Science of the Total Environment 673 (2019) 92–101

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Changes in nitrogen functional genes in soil profiles of grassland underlong-term grazing prohibition in a semiarid area

Zilin Song a,b, Jie Wang a, Guobin Liu a,c, Chao Zhang a,⁎a State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Northwest A&F University, Yangling 712100, PR Chinab College of Natural Resources and Environment, Northwest A&F University, Yangling 712100, PR Chinac Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry of Water Resources, Yangling 712100, PR China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Nitrogen functional genes in soil profilesof grassland under grazing prohibitionwere studied.

• Grazing prohibition increased the abun-dances of chiA, amoA-AOA, amoA-AOB,nirK, nirS, and nifH genes.

• The chiA and nifH abundance decreasedwith soil depth while nirK and nirS in-creased.

• Changes of nitrogen functional genesresponded differently to plant and soilproperties.

• Grazing prohibition improved the mi-crobial N turnover potential ofgrassland.

⁎ Corresponding author.E-mail address: zhangchaolynn@163.com (C. Zhang).

https://doi.org/10.1016/j.scitotenv.2019.04.0260048-9697/© 2019 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 November 2018Received in revised form 1 April 2019Accepted 2 April 2019Available online 03 April 2019

Editor: Charlotte Poschenrieder

Grazing prohibition has been used to restore degraded grassland ecosystems in semiarid areas; however, the impactof this measure on soil nitrogen (N) cycling is poorly understood. Furthermore, recent studies have tended to focuson the topsoil and ignored a steep gradient of nutrient accumulationwith soil depth. Here, we investigated changesinN functional genes (NFGs) involved in organic N decomposition (chiA), archaeal and bacterial ammonia oxidation(amoA-AOA and amoA-AOB), respectively, denitrification (nirK and nirS), and N fixation (nifH) in soil profiles from achronosequence of grazing prohibition (0, 10, 15, 25, and 35 years) in the semiarid grasslands of the Loess Plateau,China. The abundance of all the investigated NFGs in grassland soils after 35 years' grazing prohibition was higherthan in grazed grassland. This result suggests thatmicrobial N turnover potential is facilitated by grazing prohibition,probably through enhanced biomass production via increases in nutrient input into the soil. The higher ratio of (chiA+ nifH)/(amoA-AOA + amoA-AOB) and values of (nirK + nirS) in grazing-prohibited grasslands than in grazedgrassland suggest that prohibition of grazing not only improved microbial N storage potential but also increasedN gas emission potential. The abundances of NFGs varied along the soil profiles and responded differently to envi-ronmental factors. The chiA and nifH abundances decreased with soil depth and were associated with variation inaboveground biomass, NH4+-N, and organic carbon, while amoA-AOA, nirK, and nirS genes increased with depthand were more affected by soil organic carbon, moisture, and bulk density. Multivariate regression tree analysisdemonstrated that aboveground biomasswas the best explanatory variable for the changes inNFGs in grazed grass-land, while soil organic carbon was the best in the grazing-prohibited grasslands. Our results provide new insightinto the soil N cycling potential of degraded and restored semiarid grassland ecosystems.

© 2019 Elsevier B.V. All rights reserved.

Keywords:Grazing prohibitionNitrogen functional genesSoil profileSemiarid grassland

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93Z. Song et al. / Science of the Total Environment 673 (2019) 92–101

1. Introduction

Approximately 50% of global grasslands have experienced degrada-tion, which is caused by disturbance from anthropogenic activitiessuch as livestock grazing (Ebrahimi et al., 2016; Che et al., 2018). Prohi-bition of grazing is considered to be one of themost effectivemethods ofrestoring degraded grasslands and is intensively applied, especially inarid and semiarid regions of China (Cheng et al., 2016; Xiong et al.,2016; Mekuria et al., 2017). The Loess Plateau area is a typical semiaridregion, and livestock grazing is the major land use in this area (Chenget al., 2016). In the last century, degradation of many grasslands has oc-curred due to overgrazing. Since the launch of the Grain for Green pro-gram (GGP) in 1999 (Deng et al., 2014a), natural restoration ofdegraded grasslands via grazing prohibition in China has beenencouraged.

Grazing prohibition not only improves degraded ecological environ-ments but also affects soil ecological processes. For instance, above-ground vegetation productivity, coverage, and species diversity havebeen shown to increase with the duration of grazing prohibition(Ebrahimi et al., 2016; Norton and Young, 2016), and the carbon(C) and nitrogen (N) storage of plants also increased (Mekuria andAynekulu, 2013; Bi et al., 2018). Grazing prohibition also affects soilproperties, microbial community structure, and microbial biomass andmetabolic activity (Zeng et al., 2017; Zhang et al., 2018). For example,grazing prohibition has been shown to not only increase the diversityof soil microbes (Zhang et al., 2018), but also to influence soil microbialbiomass, activity, and community structure (Haynes et al., 2014; Wuet al., 2014; Chen et al., 2018). However, relatively little is knownabout the impact of long-term grazing prohibition on soil N turnoverpotential along the soil profile.

