Effects of simulated climate change on soil microbial biomass and enzyme activities in young Chinese fir (Cunninghamia lanceolata) in subtropical China

Acta Ecologica Sinica 37 (2017) 272–278

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Effects of simulated climate change on soil microbial biomass and enzyme activities in young Chinese fir (Cunninghamia lanceolata) in subtropical China Jintao Gao a,b, Enxi Wang a,b, Weiling Ren a,b, Xiaofei Liu a,b, Yuehmin Chen a,b,c,⁎, Youwen Shi d, Yusheng Yang a,b,c a

School of Geographical Sciences, Fujian Normal University, Fuzhou 350007, China State Key Laboratory of Subtropical Mountain Ecology (Funded by Ministry of 9 Science and Technology and Fujian Province), Fujian Normal University, Fuzhou 10 350007, China c Institute of Geography, Fujian Normal University, Fuzhou 350007, China d The Forestry Bureau of Sanming Sanyuan District, Sanming 365000, China b

a r t i c l e

i n f o

Keywords: Soil microbial biomass Soil enzyme activities Global warming Nitrogen addition interaction Chinese fir

a b s t r a c t Global warming and nitrogen deposition have been responsible for numerous environmental disturbances, and have attracted much attention from researchers, government agencies and international community. Recent studies indicate that the trend of global warming and nitrogen deposition will continue over the next few decades. These changes not only affect the growth of aboveground vegetation, but also change the belowground soil environment, and thus directly or indirectly affect the microbial process. The microbial biomass and soil enzymes play significant roles in terrestrial environments, particularly through the decomposition of soil organic matter, dynamic fluctuation between carbon sink and source, and the transformation of soil nutrient. However, little is known about that how global warming and nitrogen deposition will affect the soil microbial and soil enzymes in the subtropical zone. In the present study, we aim to evaluate the responses of the microbial biomass and soil enzyme activity to shortterm simulated warming and nitrogen deposition in young Chinese fir (Cunninghamia lanceolata) in Sanming Fujian province in subtropical China. The results showed that soil warming increased microbial biomass carbon content and improved the activity of acid phosphatase and lignin enzymes significantly (P b 0.05). Additionally, microbial biomass carbon content was significantly higher than that of the control after the application of nitrogen fertilizer. Besides, nitrogen addition also significantly raised the C/N ratio of microbial biomass. It also reduced the activity of lignin and cellulose hydrolysis. The combination of warming and nitrogen treatment was more effective than individual warming and nitrogen treatments, increasing the content of soil microbial biomass carbon and nitrogen, decreasing the activity of lignin hydrolytic enzymes and chitinase, and leading to further acidification of the soil. Redundancy analysis (RDA) showed that moisture and pH are the major determinants of soil enzyme activity at the 0–10 cm depth. However, at the 10–20 cm depth, the major determiner is microbial biomass. In summary, the simulated warming and nitrogen deposition affected soil microbial biomass and enzyme activity significantly in the short-term, and the interaction of the two factors was significant. That suggests that the climate change could have a profound effect on soil microbial processes. Therefore, the effects of simulated warming and nitrogen deposition on microbial biomass and soil enzyme activity and the mechanism of their interaction with soil, microorganisms and plants need to be studied further, in order to reveal the responses and feedback mechanisms of Chinese fir plantations to global climate change in subtropical China. © 2017 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

1. Introduction Climate warming and nitrogen deposition are the two main features of the global climate change, thus provoking a series of environmental problems to the terrestrial ecosystem, which has become a concern of ⁎ Corresponding author at: School of Geographical Sciences, Fujian Normal University, Fuzhou 350007, China. E-mail address: [email protected] (Y. Chen).

http://dx.doi.org/10.1016/j.chnaes.2017.02.007 1872-2032/© 2017 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

traitor, research scientists, government agencies and the international community [1]. According to climate models, IPCC (2013) predicted that global surface will mean increase temperature 1.8–4.0 °C at the end of the 21st century [2]. Nitrogen deposition will still continue to increase within the coming decades in the worldwide areas under the influence of human activities [3,4]. Nitrogen deposition increase and the warming will undoubtedly directly or indirectly impact soil microbial biomass and soil enzyme activity. Moreover, soil microbial biomass and soil enzyme activity exert an important role, such as fixation and

