Alteration of the soil bacterial community during parent material maturation driven by different fertilization treatments

Soil Biology & Biochemistry 96 (2016) 207e215

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Alteration of the soil bacterial community during parent material maturation driven by different fertilization treatments Li Sun a, 1, Weibing Xun a, b, 1, Ting Huang c, Guishan Zhang b, Jusheng Gao d, Wei Ran a, Dongchu Li d, Qirong Shen a, Ruifu Zhang a, b, * a Jiangsu Key Lab and Engineering Center for Solid Organic Waste Utilization, National Engineering Research Center for Organic-based Fertilizers, Nanjing Agricultural University, Nanjing, 210095, PR China b Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, 100081, PR China c Hanlin College, Nanjing University of Chinese Medicine, Taizhou, 225300, PR China d Qiyang Red Soil Experimental Station, Chinese Academy of Agricultural Sciences, Qiyang, 426182, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 August 2015 Received in revised form 16 February 2016 Accepted 19 February 2016 Available online 3 March 2016

Soil parent materials are potential arable land resources that have great value for utilization. Soil bacteria play vital roles in soil formation, and soil parent material provides the basic nutritional environment for the development of the microbial community. Due to the extremely limited available nutrients in most parent materials, fertilization management is important for providing necessary available nutrients and for enhancing the maturation process of the parent materials. After 30 years of artificial maturation driven by different fertilization treatments, the soil development of three different parental materials was evaluated, and the bacterial community compositions were investigated using a high-throughput nucleic acid sequencing approach. The results showed that fertilization management increased the soil fertility and microbial biomass and enhanced soil parent material maturation compared with cultivation alone. Supplying available nutrients via chemical fertilization was more effective than cultivation alone for soil nutrient accumulation, microbial biomass promotion, and copiotrophic bacterial enrichment during soil parent material development. The soil bacterial community structure was determined by both parent material and fertilization strategies. Compared to straw returning, chemical fertilization-driven parent material maturation decreased soil bacterial diversity and significantly changed the soil bacterial community structure. However, compared to chemical fertilization, straw returning had a less negative effect on soil bacterial diversity, but was not as efficient in resolving the nutrient limitation during soil parent material maturation. This study provided insight into the maturation of soil parent materials for agriculture production to support the ever constant need for food by an increasing human population. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Soil parental material Bacterial community Soil maturity Fertilization

1. Introduction There is a variety of parent materials distributed in the subtropical region of China and reclaim this kind of land is in urgent need to meet an increasing demand for agricultural products (Xu and Cai, 2007). However, soil parent material is inherently less

* Corresponding author. College of Resources & Environmental Science, Nanjing Agricultural University, 210095, Nanjing, PR China. Tel.: þ86 25 84396477; fax: þ86 25 84396260. E-mail address: [email protected] (R. Zhang). 1 Both authors contributed equally to this paper. http://dx.doi.org/10.1016/j.soilbio.2016.02.011 0038-0717/© 2016 Elsevier Ltd. All rights reserved.

fertile than fully formed soil and cannot support high amounts of food production (Li et al., 2014). Exploiting and utilizing soil parent material is an effective measure to alleviate the shortage of arable land resources and develop agricultural production. Nutrient accumulation processes are affected by parent materials (Anderson, 1988) and nutrient concentrations in parent materials, that is often limited, is needed to be increased to increase crop yields (Robertson and Vitousek, 2009). Fertilizer application and crop straw return are two effective methods to improve soil nutrient concentrations and availability (Marschner et al., 2003). Straw incorporation is an important strategy to improve soil quality and has a long-term positive effect on crop yield (Tejada et al., 2008; Han and He,