N, as one of the critical factors for degraded grassland restoration(Nannipieri and Paul, 2009), is the predominant limiting factor forplant primary productivity and microbial function in terrestrial ecosys-tems (Hayden et al., 2010; Levy-Booth et al., 2014). In natural ecosys-tems, the dynamics of N are mainly driven by N cycling microbes suchas diazotrophs, nitrifiers, and denitrifiers (Pajares and Bohannan,2016; Zhou et al., 2017); thus, changes in the structure of N cycling mi-crobial communities are closely related to soil-atmospheric N cycle pro-cesses and soil ecosystem function (Bender et al., 2016). Conversely,nutrient availability, particularly C and N, also affects microbial proper-ties, including diversity and community composition (Zhang et al.,2019a). Soil N cyclingmicrobes in various ecosystems have been exten-sively investigated using molecular marker methods (Levy-Booth et al.,2014; Li et al., 2018). These genes code the enzyme subunits that are es-sential to the corresponding N cycling processes. For instance, bioavail-able N in soil is usually fixed from the atmosphere by diazotrophicmicrobes (mostly the nifH gene), and the decomposition of organicmat-ter is closely related to the chiA gene (Pajares and Bohannan, 2016). Or-ganic matter decomposition and N fixation for the acquisition ofbioavailable N, are both based on the availability of soil organic matterand energy (Tang et al., 2018). The amoA genes participate in the firststep of autotrophic nitrification, in which ammonia-oxidizing bacteria(amoA-AOB) and ammonia-oxidizing archaea (amoA-AOA) obtain en-ergy by transforming oxidized ammonia to hydroxylamine, and fix or-ganic C (Pajares and Bohannan, 2016; Wang et al., 2017; Che et al.,2018). The reduction of nitrite to nitric oxide during denitrification iscatalyzed by two different nitrite reductases, encoded by the nirK ornirS genes. Moreover, the proportion of (chiA + nifH)/(amoA-AOA +amoA-AOB) and the value of (nirK + nirS), which usually representthe N storage and emission potential of soil microbes, respectively,have been studied in continental ecosystems (Li et al., 2018; Tanget al., 2018). Previous studies concerning grazing effects on grasslandshave revealed differences in the abundances of N cycling genes (Xieet al., 2014; Ding et al., 2015). However, these studies have tended tofocus on the topsoil and ignored a steep gradient of nutrient accumula-tion with soil depth. Given the essential roles of these genes in

bioavailable N cycling, discerning their responses to long-term grazingprohibition, especially at different soil depths, is important for increas-ing our understanding of the mechanisms of grassland restoration.Such knowledge can also enhance our capacity to forecast the responsesof grassland ecosystems to anthropogenic disturbance.

In this study, the influence of long-term grazing prohibition on soilmicrobial N turnover potential and its association with plant and soilvariables was determined via the investigation of changes in the abun-dances of N functional genes (NFGs) in different soil layers (0–10,10–20, 20–40, 40–60 cm) of five semiarid grasslands with achronosequence of grazing prohibition (0, 10, 15, 25, and 35 years) inthe Loess Plateau of China. We used chiA to represent resource acquisi-tion fromorganic N decomposition, amoA-AOA and amoA-AOB to repre-sent ammonia oxidation of nitrification, and nirK and nirS to representdenitrification, and nifH to represent N fixation (Fig. S1). We hypothe-sized that i) grazing prohibition improves microbial N turnover poten-tial, characterized by an elevated abundance of NFGs during theprohibition process due to enhanced biomass production and increasedsoil nutrient content; ii) nifH and chiA genes associated with N acquisi-tion accumulate in the topsoil owing to themore abundant organicmat-ter content in the topsoil than in the deeper soil layers, while nirK andnirS genes, involved in denitrification, increase in deeper soil becauseof increasing anoxicity caused by high bulk density; iii) long-term graz-ing prohibition improves the capacity of microbes for N storage poten-tial and facilitates N emission potential.

2. Material and methods

2.1. Description of study area

The study area was located at the Yunwushan National NatureGrassland Reserve (106°21′–106°27′E, 36°10′–36°17′N) in GuyuanCity, Ningxia Hui Autonomous Region, PR China. This reserve has atotal area of 66.6 km2, including a core conservation area (10 km2), abuffer conservation area (13km2) and an experimental area(43.60 km2). The elevation of the area is 1800–2100 m a.s.l. and it hasa typical semiarid moderate temperate climate (Cheng et al., 2016).The annual average temperature is about 7.0 °C. Mean annual precipita-tion is 425 mm, which mainly falls from July to September. The annualevaporation is about 1500mm, ranging from 1017 to 1739mm. Annualdaylight hours are about 2200 h. The soil type is montane grey-cinnamon soil according to the Chinese soil general classification sys-tem, which is similar to Haplic Calcisol in the FAO/UNESCO system.The vegetation is dominated by Gentianaceae, Stipa, and Potentilla;Stipa grandis, Thymusmongolicus, Stipa bungeana, Potentilla acaulis, Arte-misia sacrorum, and Androsace erecta are common plants (Jing et al.,2014). Since 1982, different grazing prohibition treatments have beenestablished at different times in this area (Cheng et al., 2016).

2.2. Experimental design and sampling

Five grasslands with different durations of grazing prohibition wereselected in August 2017. Grazing has been prohibited at these sites since1982, 1992, 2002, and 2007, corresponding to grazing prohibition for 35(GP35), 25 (GP25), 15 (GP15), and 10 years (GP10), respectively. Beforegrazing prohibition, these siteswere heavily grazed by sheep (50 sheep/ha). Meanwhile, a site that has been continuously grazed (50 sheep/ha)was used as a control (grazed grassland). Detailed information for allsites is shown in Table 1.

Three replicated plots (50 m × 100 m) were randomly establishedwithin the grazed and grazing-prohibition sites, and distances betweeneach plot were 80–100 m. After litter was removed, soil samples werecollected from depths of 0–10, 10–20, 20–40, and 40–60 cm from eachplot using a stainless steel corer with a diameter of 5 cm. Sampleswere randomly collected from nine points with an S-shape patternwithin each plot andmixed as one sample. After the roots, litter, debris,

Table 1Description of the experimental sites.