J. Gao et al. / Acta Ecologica Sinica 37 (2017) 272–278

decomposition of organic matter, carbon source and sink, soil nutrient cycling and transformation process, which cannot be replaced [5]. And due to the complexity and heterogeneity of the soil ecosystem, global climate change on soil microbial biomass and soil enzyme activity ring is still in great uncertainty [6]. Currently, the field warming control experiment is primarily concentrated in the middle and high latitude area grassland, farmland, tundra and forest ecosystem [7,8]. In the tropical and subtropical regions of the south, there is no field soil warming experiment, nor the interaction between temperature and climate change factors [9]. There is no denying the fact that simulating the interaction of climate experiment to understand individual effect of each factor and interaction effects can be integrated to reflect forest ecosystem soil microbial transformation and nutrient cycling process of global climate change response and feedback situation. The humid subtropical in China is inclusive at the same latitude with rare oasis distribution area of the world's largest and most typical evergreen broad-leaved forest, which is the global subtropical biodiversity center. Because the heroic endeavor to create artificial forest, the area contributed to the forest carbon sink capacity of 65%. The Chinese fir forest is one of the most important plantations in the area, accounting for the area of plantation in the world 6.5% [10], accounting for 19% of the area of artificial forest in China with the volume of 25% in the forestry production and carbon sequestration in our country, which has a pivotal position. However, this region is confronted with rapid global climate change and the change of soil microbial response to environmental changes will inevitably exert an important influence on the growth of Chinese fir plantations. But researches on the region under the background of global change on Chinese fir plantation soil microbial research is still scarce, especially that the combined effects of climate on the Chinese fir plantation soil microbial biomass and soil enzyme activity influence research traitor are reported rarely. Therefore, research on early on humid subtropical Chinese fir plantation ecosystems is based on related working and on the soil of Chinese fir plantation seedlings to simulate to increase moderate nitrogen deposition [11]. It combined processing of field control experiment to research traitor in Chinese fir plantation soil microbial quantity and soil enzyme activity of elevated temperature and nitrogen increased response in order to provide scientific basis for soil nutrient cycling response and feedback of artificial forest under the background of global change. 2. Materials and methods 2.1. Study site The experiment was conducted at the Fujian Normal University's Forest Ecosystem and Global Change Research Station in Chenda Town, Sanming City in the Fujian Province of China (26° 19′ N,117° 36′ E). This site is situated in a latosol soil; the regional climate is subtropical monsoon. The average annual precipitation, temperature, and evaporation are 1749 mm, 19.1 °C and 1585 mm respectively. The elevation is 300 m above sea level. 2.2. Experimental design and sampling The experiment was a randomized complete block factorial design, with warming and N fertilization as fixed factors. There were four treatments (five replicates) in this study: (1) control (CT); (2) nitrogen addition (N); (3) warming (W); and (4) warming + nitrogen addition (WN). There were 20 2 m × 2 m mini-plots, and indigenous soil in the plots was replaced, to a depth of 60 cm, with sieved topsoil from a same forest area. PVC pipes (200 cm width, 60 cm depth) were buried vertically in each plot for planting four Chinese fir seedlings together. In November 2013, 120 healthy, uniform Chinese fir seedlings were selected based on plant basal diameter, height, and fresh weight. Four seedlings were randomly transplanted into each mini-plot.

