Soil Microbial Responses to Experimental Warming and Nitrogen Addition in a Temperate Steppe of Northern China

Pedosphere 24(4): 427–436, 2014 ISSN 1002-0160/CN 32-1315/P c 2014 Soil Science Society of China  Published by Elsevier B.V. and Science Press

Soil Microbial Responses to Experimental Warming and Nitrogen Addition in a Temperate Steppe of Northern China∗1 SHEN Rui-Chang1,2 , XU Ming3,∗2 , CHI Yong-Gang1,2 , YU Shen4 and WAN Shi-Qiang5 1 Key Laboratory of Ecosystem Network Observation and Modeling, Institute of Geographical Sciences and Natural Resources Research, Chinese Academy of Sciences, 11A Datun Road, Beijing 100101 (China) 2 University of Chinese Academy of Sciences, Beijing 100049 (China) 3 Center for Remote Sensing and Spatial Analysis, Department of Ecology, Evolution and Natural Resources, Rutgers University, 14 College Farm Road, New Brunswick NJ 08901 (USA) 4 Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021 (China) 5 Key Laboratory of Plant Stress Biology, College of Life Sciences, Henan University, Kaifeng 475004 (China)

(Received July 23, 2013; revised May 21, 2014)

ABSTRACT The responses of soil microbes to global warming and nitrogen enrichment can profoundly affect terrestrial ecosystem functions and the ecosystem feedbacks to climate change. However, the interactive effect of warming and nitrogen enrichment on soil microbial community is unclear. In this study, individual and interactive effects of experimental warming and nitrogen addition on the soil microbial community were investigated in a long-term field experiment in a temperate steppe of northern China. The field experiment started in 2006 and soils were sampled in 2010 and analyzed for phospholipid fatty acids to characterize the soil microbial communities. Some soil chemical properties were also determined. Five-year experimental warming significantly increased soil total microbial biomass and the proportion of Gram-negative bacteria in the soils. Long-term nitrogen addition decreased soil microbial biomass at the 0–10 cm soil depth and the relative abundance of arbuscular mycorrhizal fungi in the soils. Little interactive effect on soil microbes was detected when experimental warming and nitrogen addition were combined. Soil microbial biomass positively correlated with soil total C and N, but basically did not relate to the soil C/N ratio and pH. Our results suggest that future global warming or nitrogen enrichment may significantly change the soil microbial communities in the temperate steppes in northern China. Key Words: arbuscular mycorrhizal fungi, global warming, Gram-negative bacteria, nitrogen enrichment, microbial biomass, microbial community Citation: Shen, R. C., Xu, M., Chi, Y. G., Yu, S. and Wan, S. Q. 2014. Soil microbial responses to experimental warming and nitrogen addition in a temperate steppe of northern China. Pedosphere. 24(4): 427–436.

Global temperature and nitrogen deposition are projected to increase significantly in the coming decades (Solomon et al., 2007; Gruber and Galloway, 2008), which may produce profound impacts on soil microbial communities in various ecosystems (Frey et al., 2008; Zhang et al., 2008; Zak et al., 2011; Zhou et al., 2012). The shifts in soil microbial communities can enhance ecosystem feedbacks to climate change through microbial decomposition of soil organic matters, resulting in more greenhouse gas (GHG) emissions to the atmosphere (Conrad, 1996; Schimel and Gulledge, 1998; Bardgett et al., 2008). Therefore, examining the responses of soil microbial communities to global warming and nitrogen enrichment are critical to the projection of future changes in ecosystem func∗1 Supported

tions and atmospheric GHG concentration because of the massive organic carbon pools in the soils in terrestrial ecosystems (Schimel and Gulledge, 1998; Jobb´agy and Jackson, 2000; Singh et al., 2010). Scientists have put much effort into studying the impacts of warming and nitrogen enrichment on soil microbial communities individually. Many experiments, such as laboratory soil incubation experiments (Zogg et al., 1997; Feng and Simpson, 2009), seasonal temperature gradient-based experiments (Waldrop and Firestone, 2006; Bj¨ork et al., 2008), soil translocation experiments (Budge et al., 2011; Vanhala et al., 2011), and in-situ ecosystem warming experiments (Zhang et al., 2005; Rinnan et al., 2007), have reported the warming effects on soil microbial community biomass

by the National Key Research and Development Program (973 Program) of China (No. 2012CB417103) and the Forestry Department of Qinghai Province, China (No. Y22LO300AJ). ∗2 Corresponding author. E-mail: [email protected].