Sites Latitude (N) Longitude (E) Altitude (m) Slope gradient (°) Slope aspect Dominant species

Grazed 36°17′06″ 106°23′28″ 2017 18 E26°N Potentilla bifurca Linn., Stipa przewalskyi Roshev., Carex tangiana Ohwi.GP10 36°16′57″ 106°23′28″ 2034 20 E38°N Leymus secalinus Tzvel., Carex tangiana Ohwi, Stipa grandis P. Smirn.GP15 36°16′21″ 106°23′15″ 2025 20 E32°N Carex tangiana Ohwi., Carex tangiana Ohwi, Stipa grandis P. Smirn.GP25 36°16′3 ″ 106°23′27″ 2070 18 W21°N Stipa grandis P. Smirn. Artemisia sacrorum Ledeb, Oxytropis bicolor.GP35 36°15′05″ 106°23′10″ 2071 21 E29°N Stipa przewalskyi Roshev., Carex tangiana Ohwi.

94 Z. Song et al. / Science of the Total Environment 673 (2019) 92–101

and stoneswere removed, the collected soil was passed through a 2mmmesh and then divided into two subsamples. One subsample was im-mediately stored at −80 °C for DNA analysis, and the other was air-dried for physicochemical analysis.Moreover, in each plot,we randomlyselected three subplots (1 × 1 m) to measure cover, species number,aboveground biomass and root biomass (RB), and we used a rootauger (10 cm inside diameter) for the measurement of root biomassin the different soil layers. Roots were composited based on depth,and were subsequently oven-dried at 60 °C for 36 h to weigh the rootbiomass (refer to Zhang et al. (2018) for detailed methods).

2.3. Physicochemical characteristics of soil

Soil moisture was expressed as the ratio of water weight to soil dryweight and bulk density was expressed as the ratio of soil dry weightto soil volume. Organic Cwasmeasured using the dichromate oxidationmethod. Total N was measured using the Kjeldahl method (Kjeltec2300, Denmark). Available phosphorus (P) was determined using the0.5 mol L−1 NaHCO3melting (pH = 8.5) molybdenum antimony color-imetric method. NH4+-N and NO3−-N were measured using continuousflow auto-analysis (AutAnalyel, Bran+Luebbe GmbH, Norderstedt,Germany) after extraction in 2MKCl, with a ratio of 1:5. Soil pHwas de-termined using a combination pHmeter (soil:water = 1:2.5; Metrohm702, Herisau, Switzerland).

2.4. Microbial DNA extraction and real-time quantitative PCR

Microbial DNAwas extracted from0.25 g of homogenized frozen soilusing a FastDNA® SPIN kit (MP Biomedicals, Santa Ana, CA, USA) ac-cording to the manufacturer's instructions. The concentration and pu-rity of extracted DNA was independently checked using a NanoDrop®ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE,USA) at 260 nm. All soil samples were stored at −80 °C until used foranalysis.

The abundance of NFGs was estimated using an ABI 7500 real-timequantitative PCR system (Applied Biosystems, Foster City, CA, USA).The target genes included amoA-AOA amoA-AOB, nirK, nirS, nifH, andchiA. The standard curves for each target gene were generated usingreal-time PCR with ten-fold serially-diluted plasmids of each gene(Tang et al., 2018). Samples, standard series, and negative controlswere run in triplicate in 96-well plates. The real-time quantitative PCRreaction mixture (10 μL) contained 1 μL template DNA, 0.2 μL forwardand reverse primers (20 μM), 5 μL SYBR® Premix ExTaq™ (Takara),

Table 2Changes in plant properties during process of the grazing-prohibition.

Properties – Grazed GP10

Cover – 53.2 ± 1.9 B 94.7 ± 2.6Aboveground biomass – 148.0 ± 11.4 D 440.4 ± 60

Root biomass 0–10 cm 4.26 ± 1.11 Da 8.50 ± 1.9210–20 cm 2.61 ± 0.26 Bb 1.81 ± 0.5820–40 cm 1.31 ± 0.20 Bc 1.33 ± 0.0640–60 cm 0.65 ± 0.03 Ad 0.92 ± 0.08

Result is reported as themean±SE (n=3). Different uppercase in the row indicated the significlowercase in the column indicated the significant difference at the P b 0.05 along soil depth at

and 3.6 μL sterile distilledwater. Meanwhile, to ensure specificity of am-plification, we performed amelting curve analysis following each assay.Specific primers for the target genes and thermal cycling conditions areshown in Table S1. The unit of abundance of the NFGs is copies g−1 drysoil.

2.5. Statistical analyses

The abundance of total NFGs in our study was calculated by the sumof the abundance of genes detected. The ratios of (chiA+ nifH)/(amoA-AOA + amoA-AOB) and values for (nirK + nirS) were used to indicatesoil microbial N storage (Li et al., 2018) and gaseous N emission poten-tial (Li et al., 2018; Tang et al., 2018), respectively. A one-way analysis ofvariancewasused to evaluate the effects of grazingprohibition time andsoil depth on plant and soil properties and abundance of NFGs, followedby post hoc comparisons using least significant difference test. Signifi-cance was established at P b 0.05. Redundancy analysis (RDA) was per-formed to examine the correlations between soil properties and NFGsusing the “vegan” package in R v.2.9.2 software (R Development CoreTeam, 2009). Moreover, a multivariate regression tree (MRT) analysiswas applied to determine the best predictor variables for NFGs usingthe “mvpart” package (De'Ath, 2002). To quantify and visualize the ef-fects of environment factors on the N storage potentials and gaseous Nemission potentials, an aggregated boosted tree (ABT) analysiswas con-ducted using the “gbmplus” package in R v.2.9.2 softwarewith 500 treesused for boosting. All analyses not specifically mentioned were per-formed using R v.3.4.3 software (R Development Core Team, 2017).