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Beginning in March 2014, artificial warming and nitrogen addition were conducted. Heating cables were used to generate a warmed environment and were buried in a spiral pattern 10 cm below the ground. Soil temperature was measured in each plot using temperature sensors (T109; Campbell Scientific Inc., Logan, UT, USA) buried continuously between heating cables. The warming cables significantly increased (5 °C) the soil temperature in the warmed plots at the 10 cm depth, and the effects of the cables over the soil surface were equal to the control. The control plot and unwarmed plots had a “dummy” heater the same shape and size as the cables in order to simulate the shading effects of the heater. Detail of the experimental warming system design has been reported previously [11]. During this same period, two N levels were applied (0 and 80 kg N ha−1 year−1, added as ammonium nitrate (NH4NO3)). Nitrogen was added twelve times each year. After one year, soil (0–10 cm) was collected from five random points in each plot using 5 cm soil cores. Soil samples were immediately transported to the laboratory and stored at 4 °C until the analyses. The soil was cleared of roots and all organic debris, air-dried and analyzed for soil pH, soil organic carbon (SOC), total nitrogen (N) and available phosphorus (P). Air-dried soil samples were ground and passed through a 2 mm sieve. Soil pH was determined using a pH meter with a soil:water ratio of 1:2.5. Soil organic C and soil total N was measured in a single analysis using a CN auto analyzer (Elementar Vario MAX, Germany). A 3 g air-dried soil sample was mixed with 30 mL of the Mehlich-3 extracting solution [12], shaken immediately for 5 min, and centrifuged for 5 min at 8000 rpm. The supernatant was used to determine the available P (Skalar san ++). Stand characteristics and soil properties are provided in Table 1. 2.3. Soil microbial biomass analysis Soil microbial biomass C (MBC), N (MBN) and P (MBP) were measured by the chloroform fumigation–extraction method as described previously [13]. Briefly, two portions of 10 g field moist soil samples were weighed, and one portion of them was fumigated with chloroform for 24 h and extracted with 0.5 mol·L−1 K2SO4 in an end-to-end shaker for 1 h, then the supernatants were filtered through a Whatman no. 42 paper. The other proportion of the soil was directly extracted as above. The amounts of total C in the fumigated and un-fumigated soil extracts were determined using a TOC-VCPH/CPN analyzer. MBP was extracted with 0.5 mol·L−1 NaHCO3 and rinsed in deionized water and the phosphate recovered by shaking for 1 h in 50 mL of 0.25 mol·L H2SO4. The amounts of total N and P was detection by Skalar san ++. MBC,MBN and MBP were calculated as the difference between the fumigated and unfumigated samples and corrected for unrecovered biomass using kC, kN and kP factor of 0.38, 0.45 and 0.4 respectively [14]. 2.4. Measurements of soil enzyme activity We used a procedure adapted from Saiya-Cork and Sinsabaugh [15] (2002) to analyze soil enzyme. Activities of soil enzymes involved in C, N and P cycling were measured. These enzymes included four hydrolytic enzymes: β-glucosidase (βG), cellobiohydrolase (CBH), Nacetylglucosaminidase (NAG), and acid phosphomonoesterase (AP) and two oxidase enzymes: phenol oxidase (PHO) and peroxidase (PEO). We marked with 4-methylumbelliferone (MUB) as substrate labeled hydrolytic enzymes activity and the activity of lignin enzyme was estimated using L-dihydroxyphenylalanine (L-DOPA) as substrate. Briefly, 1 g fresh soil sieved at 2 mm was mixed with 125 ml 50 mmol·L−1 acetate buffer (pH = 5.0), then used a magnetic stirrer stirring 5 min to homogenize and took 200 μL to a 96 well plate by pipettor. The mixture was then incubated in the dark for 4 h (hydrolytic enzymes) and 18 h (oxidase enzymes) at 20 °C. Following incubation, we measured sample fluorescence using 365-nm excitation and 450-nm emission filters (hydrolase enzymes) or absorbance at 450 nm (lignin enzymes) on a SpectraMax M5 Microplate Reader (MDS Analytical Technologies,

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Table 1 Basic physicochemical characteristic of soil at different treatments (mean ± SD, n = 5). Depth

Treatment

pH

0–10 cm

CT W N WN CT W N WN

4.7 4.5 4.5 4.4 4.9 4.8 4.8 4.6

10–20 cm

± ± ± ± ± ± ± ±

0.1a 0.1b 0.1b 0.1b 0.1a 0.0a 0.1a 0.1b

Moisture(%)

SOC(g kg−1)

Total N(g kg−1)

Available P (mg kg−1)

21.3 19.1 20.4 17.5 19.7 19.3 19.6 15.3

14.3 ± 0.1a 12.7 ± 0.3a 13.6 ± 0.2a 14.0 ± 0.2a 10.2 ± 1.3a 8.8 ± 2.4a 10.1 ± 2.1a 11.0 ± 2.0a

1.4 1.2 1.4 1.3 1.1 1.0 1.1 1.2

1.8 1.7 1.0 1.5 1.5 1.1 1.0 1.2

± ± ± ± ± ± ± ±

0.5a 1.1b 0.3a 0.9c 2.0a 1.9a 2.2a 2.8b

± ± ± ± ± ± ± ±

0.1a 0.2a 0.4a 0.1a 0.1a 0.2a 0.2a 0.2a

± ± ± ± ± ± ± ±

0.2a 0.1a 0.2b 0.2a 0.3a 0.3ab 0.1b 0.4ab

CT: Control, W: Warming, N: Nitrogen addition, WN: Warming + nitrogen addition. Different small letters in the same column in the same soil layer (P b 0.05).

and nitrogen addition was significant. This treatment on soil moisture influence groups tips more. There were no significantly differences of soil organic carbon and total nitrogen at two depths of all treatments. Nitrogen addition significantly reduced the available phosphorus at two depths. While warming and warming + nitrogen addition decreased available phosphorus, but the effect was not significant, which showed that the response of available phosphorus to nitrogen addition was more obvious than that of the warming (Table 1).

