, K., Fei, S.L., 2015. Evaluating the evolution of forest restoration research in a changing world: a “big literature” review. New For. 46, 669–682. Orr, C.H., James, A., Leifert, C., Cooper, J.M., Cummings, S.P., 2011. Diversity and activity of free-living nitrogen-fixing bacteria and total bacteria in organic and conventionally managed soils. Appl. Environ. Microbiol. 77, 911–919. Qiu, L.P., Zhang, X.C., Cheng, J.M., Yin, X.Q., 2010. Effects of black locust (Robinia pseudoacacia) on soil properties in the loessial gully region of the Loess Plateau, China. Plant Soil 332, 207–217. Quanhou, D., Guobin, L., Xue, S., Yu, N., Zhang, C., 2008. Dynamics of soil water-stable aggregates in the restoration process of artificial Robinia pseudoacacia under erosion environment. Bull. Soil Water Conserv. 28, 56–59. Ren, C.J., et al., 2016. Responsiveness of soil nitrogen fractions and bacterial communities to afforestation in the loess hilly region (LHR) of China. Sci. Rep. 6. Ren, C., et al., 2017. Response of microbial diversity to C:N:P stoichiometry in fine root and microbial biomass following afforestation. Biol. Fertil. Soils 1–12. Rice, S.K., Westerman, B., Federici, R., 2004. Impacts of the exotic, nitrogen-fixing black locust (Robinia pseudoacacia) on nitrogen-cycling in a pine-oak ecosystem. Plant Ecol. 174, 97–107. Roper, M.M., Smith, N.A., 1991. Straw decomposition and nitrogenase activity (C2H2 reduction) by free-living microorganisms from soi: effects of pH and clay content. Soil Biol. Biochem. 23, 275–283. Schimel, J.P., Bennett, J., 2004. Nitrogen mineralization: challenges of a changing paradigm. Ecology 85, 591–602. Schutte, U.M.E., et al., 2010. Bacterial diversity in a glacier foreland of the high Arctic. Mol. Ecol. 19, 54–66. Sha, X., Liu, G.B., Dai, Q.H., Wei, W., 2007. Evolution of soil microbial biomass in the restoration process of artificial Robinia pseudoacacia under erosion environment. Acta Ecol. Sin. 27, 909–917. Shen, C.C., et al., 2013. Soil pH drives the spatial distribution of bacterial communities along elevation on Changbai Mountain. Soil Biol. Biochem. 57, 204–211. Vitousek, P.M., Menge, D.N.L., Reed, S.C., Cleveland, C.C., 2013. Biological nitrogen fixation: rates, patterns and ecological controls in terrestrial ecosystems. Philos. Trans. R. Soc. B 368. Vos, B.D., Meirvenne, M.V., Quataert, P., Deckers, J., Muys, B., 2005. Predictive quality of pedotransfer functions for estimating bulk density of forest soils. Soil Sci. Soc. Am. J. 69, 500–510. Wang, B., Liu, G., Xue, S., 2012a. Effect of black locust (Robinia pseudoacacia) on soil chemical and microbiological properties in the eroded hilly area of China's Loess Plateau. Environ. Earth Sci. 65, 597–607. Wang, B., Liu, G.B., Xue, S., 2012b. Effect of black locust (Robinia pseudoacacia) on soil chemical and microbiological properties in the eroded hilly area of China's Loess Plateau. Environ. Earth Sci. 65, 597–607. Williams, M.A., Jangid, K., Shanmugam, S.G., Whitman, W.B., 2013. Bacterial communities in soil mimic patterns of vegetative succession and ecosystem climax but are resilient to change between seasons. Soil Biol. Biochem. 57, 749–757. Xiaogang, D., Ming, T., Hui, C., Haihan, Z., Yongan, Z., 2008. Mycorrhizae and diversity of microbial community in rhizosphere soils of Robinia pseudoacacia at different ages on the Loess Plateau. Sci. Silvae Sin. 78–82.