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and composition in various biomes across the world. It is shown that soil microbial communities of a forest in Petersham, USA (Frey et al., 2008), a grassland in western Alberta, Canada (Feng and Simpson, 2009), a subarctic heath in the Swedish Lapland (Rinnan et al., 2007), and a tundra in Antarctica (Yergeau et al., 2012) all would be greatly modified by global warming. As regards to nitrogen deposition, it is anticipated that the effects of nitrogen addition on soil microbes are related with the experimental duration, the total amount of nitrogen added and the plant community (Treseder, 2008; Liu and Greaver, 2010; Liu et al., 2010). Low-level and short-term nitrogen addition can relieve the nitrogen deficiency in soils and increase soil microbial biomass (Vitousek and Howarth, 1991; Allison et al., 2009). However, intensive and long-term nitrogen deposition may have negative effects on soil microorganisms by acidifying soil, increasing osmotic pressures, depleting soil minerals, and increasing aluminum toxicity (Aber et al., 1998; Treseder, 2008; Niu et al., 2011; Li et al., 2012). Furthermore, nitrogen addition can change plant community structure and carbon allocation by, e.g., decreasing the proportion of the legumes and modifying the plant shoot to root ratio, which would also modified soil microbial community (Xia and Wan, 2008; Liu and Greaver, 2010). So far, few studies have reported soil microbial responses to the combined effects of global warming and nitrogen enrichment and no consistent conclusions have been drawn from the limited results. For example, Bell et al. (2010) showed that warming and nitrogen addition had no interactive effect on soil microbial community in a temperate old field in Canada. Ma et al. (2011) found that combined warming and nitrogen addition significantly decreased the ratio of bacterial to fungal biomass in a grassland in northeastern China; however, Gutknecht et al. (2012) have recently suggested that global warming and nitrogen enrichment have positive interactive effects on this ratio at the Jasper Ridge Biological Preserve in California, USA. Hence, much more multi-factor research is needed to fully understand how global warming and nitrogen enrichment affect soil microbial communities. Temperate steppe are one of the largest ecosystems by area in China and it is pivotal to maintain the ecosystem functions, such as carbon and water cycling and biodiversity, in northern China (Kang et al., 2007; Han et al., 2009; Fang et al., 2010). Global change may have profound impacts on the steppe ecosystem because the temperature and nitrogen availability in this region may continue to increase in the 21st century according to the projection of global change models

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(Dentener et al., 2006; Ding et al., 2007; Solomon et al., 2007). Understanding the impacts of global change on the steppe ecosystem is necessary for effectively managing the ecosystem in the near future. Taking the opportunity of a long-term field experiment in a temperate steppe of northern China (Wan et al., 2009; Xia et al., 2009), the current study aimed to investigate the individual and interactive effects of experimental warming and nitrogen addition on soil microbial biomass and community structure, and to determine the relationships between soil microbial biomass and soil properties in the steppe ecosystem. MATERIALS AND METHODS Study area This study was conducted at the Duolun Ecological Station (42◦ 02 N, 116◦ 17 E, 1 324 m above sea level), in Inner Mongolia, China. The study area is a temperate steppe with a typical temperate monsoon climate. Mean annual temperature is 2.1 ◦ C with the minimum and maximum temperatures being −17.5 ◦ C in January and 18.9 ◦ C in July, respectively. The mean annual precipitation is 383 mm, 90% falling between May and October. This area received 325.6 mm rainfall during the entire growing season of 2010 (May to October), which was very close to the long-term average precipitation of 345 mm. The soils of the study area are very sandy with 62.75% sand, 20.30% silt, and 16.95% clay. The mean bulk density of the soils is 1.31 g cm−3 . The dominant plant species are perennial grasses and herbs, including Stipa krylovii, Artemisia frigida, and Leymus chinensis. There are also some legumes existing, such as Astragalus galactites, Astragalus scaberrimus, Gueldenstaedtia stenophylla, and Melilotoides ruthenica. Experimental design and soil sampling The long-term field experiment began in 2006 (Wan et al., 2009; Xia et al., 2009), including four treatments: control (CK), diurnal experimental warming (EW), nitrogen addition (ND), and diurnal warming plus nitrogen addition (WN). Each treatment had six replicate plots and the size of each plot was 3 m × 4 m. The warming treatment was achieved using MSR-2420 infrared radiators (Kalglo Electronics Inc., Bethlehem, USA), which were suspended 2.25 m above the ground. In each control or nitrogen addition plot, one ‘dummy’ heater with the same shape and size was used to simulate the shading effects of the infrared radiator. The warming treatment started on April 23, 2006, with significantly increased mean soil temperature by 1.79 ◦ C