USA). All enzymes are shown in Table 2. The substrate concentration and incubation time of the enzyme were determined by pre experiment. 2.5. Statistical analysis Excel 2010 (Microsoft Office 2010), SPSS16.0 (SPSS, Inc., Chicago, IL) and Canoco 5.0 (Ithaca, NY, USA) were used in the data processing and statistical analysis. One-way ANOVA and Duncun were used to compare the differences of microbial biomass and enzyme activities among different treatments. Two-way ANOVA was used to analyze the effect of warming, nitrogen addition on the soil microbial biomass. The statistical significance was accepted at a P b 0.05 level. Pictures were draw using Origin 9.0 (OriginLab, Hampton, USA).

3.2. Soil microbial biomass and its ratio Warming and nitrogen addition caused the MBC, MBN and MBP in different changes and there were significant warming and nitrogen addition interaction on microbial biomass (P b 0.001). Compared with control treatments, warming significantly increased MBC whereas MBN was the opposite and MBP was not sensitive to temperature rise. Similarly, nitrogen addition also significantly increased MBC. While nitrogen addition slightly and no significantly increased MBN and MBP at the 0–10 cm depth (P b 0.05), but both of them decreased significantly at the 10–20 cm depth. Warming + nitrogen addition significantly decreased MBC and MBN and the interaction is obvious (P b 0.001). Warming + nitrogen addition decreased MBP significantly at the 0– 10 cm depth, but had no effect at the 10–20 cm depth (Table 3). Warming significantly increased MBC/MBN at two depths. Nitrogen addition and warming + nitrogen addition significantly increased MBC/ MBN at 10–20 cm depth, but had no effect at the 0–10 cm depth. MBC/

3. Results 3.1. Soil physical and chemical properties Compared with control treatment, warming decreased the water content significantly at the 0–10 cm depth, while warming and nitrogen addition decreased pH significantly respectively at the 0–10 cm depth; warming and nitrogen addition marginally but not significantly decreased soil pH and moisture at the 10–20 cm depth. The soil water content and soil pH of warming + nitrogen addition plots significantly reduced, which demonstrates that the interaction between warming

Table 2 The type, targets and substrates of soil enzymes. Enzyme

Type

Targets

Substrate

β-glucosidase (βG) Cellobiohydrolase (CBH) Phenol oxidase (PHO)

C-targeting hydrolytic C-targeting hydrolytic C-targeting oxidase

Cellulose (for glucose) Cellulose (for disaccharides) Lignin and other complex compounds

4-MUB-β-D-glucoside 4-MUB-β-D cellobioside L-DOPA

Peroxidase (PEO)

C-targeting oxidase

Lignin and other complex compounds

L-DOPA

N-acetylglucosaminidase (NAG) Acid phosphatase (AP)

N-targeting hydrolytic P-targeting hydrolytic

Chitin Phosphorous

4-MUB-N-acetyl-β-D-glucosaminide 4-MUB-phosphate

Table 3 The contents of MBC, MBN and MBP at different treatments (mg kg−1, mean ± SD, n = 5). Treatment

Two-way ANOVA

Depth

Microbial biomass

CT

W

N

WN

WE

NE

WE*NE

0—10 cm

MBC MBN MBP MBC MBN MBP

181.4 ± 18.9b 19.4 ± 2.1a 16.8 ± 1.8a 134.1 ± 26.6c 15.2 ± 2.0a 25.8 ± 2.0a

230.6 ± 16.6a 12.7 ± 2.3b 16.2 ± 2.1a 157.6 ± 14.9b 12.4 ± 1.6b 22.6 ± 4.1b

219.3 ± 17.4a 20.4 ± 2.1a 18.6 ± 0.2a 195.1 ± 23.6a 11.4 ± 1.0b 17.7 ± 0.3c

92.7 ± 11.7c 12.0 ± 2.9b 18.1 ± 1.1a 83.3 ± 14.6d 4.9 ± 0.3c 16.8 ± 1.4c

⁎⁎⁎ ⁎⁎⁎

⁎⁎⁎

⁎⁎⁎

NS ⁎⁎⁎ ⁎⁎⁎

NS NS ⁎⁎⁎ ⁎⁎⁎

NS

NS

NS NS ⁎⁎⁎ ⁎⁎⁎ ⁎

10—20 cm

WE: warming effect, NE: nitrogen addition effect, WE × NE: interaction effect of warming and nitrogen addition. NS: no significant difference. Different small letters in the same row indicate significant differences among the four treatment by One-way ANOVA (P b 0.05). ⁎ P b 0.05. ⁎⁎⁎ P b 0.001.