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2010; Ortiz Escobar and Hue, 2008). Nitrogen is often limiting in soil parent materials (Anderson, 1988), and its input can lead to higher net primary production (LeBauer and Treseder, 2008). Fertilizer and straw application provide nutrition (especially nitrogen) for plants growing in soil parent materials. Microorganisms (especially bacteria) are the foundation of the earth's biosphere and play unique roles in ecosystem functions. In agricultural soil, bacteria are indispensable maintainers of soil productivity (Smith and Paul, 1990). Bacteria contribute to soil productivity through their involvement in organic matter decomposition, humus formation, nutrient transformation, and element cycling. The latest studies have demonstrated that the microbial necromass (e.g. the remains of dead bacterial cells) could be a significant source of soil organic matter (Langerhuus et al., 2012). Moreover, microbial products initiate and enhance the formation of long-term stabilized soil organic matter (Ludwig et al., 2015). Soil microorganisms are actively involved in soil aggregation dynamics and play a key role in soil structure formation processes. Thus, microorganisms are considered architects that can have an effect on the surrounding soil environment (Vogel et al., 2014). Moreover, during the early period of soil formation, microorganisms enhance weathering through the production of organic acids, promotion of hydrolysis reactions, and the release of nutrients such as phosphorus and potassium from soil minerals (Lian et al., 2008; Uroz et al., 2009). Soil parent material provides the foundation elements that comprise the basic nutritional environment for microbial community development (Anderson, 1988; Ulrich and Becker, 2006). During soil formation, soil physicalechemical properties (i.e., soil pH and organic matter concentration) change with time and can influence soil microbial communities (Lombard et al., 2011). Moreover, soil microbial communities mediate many soil biogeochemical processes (Balser and Firestone, 2005). Therefore, there are complex interactions between soil microbial communities and the soil physicalechemical environment during the different stages of soil development (Tarlera et al., 2008). Consequently, measuring soil microbial communities can indicate the status of soil development and the effectiveness of management interventions (Harris, 2009; Li et al., 2014). Soil parent material is a key factor that determines the bacterial community of the mature soil (Ulrich and Becker, 2006). Sun et al. (2015a) reported that the use of cultivation as a management strategy to accelerate parent material maturation had an important effect on the bacterial community of the soil. Fertilization and crop straw returning are major management strategies to accelerate soil parent material maturation, but their effects on bacterial community development during the maturation of soil parent materials have generally been less studied, especially in long-term field experiments. Fertilization supplies available nutrients for both soil microbes and crop plants, which in turn enhance soil nutrient input through rhizosphere deposition. Soil parent material and fertilization (the main factors involved in soil maturation) should also be the main driving forces for soil microbial community formation during the maturation process. To investigate the bacterial community alteration during soil maturation under different fertilization regimens, we measured the soil properties, soil enzymatic activity and bacterial community composition. 2. Materials and methods 2.1. Site description and soil sampling The long-term experimental research was conducted in Qiyang (111520 3200 E, 45 260 4200 N, 150e170 m a.s.l), Hunan province, South

China, and was established in 1982 by the Chinese Academy of Agricultural Sciences (CAAS) to study how to accelerate the artificial maturation process for the rapid fertilization of parent materials. This region has a subtropical monsoon climate with an average annual temperature of 17.8  C and mean annual rainfall of 1255 mm, of which 70%e80% occurs from April to October. There are three soil parent material types in this long-term soil maturation experiment: quaternary red clay soil (Q), granite soil (G) and purple sandy shale (P). These three types represent the three major soil parent materials of the agricultural soils in Hunan province in South China (soil material information is shown in Table_SI_1). To assess the effect of chemical fertilization and crop straw returning, four treatments were selected for each parent material: control without fertilizer (CK), straw returning (SR), chemical fertilizer without straw returning (NPK) and chemical fertilizer with straw returning (NPK þ SR) (the treatments are described in Table_SI_2). The experimental plots were consisted of cement pools that were 4 m long  2 m wide  1 m deep, with open bottoms, the cement walls were about 10 cm above the soil surface to avoid the cross contamination of soils from different plots. All of the plots were cultivated in the same way using a Poaceae, Leguminosae, Cruciferae and Tuber crop rotation pattern. The soils were sampled in May, 2012, as described previously (Sun et al., 2015a). All collected samples were sieved (2 mm) and divided equally into two parts: one was frozen at 80  C for DNA extraction and the second was stored at 4  C prior to analysis. 2.2. Soil chemical analysis, soil microbial biomass and soil enzyme analysis All chemical properties were determined by routine methods (Bao, 2010). Soil pH was measured with a glass electrode (soil/ water ¼ 1:5). Soil microbial biomass C (MBC) was determined by the chloroform fumigation extraction method (Vance et al., 1987). After 24 h of fumigation, 12.5 g of soils were extracted using 0.5 M K2SO4 with a 1:5 ratio for 60 min on a rotary shaker. The amount of organic C in the extract was measured by a LiquiTOC II total organic C analyzer (Elementar, Shanghai, China). The MBC was calculated using the following equation: MBC ¼ EC  kEC, where EC was the difference between the amount of C extracted from the fumigated and non-fumigated soils and kEC was 2.64 (Vance et al., 1987; Zhong and Cai, 2007). Catalase activity was measured by back-titrating residual H2O2 with KMnO4 (Edwards et al., 1992). Two grams (<2 mm) of air-dried soils were added to a 40 mL aliquot of distilled water and 5 mL of 30% H2O2. The mixture was shaken for 10 min, and then 5 mL of 1.5 N H2SO4 was added. A 25 mL aliquot of the filtered solution was titrated with 0.02 N KMnO4. Controls were generated in the same way without the addition of H2O2. Invertase activity was determined using sucrose as the substrate (Hu et al., 2006). Five grams (<2 mm) of air-dried soil, 15 mL of sucrose solution (8%) and 5 mL of phosphate buffer (pH5.5) were added to a 50 mL triangular flask and incubated for 24 h at 37  C. After filtration, 1 mL of filtrate was added to a test tube and heated for 5 min with 3 mL of 5-dinitrosalicylic acid (DNS). The colour was measured using a colourimetric assay at 508 nm. Acid and alkaline phosphatase were assayed using disodium phenyl phosphate as the substrate (Dick et al., 2000). Briefly, 1 g of soil (<2 mm) and 5 drops of toluene were added to a 250 mL triangular flask and shaken for 15 min; then, 20 mL of 0.5% disodium phenyl phosphate was added, and the mixtures were incubated at 37  C for 24 h. Next, 40 mL of 0.3% aluminium sulphate solution was added to the mix and filtered. After filtration, 3 mL of filtrate was added to a 50 mL volumetric flask for the colour reaction measured by a colourimetric assay at 660 nm. We used acetate