SOIL MICROMIAL RESPONSE TO WARMING AND N ADDITION

at the 10 cm depth (Xia et al., 2009). Nitrogen addition was treated once a year with NH4 NO3 (10 g N m−2 year−1 ) in July. Soil samples were collected randomly from 3 of the six replicate plots of each treatment in middle August, when the plants and microbes were most active, in 2010. The soil samples were collected from two depth intervals: 0–10 and 10–20 cm. Two soil cores (3 cm in diameter) were collected at each depth and completely mixed into one composite fresh soil sample. After sieving to remove plant roots and stones through 2-mm mesh, soil samples were transported to the laboratory with a dry ice cooler within 24 h for further analyses. Soil chemical analysis Soil pH was measured with an Accumet Excel XL60 pH meter (Fisher Scientific, Fair Lawn, USA) at a soil to water ratio of 1:2.5. Soil total carbon (TC) and total nitrogen (TN) contents were measured with a Vario Max CN analyzer (Elementar, Hanau, Germany). The soil C/N ratio was calculated as the ratio of TC to TN.

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biomass equaled the sum of the biomass of GP, GN, and ACT. FUN was indicated by 16:1ω5c, 18:1ω9c, 18:2ω6,9c, and 18:3ω6,9,12c, of which 16:1ω5c represented AMF and the other three fatty acids represented SF (Zogg et al., 1997; Olsson, 1999; Gray et al., 2011). The rest of the fatty acids were called ubiquitous fatty acids (UBI) because they are widely distributed in all the microbial groups. Statistical analysis The effects of experimental warming and nitrogen addition on soil PLFAs and chemical properties were examined using two-way analysis of variance (ANOVA). The influences of soil depth on soil properties were analyzed using paired t-test. Correlation analysis was carried out to determine the relationships between soil chemical properties and microbial community indicators, such as biomass and abundance. All statistical analyses were performed using the SPSS 16.0 software (SPSS Inc., Chicago, USA). RESULTS

Phospholipid fatty acid (PLFA) analysis Soil microbial biomass Phospholipid fatty acids were double extracted with a chloroform:methanol:phosphate (0.8:2:1) buffer solution from 3 g of freeze-dried soils (Yu and Ehrenfeld, 2010). The chloroform phase of the extraction was evaporated and concentrated (close to 1 mL) with a vacuum evaporator, and separated on a silicic acid column using 5 mL chloroform, 10 mL acetone, and 5 mL methanol in sequence. The phospholipid was then concentrated to about 1 mL, and further saponified and methylated. The fatty acid methyl esters were extracted into hexane and analyzed using the Sherlock microbial identification system (MIDI Inc., Newark, USA). The amount of each fatty acid methyl ester identified was standardized to the peak area and amount (5 μg) of the internal standard (19:0) and expressed as μg PLFA g−1 dry soil. We used the total PLFA content to indicate soil total microbial biomass and assigned PLFAs into seven microbial groups, namely bacteria (BAC), gram-positive bacteria (GP), gram-negative bacteria (GN), fungi (FUN), arbuscular mycorrhizal fungi (AMF), saprophytic fungi (SF), and Actinobacteria (ACT). Saturated and branched chain fatty acids (iso-, anteiso-) were taken as indicators for GP (Zelles, 1997, 1999). Monounsaturated, straight-chain and cyclopropane fatty acids represented GN (Zelles, 1997; Zogg et al., 1997; Biasi et al., 2005). ACT was quantified by 10Me fatty acids: 10Me16:0, 10Me17:0, and 10Me18:0 (Zelles, 1999; Frey et al., 2008). The BAC