J. Gao et al. / Acta Ecologica Sinica 37 (2017) 272–278

275 0-10 cm

32

A

Aa

16

B Aa Aa

Aa

24

12

Aa

Aa 16

Bb

Ba Bb

Ba Ca

Soil Microbial biomass C/P ratio

Soil Microbial biomass C/N ratio

Aa

10-20 cm

8

Cb

Ca

Ba

Bb

8

4

0

0

CT

W N Treatments

WN

CT

W

N Treatments

WN

Fig. 1. Effect of warming and nitrogen deposition on MBC/MBN, MBC/MBP. Notes: CT: Control; W: Warming; N: Nitrogen addition; WN: warming + nitrogen addition. Different small letters in the same column in the same soil layer (P b 0.05).

3.3.1. C cycle related enzymes β-glucosidase (βG) and cellobiohydrolase (CBH) are closely related to cellulose decomposition. BG is an important indicator of the change of soil organic carbon, which is considered to be the

0-10 cm Aa

100

75

Aa

Aa

Ba

Cb

Db

12

Bb

Bb

8

Cb 25

4

Bb 0

C Aa ABa

21

14

Cb

Cb

1

450

Aa Bb

Bb

2

0

Aa

Ba

Db

Aa

Bb

Aa

E

Ca

7

Db 0

Aa

F

Ba

Ba

120

Ab Ba

300

Ca

BCb Ba

Cb

Cb

Cb

Cb

80

Db Db

150

40

0

CT

W

N

Treatment

WN

CT

W

N

(µmol·g

Ab

PEO

3

WN

Treatment

Fig. 2. Change of soil enzyme activities under warming and nitrogen deposition in Chinese fir.

0

AP

Aa

D ·h -1)

Aa

-1

PHO (µmol·g-1·h-1)

Ba

16

B

Aa

Ba Cb

50

0

NAG (nmol·g-1·h-1)

A

Aa

(nmol·g-1·h-1)

βG (nmol·g-1·h-1)

Aa

10-20 cm

CBH

3.3. Soil enzyme activity under warming and nitrogen addition

mainly carbon-acquisition enzymes in soil [14]. Warming and nitrogen decreased BG at the two depths. Warming marginally but significantly increased CBH at the 0–10 cm depth. While nitrogen addition and warming + nitrogen addition increased more CBH activity in subsurface than surface. In general, nitrogen addition and warming + nitrogen addition decreased cellulose decomposition rate; βG and CBH show different response to warming. Warming decreased BG activity while CBH activity was enhanced (Fig. 2). Phenol oxidase (PHO) and Peroxidase (PEO) are mainly lignin degrading enzymes in soil [15]. PHO limits the accumulation of soil organic carbon and PEO can not only oxidize hydrogen peroxide, but also accelerate the decomposition of soil organic matter and the synthesis of soil humus [16]. Warming and nitrogen addition were increased

(nmol·g-1·h-1)

MBN in warming plots was lower than above layer, while it was higher than above layer in nitrogen addition and warming + nitrogen addition (Fig. 1). Warming and nitrogen increased significantly MBC/MBP at the 10–20 cm depth, but had no effect at 0–10 cm depth. Warming + nitrogen addition significantly decreased MBC/MBP at the 0–10 cm depth (Fig. 1).

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lignin degrading enzymes activity at the 10–20 cm depth. But warming + nitrogen addition significantly decreased lignin degrading enzymes activity and PEO reached the lowest value 2.1 μmol g−1 h−1, indicating that the soil detoxification ability is the worst in warming + nitrogen addition plots, the accumulation of toxic substances may affect tree growth (Fig. 2). 3.3.2. N cycle related enzymes N-acetylglucosaminidase (NAG)mainly involved in decomposition of chitin in the soil, which is considered to be the mainly nitrogen-acquisition enzymes [14]. Compared with the control treatment, warming and nitrogen addition significantly decreased NAG activity, which mean that both warming and nitrogen addition weakened chitin decomposition(Fig. 2). 3.3.3. P cycle related enzymes Acid phosphatase (AP) affects soil organic phosphorus mineralization, which is considered as the most important phosphorus-acquisition enzymes in acid soil [14]. Warming significantly increased AP at the 010 cm depth, but no significant influence at the 10–20 cm depth; Nitrogen addition and warming + nitrogen addition significantly decreased AP at the 0–10 cm depth, while decreased significantly at the 10– 20 cm depth (Fig. 2).