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buffer (pH ¼ 5) for measuring acid phosphatase (G and Q soils) and borate saline buffer (pH ¼ 10) for measuring alkaline phosphatase (P soils).

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bacterial community and environmental factors (Hill and Gauch Jr, 1980). 3. Results

2.3. DNA extraction, PCR and sequencing 3.1. Fertilization enhanced the soil parent material maturation Soil DNA was extracted from 0.5 g of soil with the PowerSoil DNA Isolation Kit (Mo Bio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. The extracted DNA was quantified with a NanoDrop ND-2000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and stored at 20  C. The primer set 27F: 50 -AGAGTTTGATCCTGGCTCAG-30 and 533R: 50 -TTACCGCGGCTGCTGGCAC-30 plus a Roche-454 sequencing adapter, a linker sequence and a unique, error-correcting barcode sequence (Ns), as previously described by Dethlefsen et al. (2008) and Huse et al. (2008), targeting the V1eV3 hypervariable region of the 16S rRNA gene were used for PCR. Amplification reactions were performed in a 20 ml volume containing 2 mM of each primer, 0.25 mM dNTPs (Takara), 4 ml of 5  FastPfu Buffer (TransGen Biotech Co., Ltd., Beijing, China), one unit of FastPfu DNA polymerase (2.5 U/ml, TransGen) and 20 ng of soil DNA template. Amplification was initiated at 95  C for 2 min, followed by 25 cycles of denaturation at 95  C for 30 s, annealing at 55  C for 30 s, extension at 72  C for 30 s, and a final elongation at 72  C for 5 min. The PCR products were visualized on 2% agarose gels, pooled together to minimize PCR bias, and purified with a PCR Purification Kit (Axygen Bio, Union City, CA, USA). High-throughput sequencing was performed with the 454 GSFLX Titanium System sequencer at Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) after an emulsion PCR to generate single strands on beads as required for the 454 pyrosequencing. 2.4. Sequence analysis Sequence processing was performed using the bioinformatics platform Mothur (Schloss et al., 2009). Briefly, sequences with a minimum length of 450 flows were denoised using the Mothurbased reimplementation of the PyroNoise algorithm with default parameters (Quince et al., 2009). Sequences were discarded if they contained any ambiguous base calls or if they had more than two mismatches to the forward primer, one mismatch to the barcode sequence, or a homopolymer longer than 8 bp or shorter than 200 bp. After removing the barcode and primer sequences, the retained sequences were aligned against the Silva 106 reference database (Pruesse et al., 2007). After filtering, preclustering, and the removal of chimaeras, the retained sequences were used to build a distance matrix with a distance threshold of 0.2. Using the average neighbour algorithm with a cut-off of 97% similarity, the sequences were clustered into operational taxonomic units (OTUs). The representative sequence of each OTU was selected and classified using the platform Ribosomal Database Project (RDP) with a confidence threshold of 80% (Wang et al., 2007). 2.5. Data analysis The relative abundance of the OTUs was subjected to a ratio transformation. All statistics were performed using the program R (version 2.15.3). An OTU-based analysis was performed to calculate the richness and diversity in these samples with a cutoff of 3% dissimilarity. A hierarchical cluster analysis was performed using BrayeCurtis distances (Oksanen, 2010). Canonical correspondence analysis (CCA) was conducted in the vegan package of R (Oksanen et al., 2013). These analyses were conducted to compare bacterial community structures across all samples based on the OTU composition and to visualize the relationship between the