Our results showed that the soil total microbial biomass (total PLFA content) was 4.82 ± 0.19 and 2.21 ± 0.21 μg PLFA g−1 dry soil at both the 0–10 and 10– 20 cm soil depths in the control plots (Fig. 1). Experimental warming significantly (P = 0.037) increased soil total microbial biomass by 26.35% at the 0–10 cm soil depth (Fig. 1a), but not (P = 0.391) at the 10–20 cm depth (Fig. 1b). Warming significantly increased the biomass of BAC (P = 0.041) and AMF (P = 0.037) in the 0–10 cm soil layer, while other microbial groups only had insignificant increasing trends (Tables I and II). Nitrogen fertilization reduced (P = 0.016) the microbial biomass in the 0–10 cm topsoil by 22.13% (Fig. 1a), but increased (P = 0.004) the microbial biomass in the 10–20 cm soil layer by 94.16% (Fig. 1b). At the same time, nitrogen treatment also significantly decreased the biomass of GP, BAC, AMF, and UBI at the 0–10 cm soil depth, and increased the biomass of all the microbial groups at the 10–20 cm soil depth except for AMF (Tables I and II). There was no interactive effect between experimental warming and nitrogen addition on soil microbial biomass in the two soil layers (Fig. 1, Tables I and II). Soil microbial community structure Experimental warming and nitrogen addition also significantly changed soil microbial community structures in the temperate steppe of this study (Fig. 2, Ta-

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the same treatment in the surface soil. Meanwhile, nitrogen enrichment consistently decreased the proportions of AMF in both two soil layers (Fig. 2, Tables III and IV). The proportions of SF and FUN at the 10–20 cm soil depth were also significantly decreased by nitrogen addition (Tables III and IV). No significant interactive effect of experimental warming and nitrogen addition was detected on soil microbial community compositions except for the relative abundance of AMF at the 10–20 cm soil depth (Fig. 2, Tables III and IV). Specifically, warming increased the relative abundance of AMF in the unfertilized plots but decreased it in the fertilized plots at the 10–20 cm soil depth (Fig. 2). Soil chemical properties

Fig. 1 Soil total microbial biomass (total phospholipid fatty acid (PLFA) content) at the 0–10 (a) and 10–20 cm (b) soil depths without (control, CK) and with the treatments of experimental warming (EW), nitrogen addition (ND), and EW plus ND (WN) in a long-term field experiment in a temperate steppe of northern China. Values are means ± standard errors (n = 3). * and ** represent significance at P < 0.05 and 0.01, respectively.

bles III and IV). Warming increased the relative abundance of GN in both two soil layers (Fig. 2), whereas only the shift at the 10–20 cm soil depth was statistically significant (P = 0.041) due to the high variations of the biomass of the microbial groups within

Warming did not significantly (P > 0.05) change soil pH, TC, TN, and C/N ratio in the soils of the temperate steppe studied (Tables V and VI). Nitrogen addition significantly decreased soil pH by 7.26% and 3.67% at the 0–10 and 10–20 cm soil depths, respectively (Tables V and VI). Soil TC and TN at the 10–20 cm depth were also significantly increased by nitrogen fertilization (Tables V and VI). Relationships between soil microbial biomass and chemical properties Correlation analysis indicated that soil microbial biomass, including soil total microbial biomass and the biomass of microbial groups, positively correlated with soil TC and TN in both soil layers (Table VII). However, the soil microbial biomass basically did not relate to the soil C/N ratio and pH (Table VII).

TABLE I Biomass of soil microbial groups at the 0–10 and 10–20 cm soil depths with and without the treatments of experimental warming and/or nitrogen addition in a long-term field experiment in a temperate steppe of northern China Soil depth

Treatmenta)

PLFAb) content of each microbial groupc) GP

cm 0–10

10–20

GN

ACT

BAC

AMF

SF

FUN

UBI

0.76±0.09 0.94±0.06 0.56±0.07 0.77±0.17 0.37±0.02 0.40±0.02 0.54±0.13 0.59±0.04

0.93±0.10 1.16±0.06 0.66±0.07 0.90±0.18 0.45±0.03 0.50±0.02 0.67±0.16 0.69±0.05

1.04±0.01 1.35±0.10 0.89±0.07 0.94±0.14 0.48±0.02 0.52±0.01 0.89±0.18 0.98±0.09

g−1

CK EW ND WN CK EW ND WN

a) CK

1.18±0.08d) 1.33±0.01 0.85±0.05 1.06±0.19 0.50±0.07 0.56±0.03 0.86±0.16 0.96±0.04