Redundancy analyses (RDA) were used to elucidate the relationship between soil biochemical properties and soil microbial enzyme activities at the two depths, respectively (Fig. 3). Eigenvalues of RDA indicated that axes 1 and 2 explained 81.70% and 0.9% of the overall variances at the 0–10 cm depth, while they explained 41.5% and 5.9% at the 10–20 cm depth within the soil enzymes activities profiles among the treatments, respectively. The plot can be interpreted quantitatively using the length of the arrow to indicate how much variance is explained by that factor; the direction of the arrows for individual soil properties indicating an increasing concentration of that factor. The soil variable arrows pointing in approximately the same direction as the EEA profile arrows indicate a high positive correlation (the longer the arrow, the stronger the relationship) [14]. The arrows for pH and Moisture were longer than those of the other variables at the 0–10 cm depth, and explained 46.5% and 14.1% respectively, indicating that these factors accounted for the greatest proportion of variances in the soil enzymes. However, factors of MBC, MBN and MBP accounted for the greatest proportion of variances in the soil enzyme activities at the 10–20 cm depth. Particularly, MBC explained 38.6% variances in the soil enzyme activities in the subsurface (Fig. 3).

4. Discussion 3.4. Key factors driving the patterns of soil enzyme activities

1.2

A W3

CT1 CT3 CT4 CT2 CT5 CT1

CT4 CT5 CT2

WN1 WN5

N4 N1 N2 N3 N5

B

W1

W2 W4 W5

PCo2 (11.4%)

Our results showed that soil enzyme activities responded differentially to short-term warming and nitrogen addition at two depths in the humid subtropical forest. The humid subtropical forest is regarded as the global subtropical biodiversity center. To the best of my knowledge, it is the first time to report the responses of soil enzyme activities to climate change in this region.

W4

PCo2(20.5%)

1.2

The control treatment had a clear separation from the other treatments with lower ordinate scores on the PCo2, while the warming treatment differed from the other treatments along the PCo1 indicating that warming, nitrogen addition and warming + nitrogen addition have significantly effect on soil enzyme activity (Fig. 3).

WN2 WN3 WN4

W5 W2

W1

N1 N3 N5 N4 N2 WN3 WN4 WN1 WN2 WN5

-1.2

-1.2

CT3

-1.5

-1.5

2.0

PCo1 (83.3%)

1.0

0.8

W3

C

PEO

CBH

2.0

PCo1(75.2%)

MBP

D

AP MBC

MBN

PHO

BG pH

M

Moisture

pH NAG

RDA2 (5.9%)

RDA 2 (0.9%)

BG

PHO

NAG

PEO MBC

CBH

-0.6

Moisture:14.1%,F=6.1,P=0.016 pH:46.5%,F=15.6,P=0.002

MBN

-0.4

RDA 1 (81.7%)

1.0

-1.0

MBP

AP

-1.0

MBC:38.6%,F=11.3,P=0.002

RDA1 (41.5%)

1.0

Fig. 3. Correlations of soil enzyme activities to soil properties as determined by principle coordinate analysis (PCoA) and redundancy analysis (RDA)

J. Gao et al. / Acta Ecologica Sinica 37 (2017) 272–278

4.1. Warming and nitrogen addition effects on soil microbial biomass Soil microbial biomass is an important foundation of soil microbe coming into play. Because of fast turnover and high sensitivity, soil microbial biomass can reflect the minimal changes of soil fertility and soil environmental quality [16]. Our research found that the enhancement in soil MBC under warming at two depths could be mainly due to the warmer spring and the recovery of microorganism (Table 3). At this moment, the increase of soil temperature promotes the reproduction of microorganisms and improves the activity of the microorganism. Microbial decomposition of organic matter to strengthen the role, thus providing nutrients for the growth of Chinese fir [17]. Nitrogen addition also increased MBC significantly (Table 3). Nitrogen is an essential nutrient for soil microbial organic synthesis. Applying nitrogen promote the rapid growth of Chinese fir seedlings and accumulation of MBC. Unlike treatment alone, warming + nitrogen addition has shown the obvious antagonism to MBC, it is probably because the plant tolerance limit of single factor could change with the change of the other factors, which may also be warming + nitrogen addition exacerbated the soil water loss. Soil water is the important ecological factors of influence soil microbial biomass, which is generally believed that the water shortage will be inhibited the growth of microorganisms [18]. In addition, warming + nitrogen addition decreased PEO significantly, which lead to accumulate of toxic matter that affect Chinese fir and limit soil microbial activities. That may explain why MBC, MBN and MBP have a significant decline in the warming + nitrogen addition treatment plots. In addition, our study also found that applying nitrogen generally reduces the MBN. Nitrogen does not belong to the restrictive factors and lack phosphorus in the subtropical region. Long-term application of nitrogen significantly increased soil microbial biomass in European heathland limited by nitrogen, but decreased soil microbial biomass in acid grassland limited by phosphorus [19–20]. This may be due to the fact that there is a big different of microorganisms in different study site that limited by different the restricted elements. It may lead to different response of soil microbial biomass to nitrogen addition [20]. MBC/MBN and MBC/MBP are important indicators to measure the effectiveness of MBN and MBP. Warming significantly increased MBC/ MBN at the 0–10 cm depth (Fig. 1). This may be due to the differences in the demand and the use efficiency for MBC and MBN of Chinese fir seedlings at the early stage of warming. Nitrogen addition was significantly increased MBC/MBP at the 10–20 cm depth (Fig. 1), indicating that soil microbial mineralization organic phosphorus and release phosphorus reduced significantly, which means that phosphorus deficiency condition is worst in soil of nitrogen addition treatment plots. After application of nitrogen, the microbial nutrient turnover rate increased and it promotes the growth of Chinese fir seedlings. Biomass of Chinese fir increased taking a large amount of phosphorus in soil, thus leading to the soil phosphorus deficiency more severe [21]. Compared with single factor, warming + nitrogen addition decreased MBC/MBP, suggesting that it could alleviate soil relative lack of phosphorus. What Chinese fir in warming and nitrogen addition plots is slow-growing and the phosphorus in soil is underutilized can explain it. 4.2. Warming and nitrogen addition effects on soil enzyme activities Soil enzyme can be sensitive to small environmental changes, which is an important and irreplaceable participant in the material cycle and flowing of energy [22–23].Warming and nitrogen addition have different effects on soil enzymes at the two depths and their interaction was significant. In this study, we found that soil enzyme activities were driven by pH and water content in the surface, while by soil microbial biomass in the subsurface (Fig. 3). This shows that under the condition of nutrient rich in the surface, pH and water content effects on enzyme activity were greater, while nutrient comparatively not rich in the subsurface, it's MBC and other nutrient factor were main factor to determine the strength of enzyme activity.