For all three of the major red soil parent materials in South China (Q, G, and P), fertilization significantly (P < 0.05) increased the available nutrient concentrations (AN and AK) compared with the parent material (Sun et al., 2015a) and CK (Table 1). NPK and NPK þ SR significantly increased the total N (TN) and total P (TP) concentrations (P < 0.05), but only significantly increased total K (TK) concentration in the P soils (P < 0.05). All of the long-term fertilization treatments resulted in the accumulation of soil organic matter (OM) than did cultivation alone. In the G and Q soils, chemical fertilization (NPK and NPK þ SR) significantly increased the OM (P < 0.05), but in the P soil, crop straw returning (SR and NPK þ SR) contributed more to the OM accumulation during parent material maturation (Table 1). Notably, long-term chemical fertilization-driven parent material maturation also caused a decrease in soil pH. Crop straw returning alleviated soil acidification in the G and Q soils but decreased the soil pH in the alkaline P soil. 3.2. Fertilization increased the soil microbial biomass and changed the soil enzyme activity during the maturation process All of the long-term fertilization treatments increased the MBC in the three soil parent materials compared with the cultivation alone treatment (Fig. 1). In the G and P soils, NPK and NPK þ SR had significant (P < 0.05) greater MBC than CK and SR; whereas in the Q soil, SR and NPK þ SR increased the MBC more than CK and NPK. Soil enzyme activity is an important indicator of soil fertility and maturity. All of the fertilization procedures increased the invertase activity during the maturation process compared with the cultivation alone treatment, and the NPK and NPK þ SR treatments had the most significant effect (P < 0.05) (Table 2). However, chemical fertilization caused a decrease in catalase activity in the G and Q soils but had no effect on the soil catalase activity in the P soil. Fertilization significantly (P < 0.05) increased soil alkaline phosphatase activity in the P soil. In contrast, NPK decreased the soil acid phosphatase activity in the G and Q soils (Table 2). 3.3. Chemical fertilization-driven maturity significantly changed the soil bacterial community The bacterial richness and diversity of the different parent materials and mature treatments were calculated based on a subset of 4679 randomly selected sequences (Table 3). The results showed that all fertilization procedures (especially chemical fertilization) reduced the bacterial richness and diversity during the maturation of these three soil parent materials. Returning the crop straw along with the chemical fertilization could alleviate this reduction. The relative abundances of the major phyla varied with different fertilization treatments (Fig. 2). NPK and NPK þ SR significantly increased the relative abundances of Actinobacteria and Gammaproteobacteria but had an opposite effect on Chloroflexi and Gemmatimonadetes in the three parent materials. NPK also reduced the relative abundance of Alphaproteobacteria in the acidic G and Q soils but increased its relative abundance in the alkaline P soil. Alteration patterns in the soil bacterial community structure were determined by both parent material type and fertilization regimen. BrayeCurtis dissimilarity analysis (Fig. 3) was performed based on the 13,092 OTUs obtained from the 36 samples with 4679

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Table 1 Soil physicochemical characteristics of the treatments of three soil parent material types. Treatmenta

pH

G_CK G_SR G_NPK G_NPK þ SR P_CK P_SR P_NPK P_NPK þ SR Q_CK Q_SR Q_NPK Q_NPK þ SR

4.96 4.78 4.02 4.00 8.52 7.64 8.38 7.51 4.33 4.64 3.67 3.94

Organic matter (g kg1) ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.67 0.09 0.02 0.03 0.02 0.04 0.00 0.02 0.02 0.01 0.01

ab a b b a c b d b a d c

6.8 7.2 18.2 20.4 5.1 8.8 5.6 9.8 7.3 8.1 10.1 14.3

± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.5 0.6 0.1 0.4 0.1 0.2 0.6 0.7 0.1 0.5 0.6

c c b a c b c a c c b a

Total nitrogen (g kg1) 0.46 0.50 0.96 1.10 0.72 0.94 0.91 1.14 0.71 0.75 0.77 1.36

± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.02 0.03 0.01 0.04 0.01 0.03 0.02 0.13 0.01 0.03

d c b a c b b a b b b a

Total phosphorus (g kg1) 0.23 0.23 1.44 1.24 0.53 0.55 1.63 1.73 0.31 0.36 1.33 1.38

± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01

c c a b c c b a c d b a

Total potassium (g kg1) 43.0 44.7 42.9 44.2 28.5 24.6 32.1 33.1 15.2 15.4 16.5 16.5

± ± ± ± ± ± ± ± ± ± ± ±

0.8 0.5 3.3 1.8 0.1 0.8 0.2 0.1 0.1 0.7 0.2 0.1

a a a a b c a a b b a a

Available nitrogen (mg kg1) 38.1 44.6 78.9 84.6 27.4 41.9 55.3 72.0 58.3 64.4 71.3 78.5

± ± ± ± ± ± ± ± ± ± ± ±

0.1 1.9 4.2 3.1 1.5 0.8 1.1 1.5 1.9 1.1 1.1 1.5

Available potassium (mg kg1)

d c b a d c b a d c b a

73 102 106 140 72 99 154 294 103 137 213 253

± ± ± ± ± ± ± ± ± ± ± ±

1.0 1.5 2.9 1.5 0.1 0.6 0.8 2.3 1.2 1.5 2.7 2.3

d c b a d c b a d c b a

a G: quaternary red clay soil; P: purple sandy shale; Q: granite soil; CK: control without fertilizer; SR: straw returning; NPK: chemical fertilizer without straw returning; NPK þ SR: chemical fertilizer with straw returning. b Values (mean ± standard deviation, n ¼ 3) indicate the absolute amount of each characteristic. Different letters in column indicate significant differences (P < 0.05) among amendments within same soil type according to Duncan's multiple comparison.

similar structures. Bacterial community structures of the acidic G and Q soils were determined by fertilization regimens instead of soil parent material type. 3.4. Soil bacterial community alteration was associated with the soil properties

Fig. 1. Comparison of the soil microbial C concentration of different treatments in three soil parent materials. The heights show soil microbial C concentration per gram of soil (n ¼ 3). Error bars indicate the standard deviation of the mean. G: quaternary red clay soil; P: purple sandy shale; Q: granite soil; CK: control without fertilizer; SR: straw returning; NPK: chemical fertilizer without straw returning; NPK þ SR: chemical fertilizer with straw returning.

randomly selected sequences per sample and a 97% cutoff. The analysis demonstrated that the alkaline P soils were separated from the acidic G and Q soils. In the P soils, NPK and NPK þ SR resulted in similar bacterial community structures, while CK and SR also had

The CCA ordination plots showed the correlations between the soil bacterial community and the soil properties (Fig. 4). Overall, bacterial communities of the P soil were grouped together and separated from the G and Q soils in the first axis due to their relatively high pH (Fig. 4a). The bacterial communities of the NPK and NPK þ SR in the G and Q soils formed a distinct group in the second axis. This bacterial community separation could be associated with greater soil properties, such as soil AN, AP, TN, TP, and OM concentrations. In the G soils (Fig. 4b), the chemical fertilizer treatments NPK and NPK þ SR were separated from both the non-chemical fertilizer treatments (CK and SR) and each other due to the driving forces from both chemical fertilization and straw returning. The bacterial communities of CK and SR were found to be associated with greater soil pH (r ¼ 0.807, P < 0.01) but lower OM concentration (r ¼ 0.613, P < 0.01), whereas the bacterial community of NPK þ SR was associated with greater TP concentration (r ¼ 0.669, P < 0.01). The bacterial communities of the P soils (Fig. 4c) formed four distinct

Table 2 Soil phosphatase, invertase, and catalase activity in the treatments of three soil parent material types. Treatmenta G_CK G_SR G_NPK G_NPK þ SR P_CK P_SR P_NPK P_NPK þ SR Q_CK Q_SR Q_NPK Q_NPK þ SR

Phosphataseb (mg PNP g1 d1) 125 164 60 125 141 276 283 206 343 449 311 406

± ± ± ± ± ± ± ± ± ± ± ±

c

34 a 21 a 4b 23 a 27 c 25 a 24 a 40 b 15 ab 40 ab 99 b 11 ab

Invertase (mg glucose g1 d1) 2.9 2.6 17.4 15.3 6.7 20.9 81.0 100.2 0.7 5.5 18.4 20.9