3. Results

3.1. Plant and soil properties

Grazing prohibition had a positive influence on plant community,which showed higher cover and aboveground and root biomass thanthose in grazed sites (Table 2). Aboveground biomass increased withgrazing prohibition time and peaked at GP25, but decreased signifi-cantly thereafter. With the exception of the 40–60 cm layer, whereroot biomass did not differ significantly among the sites, root biomassin the 0–10, 10–20, and 20–40 cm layers increased with grazing prohi-bition time and peaked at GP25. Root biomass decreased gradually withincreasing depth at each site. Soil total N, NH4+-N, and organic C in-creased with grazing prohibition time in the same layers, and peakedat GP25 (Table S2); these variables significantly decreased with soil

GP15 GP25 GP35

A 89.2 ± 8.7 A 95.0 ± 0.6 A 92.3 ± 1.5 A.8C 729.5 ± 65.2 B 1298.2 ± 150.3 A 1019.0 ± 131.1 ACa 14.84 ± 1.81 Ba 24.49 ± 5.39 Aa 15.32 ± 0.39 BaBb 1.92 ± 0.39 Bb 7.49 ± 5.80 Ab 2.64 ± 0.64 BbBbc 1.32 ± 0.17 Bbc 2.14 ± 0.27 Ac 1.22 ± 0.05 BcAc 0.89 ± 0.19 Ac 0.67 ± 0.10 Ad 0.56 ± 0.04 Ad

ant difference at the P b 0.05 along the grazing-prohibition time at each soil layer. Differenteach site.

Table 3Summary of one-way analysis of variance for the effect of grazing prohibition time and soildepth on the abundances of NFGs.

Prohibition time Soil depth

F P F P

nifH 131.94 b0.001 51.39 b0.001chiA 372.88 b0.001 44.59 b0.001amoA-AOA 291.43 b0.001 13.48 b0.001amoA-AOB 2193.61 b0.001 17.69 b0.001nirS 52.09 b0.001 17.99 b0.001nirK 5.04 0.002 3.04 0.040Total NFGs 179.94 b0.001 11.41 b0.001

Table 5Effect of grazing prohibition time and soil depth on microbial N cycling potentials deter-mined using NFGs ratios.

Prohibitiontime

Soil depth

F P F P

(chiA + nifH)/(amoA-AOA + amoA-AOB) 250.8 b0.001 112.05 b0.001nirK + nirS 7.48 b0.001 3.29 0.030

95Z. Song et al. / Science of the Total Environment 673 (2019) 92–101

depth. TheNO3−-N content showed differentfluctuating trends in differ-ent soil layers, and was the highest at 0–10 and 10–20 cm depths in theGP15 site and at 20–40 cm and 40–60 cm depths in the GP35 site. Soilmoisture increased with grazing prohibition time but did not show aclear trend within sites along the soil profile (Table S2). The pH valuesshowed slight variation, ranging from 8.39 to 8.49 in the surface layer(0–10 cm) and from 8.19 to 8.26 in the deep layer (40–60 cm). The con-tent of available P did not change markedly with restoration time. Soilbulk density decreased with grazing-prohibition time and increasedalong the soil profile.

3.2. Abundances of N cycling functional genes, and N storage and gaseous Nemission potentials

The abundances of NFGs changed significantly with prohibition timeand with soil depth (P b 0.05, Table 3). Among them, the abundances ofchiA, nifH, and total NFGs increased with grazing prohibition time (P b0.05, Table 4), and the abundances of chiA and nifH decreased as soildepth decreased (P b 0.05). The amoA-AOA gene copies ranged from1.4 × 108 to 2.7 × 109 g−1 dry soil, and were more abundant than AOB

Table 4The abundances of NFGs in the soil profile during process of the grazing-prohibition.

Abundance (log copies−1 g dry soil) Depth (cm) Grazed GP1

chiA

0–10 2.62 ± 0.12 Da 3.210–20 2.58 ± 0.08 Ca 3.220–40 2.01 ± 0.06 Db 2.940–60 1.91 ± 0.10 Db 2.8

amoA-AOA

0–10 7.29 ± 0.08 Cb 7.5210–20 7.41 ± 0.04 Cab 7.420–40 7.46 ± 0.02 Bab 7.540–60 7.51 ± 0.02 Ba 7.62

amoA-AOB 0–10 6.76 ± 0.01 Ca 6.710–20 6.75 ± 0.03 BCa 6.720–40 6.47 ± 0.02 BCb 6.540–60 6.15 ± 0.01 Cc 6.3

nirK 0–10 7.59 ± 0.83 Cc 7.710–20 8.54 ± 0.05 Bb 8.320–40 8.62 ± 0.13 Cab 8.540–60 8.77 ± 0.06 Ba 8.8

nirS 0–10 7.31 ± 0.07 Dc 7.4910–20 7.45 ± 0.02 Bb 7.520–40 7.52 ± 0.01 Cb 7.740–60 7.75 ± 0.01 Ca 7.86

nifH 0–10 4.95 ± 0.16 Da 6.010–20 4.33 ± 0.12 Db 5.520–40 4.47 ± 0.23 Cb 5.340–60 3.85 ± 0.04 Dc 4.6

Total NFGs 0–10 31.59 ± 1.25 Da 36.10–20 32.58 ± 0.14 Ba 34.20–40 32.83 ± 0.15 Ba 36.040–60 32.13 ± 0.21 Ca 35.

Result is reported as themean±SE (n=3). Different uppercase in the row indicated the significlowercase in the column indicated the significant difference at the P b 0.05 along soil depth at

gene copies, which ranged from 3.8 × 107 to 9.5 × 108 g−1 dry soil.The abundances of amoA-AOA and amoA-AOB were lower at the GP10site than at the grazed site, and thereafter increased with duration ofgrazing prohibition. The abundance of amoA-AOA increased with soildepth, but the abundance of amoA-AOB decreased (P b 0.05). Generally,the abundance of nirS ranged from 8.1 × 107 to 3.6 × 109 g−1 dry soiland was lower than that of nirK, which ranged from 4.1 × 108 to 2.5× 1010 g−1 dry soil. The abundances of nirS and nirK were lower at theGP10 site than at the grazed site but then increased with grazing prohi-bition time, and increased with soil depth (P b 0.05). The N storage po-tential represented by (chiA + nifH)/(amoA-AOA + amoA-AOB) andgaseous N emission potential represented by (nirK+ nirS) were signif-icantly affected by grazing prohibition and soil depth (Table 5, P b 0.05).The N storage potential increased with grazing prohibition time(Fig. 1a), and the highest valuewas found at GP35; the N storage poten-tial decreased with soil depth. The gaseous N emission potential waslower at the GP10 site than that at the grazed site, but then increasedwith prohibition time (Fig. 1b) and was significantly higher at GP35.