1.18±0.05 1.68±0.32 0.92±0.05 1.17±0.16 0.54±0.07 0.72±0.04 1.06±0.22 1.25±0.12

0.48±0.05 0.57±0.07 0.43±0.03 0.53±0.08 0.23±0.03 0.27±0.01 0.45±0.10 0.47±0.01

μg dry soil 3.67±0.90 0.17±0.01 4.69±0.91 0.22±0.02 2.82±0.61 0.10±0.01 3.61±1.24 0.13±0.02 1.67±0.54 0.08±0.01 2.00±0.50 0.11±0.00 3.12±1.05 0.13±0.03 2.68±0.15 0.10±0.01

= control; EW = experimental warming; ND = nitrogen addition; WN = EW plus ND. fatty acid. c) GP = gram-positive bacteria; GN = gram-negative bacteria; ACT = Actinobacteria; BAC = bacteria; AMF = arbuscular mycorrhizal fungi; SF = saprophytic fungi; FUN = fungi; UBI = ubiquitous fatty acids. d) Values are means ± standard errors (n = 3). b) Phospholipid

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TABLE II Results of two-way analysis of variance on the effects of experimental warming and nitrogen addition on the biomass of soil microbial groups at the 0–10 and 10–20 cm soil depths in a long-term field experiment in a temperate steppe of northern China Soil depth cm 0–10

10–20

Treatmenta)

EW ND EW × ND EW ND EW × ND

P value for each microbial groupb) GP

GN

ACT

BAC

AMF

SF

FUN

UBI

0.125 0.021* 0.798 0.402 0.004** 0.827

0.077 0.067 0.513 0.200 0.004** 0.931

0.154 0.445 0.945 0.513 0.004** 0.875

0.041* 0.024* 0.735 0.288 0.003** 0.929

0.037* 0.001** 0.776 0.950 0.247 0.184

0.097 0.127 0.885 0.622 0.032* 0.893

0.076 0.053 0.926 0.680 0.046* 0.851

0.093 0.017* 0.193 0.560 0.002** 0.830

*, **Significant at P < 0.05 and 0.01, respectively. a) CK = control; EW = experimental warming; ND = nitrogen addition. b) GP = gram-positive bacteria; GN = gram-negative bacteria; ACT = Actinobacteria; BAC = bacteria; AMF = arbuscular mycorrhizal fungi; SF = saprophytic fungi; FUN = fungi; UBI = ubiquitous fatty acids.

Fig. 2 Relative abundance of soil gram-negative bacteria (GN) (a and b) and arbuscular mycorrhizal fungi (AMF) (c and d) at the 0–10 cm (a and c) and 10–20 cm (b and d) soil depths without (control, CK) and with the treatments of experimental warming (EW), nitrogen addition (ND), and EW plus ND (WN) in a long-term field experiment in a temperate steppe of northern China. Values are means ± standard errors (n = 3). * and ** represent significance at P < 0.05 and 0.01, respectively.

DISCUSSION Warming effect The results of this study demonstrate that longterm (five-year) warming significantly increases soil microbial biomass and the relative abundance of Gramnegative bacteria in the soil of a temperate steppe in northern China. The microbial biomass responses were consistent with the results Bell et al. (2010), Ma et al. (2011), and Yergeau et al. (2012). Similarly, an ear-

lier study on seasonal microbial community structure at a mid-alpine environment in Latnjajaure, northern Sweden also found that Gram-negative bacteria increased with the rising temperature (Bj¨ork et al., 2008). The microbial responses in the warming plots may result from the enhanced substrate availabiltiy in the soil (Bell et al., 2010). Previous studies have evidenced that experimental warming increases root exudates, which are a group of small organic compounds and can be easily utilized by soil microbes (Uselman

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TABLE III Relative abundance of soil microbial groups at the 0–10 and 10–20 cm soil depths with and without the treatments of experimental warming and/or nitrogen addition in a long-term field experiment in a temperate steppe of northern China Soil depth

Treatmenta)

Relative abundance of each microbial groupb) GP

cm 0–10

10–20

GN

24.56±1.71c) 22.02±0.99 22.68±0.89 22.90±0.97 22.61±0.94 21.84±0.83 22.01±0.53 22.13±1.23