± ± ± ± ± ± ± ± ± ± ± ±

0.3 c 0.9 c 0.3 a 0.1 b 1.4 d 2.0 c 8.1 b 11.6 a 0.2 d 0.3 c 0.9 b 0.5 a

Catalase (mg H2O2 g1 d1) 55.5 53.2 23.7 33.5 127 128 125 124 88.6 70.1 12.5 16.3

± ± ± ± ± ± ± ± ± ± ± ±

6.0 1.2 1.4 1.3 0.3 0.3 1.9 0.6 0.8 0.8 2.5 1.3

a a c b ab ab bc c a b d c

a G: quaternary red clay soil; P: purple sandy shale; Q: granite soil; CK: control without fertilizer; SR: straw returning; NPK: chemical fertilizer without straw returning; NPK þ SR: chemical fertilizer with straw returning. b Acid phosphatase in G and Q soils, alkaline phosphatase in P soils. c Values (mean ± standard deviation, n ¼ 3) indicate the absolute activity of each enzyme. Different letters in column indicate significant differences (P < 0.05) among amendments within same soil type according to Duncan's multiple comparison.

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Table 3 Observed OTUs, the bacterial community richness and diversity of the treatments in three soil parent materials. Treatmenta

OTUsb

Richness Acec

G_CK G_SR G_NPK G_NPK þ SR P_CK P_SR P_NPK P_NPK þ SR Q_CK Q_SR Q_NPK Q_NPK þ SR

1182 898 394 953 1461 1597 1431 1509 971 803 363 460

± ± ± ± ± ± ± ± ± ± ± ±

g

18 a 163 b 10 c 28 b 129 a 105 a 138 a 103 a 39 a 41 b 13 d 25 c

2526 1971 722 2073 3477 4141 3360 3703 2142 1647 569 873

Diversity Chaod

± ± ± ± ± ± ± ± ± ± ± ±

139 a 382 b 148 c 143 b 653 a 648 a 407 a 466 a 103 a 115 b 48 d 67 c

2022 1494 631 1643 2643 3007 2560 2834 1699 1334 549 719

± ± ± ± ± ± ± ± ± ± ± ±

Shannone 175 a 276 b 61 c 48 b 366 a 420 a 263a 338 a 95 a 76 b 20 d 84 c

6.25 5.10 4.42 5.72 6.57 6.70 6.45 6.56 5.76 5.47 4.29 4.58

± ± ± ± ± ± ± ± ± ± ± ±

0.04 1.28 0.05 0.03 0.15 0.08 0.19 0.08 0.03 0.07 0.03 0.01

Invsimpsonf a ab b a a a a a a b d c

230 83 32 84 354 388 222 281 100 94 27 36

± ± ± ± ± ± ± ± ± ± ± ±

24.3 a 70.1 b 2.8 b 4.5 b 78.8 a 38.9 a 71.9 b 25.8 bc 7.6 a 6.1 a 2.0 b 3.0 b

a G: quaternary red clay soil; P: purple sandy shale; Q: granite soil; CK: control without fertilizer; SR: straw returning; NPK: chemical fertilizer without straw returning; NPK þ SR: chemical fertilizer with straw returning. b Operation taxonomic units (97% similarity). c Abundance-based coverage estimators. d Richness estimate for an OTU definition. e Non-parametric Shannon diversity index. f The inverse Simpson index. g Values (mean ± standard deviation, n ¼ 3) indicate each index. Different letters in column indicate significant differences (P < 0.05) among amendments within same soil type according to Duncan's multiple comparison.

groups and were separated based on fertilizer addition at the first axis and straw returning at the second axis. The bacterial communities of CK and SR were found to be associated with lower AP concentration (r ¼ 0.542, P < 0.01), whereas the bacterial communities of SR and NPK þ SR were associated with lower soil pH (r ¼ 0.595, P < 0.01) but greater OM concentration (r ¼ 0.634, P < 0.01). Within the Q soils (Fig. 4d), the bacterial communities of NPK and NPK þ SR were well-grouped and separated from the CK and SR, which showed distinct differences between one another. The bacterial communities of NPK and NPK þ SR were found to be associated with greater AP concentration (r ¼ 0.846, P < 0.01) but lower soil pH (r ¼ 0.615, P < 0.01). In summary, the addition of chemical fertilizers and straw returning drove the formation of different community patterns in different soil parent materials, and fertilizer application seemed to be the stronger force influencing the bacterial community in all parent materials. The Pearson correlation coefficient was used to evaluate the correlation between soil characteristics and relative phyla abundance. The results showed that edaphic properties had close relationships with the relative abundances of bacterial phyla (Table 4). The phylum Actinobacteria, which was one of the most abundant phyla, was positively and significantly correlated with soil TN, TP, AN, and AK concentrations (P < 0.05) and had no relationship with soil OM. The sub-phylum Gammaproteobacteria had a significant positive relationship with soil AN, AK, and OM concentrations (P < 0.05) and a negative relationship with pH (P < 0.01). The other two phyla (Chloroflexi and Gemmatimonadetes) that exhibited variations between the different treatments were closely related to soil properties. These two phyla both had significant negative relationships with soil OM, AN, and AK concentrations (P < 0.05).