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Temperature is a very important factor that affects soil enzyme activities. By the characteristics of soil enzyme, global warming is to improve the ground temperature, thus being bound to profoundly affect soil enzyme activity. Temperature rise can affect soil enzyme kinetics, exerting a direct impact on the soil enzyme activity; it can also indirectly affect soil enzyme activity through influencing the soil moisture and heat conditions, microbial community structure, microbial biomass, microbial respiration and soil organic matter mineralization [24]. And too high or too low temperature will lead to soil enzyme passivation or inactivation [25]. Our study showed that warming significantly decreased βG and NAG at the 0-10 cm depth(Fig. 2). However, the results of RDA indicated that βG and NAG were more easily affected by water instead of temperature (Fig. 3). Increased precipitation would increased βG and NAG significantly and water content was more significant than temperature on influence of the two enzyme [26–28]. At the 10–20 cm depth, warming significantly increased PHO and PEO (Fig. 2), which because warming quicken the turnover rates of fine roots of Chinese fir, increased below-ground litter content, thus active microbial produced positive effect on lignin enzyme activities. Nitrogen deposition often leads to acidification of soil, which affects the efficiency of nitrogen and mineralization rate [29]. Therefore, it is bound to affect the activity of various enzymes in soil by changing the soil micro environment [30–31]. Our research found that nitrogen addition significantly decreased βG, CBH, PHO and PEO (Fig. 2). Previous studies have shown that the increase of inorganic nitrogen can inhibit the lignin degrading fungus (white rot fungi) from producing lignin degrading enzyme [32]. Lignin play the role to protect plant tissues from degradation, so lignin decomposition reduction may lead to a decrease in the carbon source used by other heterotrophic microbe, which inhibit growth of microorganism of producing cellulose degrading enzyme, as a result, βG and CBH reduced in nitrogen addition plots [33–35]. Warming + nitrogen addition shown clear interactions and interaction was more significant compared with effect of single factor. Warming + nitrogen addition significantly reduced the lignin degrading enzyme activity. The Lignin degrading enzymes are very important in the formation of humus, and the decrease of the activity of lignin degrading enzymes may reduce the formation of humus in soil [16]. PEO can not only accelerate soil organic matter decomposition and humus of synthesis, but also the oxidation of hydrogen peroxide to protect plant roots from poisoning [36]. PEO in warming + nitrogen addition plots reached the lowest activities among all treatments, which means that the ability to detoxify was the worst and the accumulation of hydrogen peroxide and other toxic substances will affect tree growth in field observation. It was discovered that Chinese fir seedlings was small and growing slow in warming + nitrogen addition plots, which accord with the results of the above. Due to the decrease of nutrients with the soil layer, the soil enzyme activity decreased with the increase of soil layer [37–38]. But the difference was that soil enzyme activity at the 10–20 cm depth (except PHO), were significantly higher than that at the 0–10 cm depth in nitrogen treatment plots (Fig. 2). The reason may be MBC/MBN increased significantly compared with surface leading to nutrient composition changes. Relatively acidic and moisture deficiency soil condition may cause part of microbial species and structure changes so as to exert a positive influence on soil enzyme activity. In addition, nitrogen addition promoted the growth of Chinese fir, the roots of Chinese fir may also promote soil enzyme release. Our results were in contrast with some previous studies that nitrogen addition promoted soil enzyme activities greater in surface [39]. These contradictory results could indicate the different time of processing and the complexity of terrestrial ecosystems. 5. Conclusion After a short-term experiment, warming + nitrogen addition, compared with single factor of climate change, significantly reduced soil pH,