CK EW ND WN CK EW ND WN

24.60±1.30 27.18±3.78 24.61±0.77 25.58±0.73 24.45±1.01 27.76±1.00 26.91±0.83 28.68±1.24

ACT

BAC

AMF

SF

FUN

UBI

9.94±0.67 9.45±1.40 11.46±0.20 11.51±0.79 10.41±0.41 10.62±0.33 11.34±0.47 10.96±0.59

% 59.09±0.73 58.65±2.14 58.75±1.79 59.99±1.26 57.46±1.69 60.23±0.48 60.27±0.38 61.77±1.69

3.63±0.09 3.57±0.26 2.55±0.17 2.84±0.24 3.47±0.39 4.09±0.25 3.17±0.14 2.31±0.21

15.59±1.32 15.42±0.43 14.90±1.62 16.59±0.88 16.93±0.64 15.40±0.23 13.62±0.64 13.52±0.74

19.22±1.35 19.00±0.20 17.45±1.45 19.43±0.89 20.41±0.42 19.49±0.35 16.80±0.74 15.83±0.71

21.69±0.67 22.35±2.33 23.79±0.48 20.57±0.86 22.13±1.28 20.28±0.34 22.93±0.58 22.39±1.01

a) CK

= control; EW = experimental warming; ND = nitrogen addition; WN = EW plus ND. = gram-positive bacteria; GN = gram-negative bacteria; ACT = Actinobacteria; BAC = bacteria; AMF = arbuscular mycorrhizal fungi; SF = saprophytic fungi; FUN = fungi; UBI = ubiquitous fatty acids. c) Values are means ± standard errors (n = 3). b) GP

TABLE IV Results of two-way analysis of variance on the effects of experimental warming and nitrogen addition on the relative abundance of soil microbial groups at the 0–10 and 10–20 cm soil depths in a long-term field experiment in a temperate steppe of northern China Soil depth cm 0-10

10–20

Treatmenta)

EW ND EW × ND EW ND EW × ND

P value for each microbial groupb) GP

GN

ACT

BAC

AMF

SF

FUN

UBI

0.358 0.687 0.275 0.724 0.887 0.647

0.414 0.726 0.703 0.041* 0.141 0.464

0.824 0.076 0.739 0.808 0.199 0.559

0.798 0.736 0.604 0.122 0.117 0.621

0.628 0.002** 0.343 0.624 0.004** 0.023*

0.524 0.844 0.441 0.221 0.003** 0.258

0.433 0.557 0.342 0.130 < 0.001** 0.955

0.357 0.912 0.175 0.217 0.139 0.483

*, **Significant at P < 0.05 and 0.01, respectively. a) CK = control; EW = experimental warming; ND = nitrogen addition. b) GP = gram-positive bacteria; GN = gram-negative bacteria; ACT = Actinobacteria; BAC = bacteria; AMF = arbuscular mycorrhizal fungi; SF = saprophytic fungi; FUN = fungi; UBI = ubiquitous fatty acids. TABLE V Some soil chemical properties at the 0–10 and 10–20 cm soil depths with and without the treatments of experimental warming and/or nitrogen addition in a long-term field experiment in a temperate steppe of northern China Soil depth

Treatmenta)

Soil chemical propertyb) TC

cm 0–10

10–20

CK EW ND WN CK EW ND WN

g 21.62±0.38c) 21.50±1.18 18.51±1.08 22.26±1.48 16.06±1.89 14.49±0.43 19.90±4.13 22.02±1.90

kg−1

TN

C/N

pH

2.34±0.28 2.65±0.17 2.08±0.06 2.47±0.34 1.62±0.20 1.45±0.03 1.91±0.30 2.39±0.12

9.48±1.06 8.13±0.10 8.94±0.71 9.24±0.85 9.92±0.05 9.96±0.12 10.24±0.69 9.21±0.42

7.21±0.10 7.38±0.08 6.69±0.07 6.79±0.12 7.54±0.10 7.61±0.10 7.27±0.32 6.63±0.16

dry soil

a) CK

= control; EW = experimental warming; ND = nitrogen addition; WN = EW plus ND. = total C; TN = total N; C/N = total C/total N. c) Values are means ± standard errors (n = 3). b) TC

et al., 2000; Hamilton and Frank, 2001). In addition, Gram-negative bacteria are widely recognized to be the

most powerful competitors chasing these additional substrates because they prefer recent plant materials as