4. Discussion Chemical fertilization was more efficient in improving soil total and available nutrients and accelerating the maturation process due to the limitation of available nutrients in the soil parent material. Crop straw returning consistently increased soil C and N concentrations (Børresen, 1999; Thomsen and Christensen, 2004)

and was an effective means for improving soil quality (Børresen, 1999; Bhogal et al., 2009; Ji et al., 2012; Liu et al., 2014). Nitrogen addition has been demonstrated to cause significant soil acidification (Moody and Aitken, 1997; Zhao and Xing, 2009). During the parent material maturation process, chemical fertilization decreased soil pH in all these three soils, this decreasing is more serious for the acidic G and Q soils, whereas straw returning only induced soil pH decreasing in the alkaline P soil. Catalase is an oxidoreductase that is associated with aerobic microbial activity and is involved in microbial redox metabolism (Garcıa-Gil et al., 2000). Chemical fertilization-driven maturation inhibited catalase activity in the acidic G and Q soils, which could be due to the very acidic soil pH of these treatments. Some studies have shown that pH can affect catalase activity, ultimately resulting in a dramatic reduction at low values (Mares et al., 1994). However, long-term chemical fertilization increased soil invertase activity in all three soils, which was a reflection of the soil fertility and soil microbial biomass (Gu et al., 2009a). Thus, chemical fertilizers promoted soil biological activity. All of the fertilization management procedures (SR, NPK, and NPK þ SR) promoted significant increases in the soil microbial biomass compared with cultivation alone (CK). The increase in the soil microbial biomass due to fertilizer and straw returning could be a result of better crop growth, a higher release of root exudates and larger amounts of root residues left in the soil, which positively influenced microbial processes and development (Gu et al., 2009b). The soil parent materials were extremely lean and lacked available nutrients (Sun et al., 2015a), although straw incorporation had a positive effect on soil fertility. Therefore, chemical fertilizer application could support soil nutrient availability more directly and efficiently during the early stage of soil maturation, which had an important influence on soil microorganism enrichment. Long-term fertilizer application had significant effects on the bacterial community. A previous study showed that long-term chemical fertilization caused a decrease in bacterial ́diversity in the soil (Sun et al., 2015b). In our study, long-term chemical fertilizer application alone not only caused a negative effect on bacterial diversity but also significantly decreased bacterial richness; this decline could be alleviated by straw returning. Due to the

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Fig. 2. Relative abundance (n ¼ 3) of the dominant bacteria phyla in different nutrient amendments. Relative abundances are based on the proportional frequencies of the DNA sequences that could be classified at the phylum level. We selected the top 11 abundant phyla (subphyla) to show in the bar chart. (a) Relative abundance of the dominant bacteria phyla in the granite soil type (G). (b) Relative abundance of the dominant bacteria phyla in the purple sandy shale soil type (P). (c) Relative abundance of the dominant bacteria phyla in the quaternary red clay soil type (Q). Error bars indicate the standard deviation of the mean. G: quaternary red clay soil; P: purple sandy shale; Q: granite soil; CK: control without fertilizer; SR: straw returning; NPK: chemical fertilizer without straw returning; NPK þ SR: chemical fertilizer with straw returning.

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Fig. 3. Hierarchical cluster dendrogram of the bacterial community from soils of four treatments in three soil types using BrayeCurtis dissimilarity. G: quaternary red clay soil; P: purple sandy shale; Q: granite soil; CK: control without fertilizer; SR: straw returning; NPK: chemical fertilizer without straw returning; NPK þ SR: chemical fertilizer with straw returning.