3.3. Possible drivers of N cycling functional genes

Due to its narrow range (pH 8.19–8.51) during grazing prohibition,pH was not considered as one of the environmental factors affecting

0 GP15 GP25 GP35

6 ± 0.11 Ca 3.89 ± 0.19 BCa 4.59 ± 0.14 Ba 6.05 ± 0.07 Aa6 ± 0.05 Ba 3.95 ± 0.10 Ba 4.82 ± 0.05 ABa 5.76 ± 0.20 Aab7 ± 0.08 Cab 3.51 ± 0.07 BCab 4.19 ± 0.08 Bab 5.16 ± 0.06 Abc1 ± 0.07 Cb 3.27 ± 0.12 BCb 3.90 ± 0.07 Bb 4.78 ± 0.32 Ac± 0.02 BCab 7.76 ± 0.04 Bb 8.10 ± 0.13 Ac 8.23 ± 0.08 Ac5 ± 0.01 Bb 7.77 ± 0.05 Bab 8.34 ± 0.01 Ab 8.31 ± 0.02 Ab5 ± 0.03 Bab 7.83 ± 0.01 Bab 8.32 ± 0.02 Ab 8.38 ± 0.04 Aa± 0.10 BCa 7.96 ± 0.02 ABa 8.41 ± 0.01 Aa 8.43 ± 0.01 Aa

7 ± 0.03 Ca 7.31 ± 0.06 Ba 7.85 ± 0.04 Aa 7.97 ± 0.02 Aa3 ± 0.01 Ca 7.18 ± 0.03 Bb 7.74 ± 0.01Aa 7.88 ± 0.01 Aab8 ± 0.06 Cb 7.16 ± 0.02 Bb 7.57 ± 0.02 Ab 7.77 ± 0.02 Ab5 ± 0.01 Cc 7.03 ± 0.02 Bc 7.53 ± 0.01 Ab 7.45 ± 0.01 Ac7 ± 0.69 BCc 7.79 ± 0.08 Bc 8.36 ± 0.20 Ab 8.65 ± 0.36 Ac2 ± 0.32 Bb 8.38 ± 0.69 Bb 9.47 ± 0.31 Aa 9.30 ± 0.08 Ab8 ± 0.11Cab 9.40 ± 0.06 Ba 9.68 ± 0.11 ABa 9.86 ± 0.11 Aa3 ± 0.62 Ba 9.51 ± 0.24 Aa 9.82 ± 0.41 Aa 9.52 ± 0.01 Ab± 0.26 CDb 7.64 ± 0.15 BCc 7.82 ± 0.08 Bb 8.23 ± 0.08 Ac2 ± 0.20 Bb 7.70 ± 0.09 ABbc 7.91 ± 0.02 Bb 8.43 ± 0.02 Ab3 ± 0.10 Cab 7.92 ± 0.02 Ba 7.91 ± 0.04 Bb 8.50 ± 0.05 Aa± 0.09 BCa 7.79 ± 0.02 BCab 8.30 ± 0.010 Ba 8.55 ± 0.01 Aa

8 ± 0.12 Ca 6.61 ± 0.07 Ba 6.85 ± 0.10 Ba 7.84 ± 0.32 Aa8 ± 0.20 Cb 6.46 ± 0.07 Ba 6.60 ± 0.01 Bab 7.39 ± 0.19 Ab2 ± 0.20 Bb 5.46 ± 0.06 Bb 6.67 ± 0.18 Aab 6.79 ± 0.30 Ac1 ± 0.29 Cc 5.19 ± 0.43 Bb 6.59 ± 0.05 Ab 6.21 ± 0.22 Ad12 ± 0.54 Ca 37.79 ± 0.60 BCa 40.44 ± 0.54 Ba 44.33 ± 0.41 Aa97 ± 1.18 Ba 37.77 ± 0.15 ABa 40.65 ± 0.14 Aa 41.84 ± 0.25 Ab7 ± 0.54 ABa 37.63 ± 0.54 ABa 40.33 ± 0.51 Aa 40.56 ± 0.52 Ab38 ± 0.45 Ba 38.90 ± 0.03 Aa 39.47 ± 0.08 Aa 38.58 ± 0.31 Ab

ant difference at the P b 0.05 along the grazing-prohibition time at each soil layer. Differenteach site.

0

2

16

20

24

bc

b b bc cb b

abb b

ab b

cb b

aaaa

Krin +Srin

Prohibition time: P

nifH nirK

nirS

chiA

RB

SOC TN

-0.10

-0.05

0.00

0.05

0.10

-0.2 -0.1 0.0 0.1

(b) GP10

amoA-AOA

amoA-AOB

NH4+NO3-

RDA1 (66.8%)

RD

A2(1

7.1%

)

SM

BD

nifH

nirK

nirS

amoA-AOA

amoA-AOB

chiA

Cover

AB

RB

SOC

TNNO3-

NH4+

-0.10

-0.05

0.00

0.05

-0.1 0.0 0.1RDA1 (75.6%)

RD

A2

(18.