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TABLE VI Results of two-way analysis of variance on the effects of experimental warming and nitrogen addition on some soil chemical properties at the 0–10 and 10–20 cm soil depths in a long-term field experiment in a temperate steppe of northern China P value for soil chemical propertyb)

Treatmenta)

Soil depth cm 0–10

EW ND EW × ND EW ND EW × ND

10–20

TC

TN

C/N

pH

0.140 0.321 0.119 0.912 0.050 0.477

0.180 0.379 0.862 0.444 0.012* 0.132

0.511 0.722 0.313 0.259 0.606 0.226

0.197 < 0.001** 0.718 0.176 0.011* 0.105

*, **Significant at P < 0.05 and 0.01, respectively. a) CK = control; EW = experimental warming; ND = nitrogen addition. b) TC = total C; TN = total N; C/N = total C/total N. TABLE VII Pearson correlation coefficients between biomass of soil microbial groups and chemical properties at the 0–10 and 10–20 cm soil depths in a long-term field experiment in a temperate steppe of northern China Soil depth cm 0–10

10–20

Soil propertya)

TC TN C/N pH TC TN C/N pH

Biomass of soil microbial groupb) Total PLFAsc)

GP

GN

ACT

BAC

FUN

SF

AMF

UBI

0.648* 0.684* −0.351 0.561 0.745** 0.769** 0.032 −0.448

0.612* 0.686* −0.411 0.474 0.700* 0.746** −0.032 −0.450

0.628* 0.687* −0.362 0.616* 0.653* 0.715** −0.087 −0.497

0.292 0.311 −0.170 0.088 0.782** 0.781** 0.110 −0.401

0.659* 0.726** −0.402 0.568 0.702* 0.748** −0.033 −0.471

0.723** 0.563 −0.108 0.435 0.733** 0.707* 0.184 −0.312

0.736** 0.542 −0.070 0.326 0.769** 0.768** 0.119 −0.395

0.523 0.532 −0.239 0.782** 0.433 0.302 0.432 0.109

0.381 0.535 −0.389 0.544 0.820** 0.818** 0.105 −0.448

*, **Significant at P < 0.05 and 0.01, respectively. a) TC = total C; TN= total N; C/N = total C/total N. b) GP = gram-positive bacteria; GN = gram-negative bacteria; ACT = Actinobacteria; BAC = bacteria; AMF = arbuscular mycorrhizal fungi; SF = saprophytic fungi; FUN = fungi; UBI = ubiquitous fatty acids. c) Phospholipid fatty acids.

substrates (Fierer et al., 2003; Kramer and Gleixner, 2006). Therefore, Gram-negative bacteria have a competitive advantage over other microbial groups. Furthermore, earlier studies at the same site of this study also confirmed that the experimental warming enhanced plant photosynthesis and growth (Niu and Wan, 2008; Niu et al., 2008). Niu et al. (2008) found that experimental warming increased daily mean assimilation rate in Pennisetum centrasiaticum and Agrostis capillaris by 30% and 43%, respectively. The additional photosynthates might increase the soil substrate availability through root exudation in the warming plots (H¨ogberg and Read, 2006). It should be noted that warming did significantly decrease soil moisture in this study according to Xia et al. (2009), who found that experimental warming at our site significantly reduced volumetric soil moisture by 5.0%. Many studies have shown that drought can

depress the growth of soil microbes and suggested that the warming-induced decrease in soil water does not counteract the warming-enhanced carbon availability effect on soil microbial communities (Liu et al., 2009; Sheik et al., 2011). Sheik et al. (2011) also found that experimental warming increased soil microbial biomass by 40%–150% in normal rainfall years, but decreased microbial biomass by 50%–80% during drought conditions in the US Great Plain. The depression effect of drought on soil microorganisms was also confirmed by our measurements in 2011, a severe drought year with the growing-season (May to October) precipitation of merely 229.3 mm (only 66.5% of the long-time average) (data not shown). Nitrogen addition effect In the current study, it was found that long-term nitrogen addition oppositely influenced soil microbial