Fig. 4. The canonical correspondence analysis (CCA) of bacterial community and soil properties. (a) The CCA of bacterial community and soil properties for all individual samples from three different soil parent materials. (b) The CCA of bacterial community and soil properties for treatments of the granite soil parent material (G). (c) The CCA of bacterial community and soil properties for treatments of the purple sandy shale parent material (P). (d) The CCA of bacterial community and soil properties for treatments of the quaternary red clay parent material (Q). G: quaternary red clay soil; P: purple sandy shale; Q: granite soil; CK: control without fertilizer; SR: straw returning; NPK: chemical fertilizer without straw returning; NPK þ SR: chemical fertilizer with straw returning.

infertile situation in the soil parent materials, the bacterial community would be sensitive to nutrient addition. The microbial community composition was associated with factors determined by the parent soil type (Bowles et al., 2014). In our study, soil type had a primary impact on the bacterial community structure, and long-term straw and chemical fertilizer addition both

had significant influences on bacterial community variation during the maturation process. The results of hierarchical cluster analysis based on the OTU composition showed that the bacterial communities of NPK and NPK þ SR differed from CK and SR Thus, chemical fertilizer, due to it effect on pH, was the uppermost factor leading to soil bacterial community variation compared with straw returning.

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Table 4 Pearson's correlation between soil characteristics and the relative abundance of abundant phyla.

Acidobacteria Actinobacteria Chloroflexi Firmicutes Bacteroidetes Planctomycetes Gemmatimonadetes Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Gammaproteobacteria

pH

Organic matter

Total nitrogen

Total phosphorus

Total potassium

Available nitrogen

Available potassium

0.762** e e 0.513** 0.759** 0.535** e e 0.343* 0.877** 0.478**

e e 0.461** e 0.348* 0.657** 0.519** e e 0.425** 0.799**

e

0.663** 0.684** 0.345* 0.528** 0.491** e 0.551** e 0.363* 0.508** e

e e e

e

e

0.398*

e e e e e 0.345* e e e

0.376*

e

0.608** 0.337* 0.718** 0.522** e e

0.415* 0.438** e e 0.357* 0.348* e e 0.420* 0.642**

0.703** 0.468** e e e 0.351* e e e 0.330*

Values displayed in the table indicate significant correlations. Asterisks (*) indicate significance: *P < 0.05, **P < 0.01.

Acidobacteria, Actinobacteria, Proteobacteria, Chloroflexi, Firmicutes, Bacteroidetes, Planctomycetes, and Gemmatimonadetes were the most common phyla, but there was some variety in their relative abundance. The phyla Proteobacteria (Alphaproteobacteria in P soils and Gammaproteobacteria in all soils) and Actinobacteria were present at a higher proportion in chemically fertilized soils. Proteobacteria and Actinobacteria were recognized as copiotrophic taxa (taxa that thrived in conditions of elevated C and N availability and exhibited relatively rapid growth rates) (Eilers et al., 2010; Fierer et al., 2012, 2007), while Acidobacteria was considered to be an oligotrophic taxon with a slower growth rate and ability to metabolize nutrient-poor and recalcitrant C substrates (Fierer et al., 2012). In our study, there was a decline in the abundance of the phyla Acidobacteria (in P soils), Gemmatimonadetes and Chloroflexi in soils that received chemical fertilizer treatments. This finding demonstrated that available nutrients facilitated thriving by copiotrophic taxa and had negative effects on oligotrophic taxa. In contrast, there were only minor variations in the copiotrophic taxa and oligotrophic taxa in the different parent materials. Compared with the effects of chemical fertilizer, the effects of straw returning on the bacterial phyla composition were reduced due to the indirect and slow provision of nutrients. 5. Conclusion Fertilization management enhanced soil parent material maturation compared with cultivation alone. Chemical fertilizers were more effective than straw returning for soil nutrient accumulation, microbial biomass promotion, and copiotrophic bacteria enrichment during soil parent material maturation. Chemical fertilization-driven parent material maturation decreased the soil bacterial diversity and richness and significantly changed the soil bacterial community structure. Straw returning also had a positive effect on soil parent material maturation and a less negative effect on soil bacterial diversity, but straw returning was not as efficient at eliminating the nutrient limitation during soil parent material maturation. This study provided insights into the rapid fertilization and maturation of soil parent materials for agriculture production to support the even increasing food requirements of an increasing human population. Acknowledgement The authors thank the staff at the Qiyang Red Soil Experimental Station for managing the field experiments and helping with the collection of soil samples. This research was financially supported by the National Key Basic Research Program of China (973 program, 2015CB150505), the Fundamental Research Funds for the Central Universities (KYTZ201404). RZ and QS were also supported by the

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