3%)

(a) Grazed

SM

nifH

nirK

nirSamoA-AOA

amoA-AOB

chiA

RB

SOC

TN

AP

SM

BD

-0.05

0.00

0.05

-0.2 -0.1 0.0 0.1RDA1 (73.9%)

RD

A2

(14

.8%

)

(c) GP15

NH4+NO3-

nifH

nirK

nirS

chiA

SOC

TN

NO3-

AP

SM

BD

-0.12

-0.08

-0.04

0.00

0.04

-0.1 0.0 0.1RDA1 (72.2%)

RDA2

(20.

9 %)

(d) GP25

amoA-AOA

amoA-AOB

NH4+

nifH

nirK

nirS

chiA

RB

SOCTN

NO3-NH4+

SM

BD

-0.05

0.00

0.05

0.10

0.15

-0.2 -0.1 0.0 0.1RDA1 (72.9%)

RD

A2(2

0.1%

)

(e) GP35

amoA-AOA

amoA-AOB

Fig. 2. RDA ordination plot for the relationship between the NFGs abundances and the environment factors in the different grazing prohibition sites along soil profile. AB: abovegroundbiomass; RB: root biomass; BD: bulk density; SM: soil moisture; AP: available P; TN: total nitrogen; SOC: soil organic carbon. Only the environmental variables which weresignificantly (p b 0.05) correlated with NFGs were shown in figure.

97Z. Song et al. / Science of the Total Environment 673 (2019) 92–101

decomposition was lower in grazed grassland soil than in grazing-prohibited grassland soil (Dungait et al., 2013).

The capacity for asymbiotic N2 fixation by diverse diazotrophs, asso-ciated with nifH gene activity, should be present in most soils(Bürgmann et al., 2003). As was expected, the abundance of the nifHgene increased with duration of grazing prohibition (Table 4). The pos-itive relationships between aboveground biomass and soil organic Cconcentrations and nifH abundances (Fig. 2) suggest that N2-fixing mi-crobial activity was likely associated with more decomposing plant lit-ter and organic matter. The fixation of biological N is mainly achievedby symbiotic bacteria in association with plants (Hayden et al., 2010;Wang et al., 2018). In our study, bulk rather than rhizosphere soils

were sampled, thus, the nifH genes detected were expected to be pre-dominantly from free-living N-fixing bacteria. This expectation is sup-ported by the finding that free-living N fixers are likely to be essentialfor ecosystem regeneration (Li et al., 2018).

In line with previous studies reporting positive effects of grazing onthe abundance of amoA genes (Xie et al., 2014; Ding et al., 2015), wefound higher abundances of amoA-AOA and amoA-AOB in grazed grass-land than in grassland where grazing had been prohibited for 10 years.As the prohibition time increased, the abundances of amoA-AOA andamoA-AOB increased and becamehigher than those in grazed grassland.This result indicates that long-term grazing prohibition could increasethe abundance of ammonia-oxidizing microbes. In addition, we found

R2

82.57

72.16

60.95

0(1)

60.95%

SOC< 23.37 SOC≥23.37

(2)

10.41%

SM≥0.215SM< 0.215

n=2(#3)

n=4(#4)

(3)

11.21%

RB< 13.1 RB≥13.1

n=4(#6)

n=2(#7)

R2 : 82.6 % Error : 0.174 CV Error : 0.871 SE : 0.199

(c) GP15

R2

79.26

70.10

56.64

0(1)

56.64%

SOC< 21.43 SOC≥21.43

n=3(#2)

(3)

13.46%

n=3(#4)

(7)

9.16%

AP< 9.9 AP≥9.9

n=2(#6)

n=4(#7)

R2 : 79.3 % Error : 0.207 CV Error : 0.971 SE : 0.215

(d) GP25

NO3-< 9.10 NO3

- 9.10

R2

78.58

75.47

61.59

0

(1)

61.59%

Aboveground biomass< 43.98

n=3(#7)

(2)

13.87%

SOC< 14.82 SOC≥14.82

n=3(#6)

(4)

3.11%n=3(#4)

n=3(#5)

R2 : 78.6 % Error : 0.214 CV Error : 0.905 SE : 0.307

(a) Grazed

NO3-< 4.46 NO3

- 4.46

R2

71.41

58.15

42.6

0(1)

42.60%

SOC< 20.45 SOC≥20.45

n=4(#2)

(3)

15.55%

SM< 9.62 SM≥9.62

n=2(#4)

(7)

13.26%

NH4+≥23.49 NH4

+< 23.49

n=4(#6)

n=2(#7)

R2 : 71.4 % Error : 0.286 CV Error : 1.64 SE : 0.394

(b) GP10

R2

79.31

74.20

53.35

0(1)

53.35%

SOC < 46.37 coverage≥46.37

n=3(#7)

(2)

20.85%

NH4+≥24.55

n=4(#3)

(5)

5.10%

RB≥1.81 RB< 1.81

n=3(#5)

n=2(#6)

R2 : 79.3 % Error : 0.207 CV Error : 0.702 SE : 0.197

(e) GP35

NH4+< 24.55

Fig. 3.Multivariate regression tree analysis discerns the effect of environmental parameters on NFGs during the grazing prohibition. (a) The bar plots illustrate the absolute abundance ofeach gene and the patterns of bar plots represent the dynamics of NFGs among each leaf; the numbers under the bar are the numbers of samples in each group. SOC: soil organic carbon.SM: soil moisture, RB: root biomass.