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biomass at the 0–10 and 10–20 cm soil depths. Previous nitrogen addition experiments also showed contradictory results of the responses of soil microbial biomass to nitrogen addition (Wallenstein et al., 2006; Allison et al., 2009; Cusack et al., 2011; Ramirez et al., 2012). The contradictory results could be explained by the critical nitrogen load or nitrogen saturation theories (Aber et al., 1998; Williams and Tonnessen, 2000). These theories propose that the effect of nitrogen enrichment on ecosystem function would switch from stimulation to inhibition when the ecosystem reaches a critical nitrogen loading or saturation level (Zhang et al., 2008; Bowman et al., 2012). Previous studies showed that study sites with high soil nitrogen stores were prone to be saturated (Fenn et al., 1998; Lovett et al., 2002). In our study, the total nitrogen was much higher (P = 0.040) in the upper soil layer than in the lower layer, so we could suppose that the upper soil layer would more likely be nitrogen saturated than the lower layer. Correspondingly, the decrease of soil pH at the 0–10 cm soil depth (7.26%) was much more profound than that at the 10–20 cm soil depth (3.67%), indicating that microorganisms indeed suffered more from stress in the surface soil layer than the lower soil layer. In addition, the different influences of nitrogen enrichment could also be contributed to the indirect effect of nitrogen addition on plant community. Roots in different soil layers would respond differently to nitrogen manipulation. For example, Bai et al. (2008) found that nitrogen addition decreased the life span of the roots of Leymus chinensis, a widely distributed native grass, in the 0–10 cm soil layer, but had no effect on that in the 10–20 cm soil layer after three years of nitrogen addition in the temperate steppe of this study, revealing that the roots in the 0–10 cm layer might get more injury from the long-term nitrogen addition than those in the 10–20 cm. Therefore, soil microbes in the 0–10 cm layer would be depressed by nitrogen addition because of the reduction in carbon supply. At the same time, the substrate availability in the 10–20 cm soil layer would not be influenced and soil microbes in this soil layer would mainly benefit from nitrogen enrichment. Our results that nitrogen addition significantly decreased the relative abundance of AMF are consistent with those of the vast majority of the nitrogen fertilization experiments (Bradley et al., 2006; van Diepen et al., 2007). In a meta-analysis research conducted by Treseder (2004), nitrogen fertilization averagely decreased the biomass of mycorrhizal fungi by 15%, which could be attributed to the shift in the plant community structure. Yang et al. (2011) found

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that nitrogen enrichment may significantly decrease the legume abundance and increase grass abundance in the temperate steppe of this study. The reduction in legume abundance would have negative impact on the mycorrhizal fungi. It was suggested that plant community would invest less carbon to AMF with higher available nitrogen because under that circumstance AMF would become less critical for nutrient uptake (Corkidi et al., 2002; van Diepen et al., 2007). Interactive effect of experimental warming and nitrogen addition The results of this study showed that long-term experimental warming and nitrogen fertilization had little interactive effect on soil microbial and chemical properties except for the relative abundance of AMF at the 10–20 cm soil depth. Similarly, in a previous study at the same site by Xia et al. (2009), warming and nitrogen addition did not have interaction effects on ecosystem carbon exchange processes. These results may be explained by warming and nitrogen fertilization influencing soil microbial community through different mechanisms. Specifically, experimental warming might mainly have indirect impact on soil microbes via modifying plant physiology such as root exudates, but nitrogen enrichment always directly affects soil microorganisms through changing soil chemical properties like soil pH. However, we should notice that when experimental warming and nitrogen addition were combined, their effects were counterbalanced at the 0–10 cm soil depth, manifesting the importance of multi-factor experiments in climate change research because the ongoing global change is a comprehensive process driven by many factors simultaneously. The integrated influences of the global change processes would differ with those of one single global change factors (Gray et al., 2011; Gutknecht et al., 2012). Our results also indicate that temperature rising could help soil microbes deal with the stresses that result from nitrogen enrichment. Soil microbes in N-fertilized plots would become more limited due to lack of carbon substrate (Demoling et al., 2008), while global warming induced an extra supply of labile carbon substrate that the hungry microbial community urgently needed. ACKNOWLEDGEMENT The authors thank Dr. HONG You-Wei, Ms. WANG Min, and Ms. ZHOU Guo-Hui, the Institute of Urban Environment, Chinese Academy of Sciences, for their assistance with the PLFA measurements.

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