Our data show the effects of changes in the chronosequence in sites afforested with RP on nifH microorganism communities, N fractions, and some other properties, e.g., SWC, and SBD. We found that with the changes with forest age, the tendency of dominant species varied and AN was the most sensitive N fraction to N-fixing microorganisms. Understanding the effects of nitrogen fractions and soil physical-chemical properties on nitrogen-fixing microbes plays an important role in the later study of nitrogen fixation capacity in this area. However, the measured species composition only reflects the microbial potential for nitrogen fixing and do not describe actual turnover rates in soils. Therefore, investigations of gene expression and enzyme activity remain to be performed with further study. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.catena.2018.11.009. Acknowledgements The project of National Natural Science Foundation of China (No. 41571501) and the Key Project of the National Key Research and Development Program of China (No. 2017YFC0504601) sustained our research. We are grateful to An'sai Ecological Experimental Station of Soil and Water Conservation for the assistance. We thank our colleagues Wei Zhang and Zekun Zhong for their help of field experiments. References Bolat, I., Kara, O., Sensoy, H., Yuksel, K., 2016. Influences of black locust (Robinia pseudoacacia L.) afforestation on soil microbial biomass and activity. IforestBiogeosciences and Forestry 9, 171–177. Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985. Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. Biochem. 17, 837–842. Calderoli, P.A., Collavino, M.M., Kraemer, F.B., Morrás, H.J.M., Aguilar, O.M., 2017. Analysis of nifH-RNA reveals phylotypes related to geobacter and cyanobacteria as important functional components of the N2-fixing community depending on depth and agriculturaluse of soil. Microbiology 6, e00502. Cao, Y., Chen, Y.M., 2017. Coupling of plant and soil C:N:P stoichiometry in black locust (Robinia pseudoacacia) plantations on the Loess Plateau, China. In: Trees-Structure and Function. 31. pp. 1559–1570. Caporaso, J.G., et al., 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336. Deluca, T.H., Drinkwater, L.E., Wiefling, B.A., Denicola, D.M., 1996. Free-living nitrogenfixing bacteria in temperature cropping systems: influence of nitrogen source. Biol. Fertil. Soils 23, 140–144. Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461. Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200. Food and Agriculture Organization (FAO), 1974. Soil Map of the World. volume 1 Legend, Paris. Fowler, D., et al., 2013. The global nitrogen cycle in the twenty-first century. Philos. Trans. R. Soc. B 368 (1621), 20130164. Fu, B.J., et al., 2011. Assessing the soil erosion control service of ecosystems change in the Loess Plateau of China. Ecol. Complex. 8, 284–293. Galloway, J.N., et al., 2008. Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320, 889–892. Goldfarb, K.C., et al., 2011. Differential growth responses of soil bacterial taxa to carbon substrates of varying chemical recalcitrance. Front. Microbiol. 2. Hai, B., et al., 2009. Quantification of key genes steering the microbial nitrogen cycle in the rhizosphere of sorghum cultivars in tropical agroecosystems. Appl. Environ. Microbiol. 75, 4993–5000. Han, X.H., 2018. Evaluation and Ecological Effects of Returning Farmland to Forest in Loess Hilly and Gully Region. Science Press, Beijing. Herrera, J.M., Garcia, D., 2010. Effects of Forest fragmentation on seed dispersal and seedling establishment in ornithochorous trees. Conserv. Biol. 24, 1089–1098. Jangid, K., Whitman, W.B., Condron, L.M., Turner, B.L., Williams, M.A., 2013. Soil bacterial community succession during long-term ecosystem development. Mol. Ecol. 22, 3415–3424. Jiao, J., Zhang, Z., Bai, W., Jia, Y., Wang, N., 2012. Assessing the ecological success of restoration by afforestation on the Chinese Loess Plateau. Restor. Ecol. 20, 240–249. Jin, Z., Dong, Y.S., Qi, Y.C., Liu, W.G., An, Z.S., 2013. Characterizing variations in soil particle-size distribution along a grass–desert shrub transition in the Ordos plateau of inner Mongolia, China. Land Degrad. Dev. 24, 141–146. Jones, D.L., Willett, V.B., 2006. Experimental evaluation of methods to quantify dissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soil. Soil Biol.
322
Catena 174 (2019) 316–323
Y. Xu et al.
changes in plant community and soil properties during the secondary succession of abandoned farmland in the Loess Plateau. Soil Biol. Biochem. 97, 40–49. Zheng, Y.A., et al., 2011. The importance of slope aspect and stand age on the photosynthetic carbon fixation capacity of forest: a case study with black locust (Robinia pseudoacacia) plantations on the Loess Plateau. Acta Physiol. Plant. 33, 419–429. Zhu, D.J., et al., 2018. Effects of alien species Robinia pseudoacacia on plant community functional structure in hilly-gully region of Loess Plateau, China. Chin. J. Appl. Ecol. 29, 459–466.
Xin, L.I., Rui-Ping, M.A., Shao-Shan, A.N., Zeng, Q.C., Ya-Yun, L.I., 2015. Characteristics of soil organic carbon and enzyme activities in soil aggregates under different vegetation zones on the Loess Plateau. Chin. J. Appl. Ecol. 26, 2282. Zeng, J., et al., 2016. Primary succession of nitrogen cycling microbial communities along the deglaciated forelands of Tianshan mountain, China. Front. Microbiol. 7 (e42149). Zhang, C., Xue, S., Liu, G.B., Song, Z.L., 2011. A comparison of soil qualities of different revegetation types in the Loess Plateau, China. Plant Soil 347, 163–178. Zhang, C., Liu, G.B., Xue, S., Wang, G.L., 2016. Soil bacterial community dynamics reflect
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