98 Z. Song et al. / Science of the Total Environment 673 (2019) 92–101

positive relationships between inorganic N concentration (NH4+-N andNO3−-N) and the abundances of both amoA-AOA and amoA-AOB(Fig. 2). In contrast, several previous studies reported that NO3−-N con-centrations were correlated with amoA-AOB abundance but not amoA-AOA abundance in grazed grasslands (Di et al., 2009; Wertz et al.,2012). The reason for this discrepancy could be different preferencesfor N concentrations; AOB is likely to prefer high N concentrations,while AOA would favor lower N levels (Verhamme et al., 2011). In ourstudy, the N concentrations ranged from 1.6 to 3.6 mg kg−1 and3.1–16.3 mg kg−1 for NH4+-N and NO3−-N, respectively, which weremuch lower than values obtained by Di et al. (2009) and Verhammeet al. (2011) (up to 180 mg kg−1). Therefore, in grasslands from theLoess Plateau, the soil N concentrationsmay be adequate for the growthof AOA and to a lesser extent AOB,while the grazing-induced increase insoil N availability partly enhances the competitiveness of AOB.

The increased abundances of nirK and nirS genes observed in thegrazing-prohibited grassland soils (GP15, GP25, and GP35) supports aprevious study, which observed that the activity of nitrate reducer com-munities increased in the later stages of vegetation succession(Kandeler et al., 2006; Tang et al., 2018). Furthermore, we identifiedthat nirKwas always more abundant than nirS in each prohibition phaseand suggest that the nirK gene should be the first candidate used as an in-dicator of themicrobial denitrification process in this semiarid ecosystem.

4.3. Changes in NFG abundances with soil depth

The vertical distribution of soil microorganisms is closely related tochanges in soil resources and energy with soil depth (Barta et al.,2010; Castellano-Hinojosa et al., 2018). The abundance of the nifHgene decreased with soil depth, consistent with previous findings that

SOC

NH4+

TN

SM

RB

NO3-

BD

AP

C:N

AB

Cover

0 5 10 15 20 25 30 35

SM

SOC

BD

RB

TN

NH4+

AP

Cover

NO3-

C:N

AB

0 4 8 12 16 20 24 28

(b) nirK+nirS

Relative influence(%)Relative influence(%)

(a) (nifH+chiA)/(amoA-AOA+amoA-AOB)

Fig. 4. Aggregated boosted tree (ABT) analysis showed the relative influence of plants and soil properties on the N storage potentials (represent by (chiA + nifH)/(amoA-AOA + amoA-AOB) and gaseous N emission potentials (represent by nirK + nirS abundance (log-transformed)) during the grazing prohibition.

99Z. Song et al. / Science of the Total Environment 673 (2019) 92–101

N2 fixing mainly occurs in the topsoil (Stone et al., 2015; Tang et al.,2018). This result supports our hypothesis. A possible reason for theprevalence of N2 fixation in the upper soil layers is the abundance ofheterotrophic decomposers, whose N requirements are high relativeto the aboveground litter being decomposed (Hayden et al., 2010).The dramatic declines in soil C and N contents (Table S2) and the posi-tive correlationwith the abundance of nifH support this statement. Sub-soils have been reported to be biologically inactive by most studies in-vestigating the potential to increase N sequestration, due to the de-creased C and N substrate for microbes in the soil profile (Stone et al.,2015). Organic N derived from the detritus of soil organisms is a majorsubstrate for microorganisms containing the chiA gene (Ning et al.,2015). In our study, the abundance of chiA decreased with soil depthandwas significantly positively correlatedwith soil C andNcontents, sug-gesting that the resource acquisition strategy for microbial growth isclosely related to the abundance of available resources. Our resultscontrasted with those of Stone et al. (2015), who found that chiA geneabundance increased with depth in the 0–140 cm soil layer of tropicalsoils. They also contrast with those of Li et al. (2018), who reported an ac-cumulation of chiA genes in the topsoil (0–20 cm)under vegetative recov-ery after abandonment of agriculture in the Karst zone of China. Thesecontrasting results could be due to different conditions in the studyareas, such as climate, soil properties, vegetation composition, and an-thropogenic management. Our results highlight the uniqueness of thesemiarid grassland ecosystem, and that the responses of NFGs involvedin N turnover processes to restoration can be ecosystem-specific.

Previous studies reported an enrichment of amoA-AOB in the surfacesoil (0–20 cm) and of amoA-AOA in the subsoils (40–60 cm) on thesemiarid Loess Plateau (Tao et al., 2018). We also found a difference inthe abundances of genes involved in ammonia-oxidization: amoA-AOBabundance decreased with depth but amoA-AOA abundance increased(Table 4). These results confirmed that ammonium oxidation was per-formed by two types of microorganisms (archaea and bacteria), whichoccupied specific resource niches in the semiarid soil profiles. Positivecorrelations between amoA-AOB abundance and soil NH4+ and organiccontents (Fig. 2) indicate that AOB are enriched in the resource-rich sur-face soil. Most AOB are typically copiotrophic and are most abundant insoils that have a relatively high concentration of substrates (Xie et al.,2014; Shah et al., 2017). In contrast, AOA appeared to be less restrictedby the availability of substrates, as theywere richest in deep layers with

relatively low nutrient concentrations and high bulk density. Soil AOAhave a lower preference for ammonia and oxygen than AOB owing totheir smaller cell size (Martens-Habbena et al., 2009; Kim et al., 2012),which allows them to prosper in oligotrophic environments.

As expected, the abundances of both nirK and nirS genes increasedwith soil depth (Table 4), consistent with previous studies(Castellano-Hinojosa et al., 2018). Denitrification occurs easily in anoxicconditions (Barta et al., 2010) and soil aeration was the major factor af-fecting the distribution of nirK- and nirS-type microorganisms (Pajaresand Bohannan, 2016; Li et al., 2018). Compared with the surface soil,deeper soil has fewer roots, resulting in higher bulk density and loweraeration, which is favorable for the denitrification process. This specula-tion was confirmed by the positive correlation between bulk densityand the abundances of nirK and nirS genes (Fig. 2). The similar increas-ing abundances of nirK and nirS with soil depth suggest that the twotypes of nitrite reducer had similar activities in the relatively nutrient-poor and anoxic environment of the lower soil.

4.4. NFG abundances as potential N cycling indicators

Ratios of NFG abundances have been used as indicators in previousstudies to explore N turnover. For example, the amoA-AOA/amoA-AOBabundance ratio was used to assess the process of ammonia oxidization(Bolan et al., 2004); the ratios of the abundances of nirK and/or nirS tonosZ have been used to evaluate the potential for N2O emissions(Henderson et al., 2010; Wang et al., 2014); and the amoA/narG abun-dance ratios and nirK + nirS abundances have been used as predictorsof NO3−-N leaching and gaseous N loss potentials, respectively (Tanget al., 2018; Li et al., 2018).We therefore deem that changes in the ratiosof NFG abundances can indicate the preferences and strategies for N-cycling processes of microbes in response to changes in environmentalconditions. In our study, we determined the ratio of (nifH + chiA)/(amoA-AOA+ amoA-AOB) and the sum of the abundances of the nirKand nirS genes to evaluate the genetic potential for microbial N storageand emissions (Li et al., 2018).

In our study, the ratio of (chiA + nifH)/(amoA-AOA + amoA-AOB)was observed to increase with grazing-prohibition time but declinedwith soil depth (Fig. 1), along with an increase in soil total N content.This result suggests a greater potential for N storage with grazing prohi-bition, especially in the surface soil. This finding agreed with that of

100 Z. Song et al. / Science of the Total Environment 673 (2019) 92–101

Deng et al. (2014b), who indicated enhanced soil N stocks in theYunwushan grassland along a similar chronosequence of prohibition.Over 60% of Nwas sequestrated in the 0–30 cmsoil layers in both grazedand no-grazing grasslands of the Loess Plateau (Qiu et al., 2013). Hence,the results of this study indicate that grassland ecosystems have the ca-pacity for N accumulation as natural restoration proceeds. The contribu-tion of organic C and NH4+-N to the changes in the ratio of (chiA+ nifH)/(amoA-AOA+ amoA-AOB) (Fig. 4a) suggest the promotion of enhancedsoil nutrient levels as a result of grazing prohibition, to a potential for Nstorage by microbes. The potential gaseous N loss was higher for thegrazing site than the GP10 site, which supports the results of Bhandralet al. (2007), who found that grazing increased the denitrification rate.This effect was closely related to an increase in soil moisture content.Soil moisture is a key factor limiting denitrification (Szukics et al.,2010; Zhong et al., 2014; Xie et al., 2014), and lower denitrificationrates have been observed when soil moisture decreases (Ding et al.,2015). We found the greatest contribution of soil moisture was to thechanges in potential gaseous N loss (Fig. 4b). After 35 years of grazingprohibition, the (nirK + nirS) of grassland was higher than that ofgrazed grassland, suggesting that long-term grazing prohibition in-creased the potential gaseousN loss of grassland, probably by increasingdenitrifier activity. However, a decreasewas found in the initial prohibi-tion stages (10 years). Previous studies reported relatively high N lossthrough denitrification was balanced by increased N fixation and or-ganic N decomposition in terrestrial ecosystem (Bolan et al., 2004;Saggar et al., 2007). These results were supported by observations of15N abundance in grassland soils (Buchen et al., 2018). An estimate ofN loss in global temperate grasslands suggests a loss of 5.6 Tg of Nthrough denitrification, for a total mean loss of approximately 2.5% ofavailable N (Saggar et al., 2013). These N export fluxes are comparableto the loss by NO3−-N leaching and emphasize the contribution of deni-trification to removingNO3−-N from grassland ecosystems (Saggar et al.,2013). Further investigation of N2O emissions in grassland ecosystemsis needed to confirm this conclusion. Although the molecular analysesdid not integrate transcription and 15N signatures in this study, the ra-tios of (chiA + nifH)/(amoA-AOA + amoA-AOB), and the (nirK + nirS)gene abundances as indicators of N storage and gaseous N emission po-tentials, respectively, can provide valuable insights into microbial Nturnover in soils under different conditions.

5. Conclusions

Long-term grazing prohibition improves microbial N turnover po-tential, characterized by the higher abundances of chiA, amoA-AOA,amoA-AOB, nirK, nirS, and nifH, as well as the higher N storage potentialand N emission potential in grazing-prohibited grassland soils than ingrazed grassland soils. In general, aboveground biomass and soil organicC were the main factors affecting the gene abundances of N cycling mi-croorganisms in grazed grassland and grazing-prohibited grasslands,respectively. The abundances of chiA and nifH decreased with soildepth and were associated with the variation in aboveground biomass,NH4+-N, and organic C, while amoA-AOA, amoA-AOB, nirK, and nirSgenes increased and were more affected by soil organic C, moisture,and bulk density. Our results provide new insight into soil N cycling po-tential in semiarid grassland ecosystems.

Acknowledgements

This work was financially supported by the National Natural ScienceFoundation of China (41771554), and the National Key Research andDevelopment Program of China (2016YFC0501707).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2019.04.026.

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Changes in nitrogen functional genes in soil profiles of grassland under long-term grazing prohibition in a semiarid area1. Introduction2. Material and methods2.1. Description of study area2.2. Experimental design and sampling2.3. Physicochemical characteristics of soil2.4. Microbial DNA extraction and real-time quantitative PCR2.5. Statistical analyses

3. Results3.1. Plant and soil properties3.2. Abundances of N cycling functional genes, and N storage and gaseous N emission potentials3.3. Possible drivers of N cycling functional genes

4. Discussion4.1. Effect of grazing-prohibition on plant and soil properties4.2. Changes in NFGs abundances with grazing-prohibition time4.3. Changes in NFG abundances with soil depth4.4. NFG abundances as potential N cycling indicators

5. ConclusionsAcknowledgementsAppendix A. Supplementary dataReferences