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water content, soil microbial biomass and lignin degrading enzymes, which aggravated soil acidification and water loss. Environmental factors affected by climate change changes were the main cause of soil microbial biomass and soil enzyme activity. In nutrient sufficient surface, pH and water is the most important factor of affecting soil enzyme activity, while in relative lack of nutrients subsurface, soil microbial biomass plays a major role. It must be said that our experiment time is shorter. And global climate change on forest soil exerts an lag influence and space-time variability. Simulation warming and nitrogen addition effect on soil microbial biomass and soil enzyme activities and on mutual mechanism of soil, microorganisms and plants remains to be studied further, so that scientifically revealing in subtropical Chinese fir plantations in response to global climate change and its feedback mechanism. Acknowledgments This research was funded by the National “973” Program of China (No. 2014CB954003), and the National Natural Science Foundation of China (31130013). We greatly thank reviewers for helpful comments on the manuscript. References [1] N.L. Zhang, J.X. Guo, X.Y. Wang, K.P. Ma, Soil microbial feedback to climate warming and atmospheric N deposition, Chin. J. Plant Ecol. 31 (2) (2007) 252–261. [2] Intergovernmental Panel on Climate Change. The Physical Science Basis, Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change, Cambridge University Press, Cambridge, 2013. [3] L.A. Martinelli, R.W. Howarth, E. Cuevas, S. Filoso, A.T. Austin, L. Donoso, V. Huszar, D. Keeney, L.L. Lara, C. Llerena, G. McIssac, E. Medina, J. Ortiz-Zayas, D. Scavia, D.W. Schindler, D. Soto, A. Townsend, Sources of reactive nitrogen affecting ecosystems in Latin America and the Caribbean: current trends and future perspectives, Biogeochemistry 79 (1–2) (2006) 3–24. [4] J.N. Galloway, A.R. Townsend, J.W. Erisman, M. Bekunda, Z.C. Cai, J.R. Freney, L.A. Martinelli, S.P. Seitzinger, M.A. Sutton, Transformation of the nitrogen cycle: recent trends, questions, and potential solutions, Science 320 (5878) (2008) 889–892. [5] Y.Y. Wei, H.J. Yin, Q. Liu, Y.X. Li, Advance in research of forest carbon cycling under climate warming, Chin. J. Appl. Environ. Biol. 15 (6) (2009) 888–894. [6] M.P. Waldrop, D.R. Zak, R.L. Sinsabaugh, Microbial community response to nitrogen deposition in northern forest ecosystems, Soil Biol. Biochem. 36 (9) (2004) 1443–1451. [7] J.M. Melillo, P.A. Steudler, J.D. Aber, K. Newkirk, H. Lux, F.P. Bowles, C. Catricala, A. Magill, T. Ahrens, S. Morrisseau, Soil warming and carbon-cycle feedbacks to the climate system, Science 298 (5601) (2002) 2173–2176. [8] C. Li, J.S. Storch, J. Marotzke, Deep-ocean heat uptake and equilibrium climate response, Clim. Dyn. 40 (5–6) (2013) 1071–1086. [9] T.E. Wood, M.A. Cavaleri, S.C. Reed, Tropical forest carbon balance in a warmer world: a critical review spanning microbial-to ecosystem-scale processes, Biol. Rev. 87 (4) (2012) 912–927. [10] S.L. Piao, J.Y. Fang, P. Ciais, P. Peylin, Y. Huang, S. Sitch, T. Wang, The carbon balance of terrestrial ecosystems in China, Nature 458 (7241) (2009) 1009–1013. [11] S.D. Chen, X.F. Liu, D.C. Xiong, W.S. Lin, C.F. Lin, L. Xie, Y.S. Yang, A preliminary study on effects of continuous active warming on soil respiration rates in Central sub-tropical forests, J. Subtrop. Res. Environ. 8 (4) (2013) 1–8. [12] M.R. Carter, E.G. Gregorich, Soil Sampling and Methods of Analysis, The Chemical Rubber Company Press, Florida, 1993 637–644. [13] R.L. Sinsabaugh, Phenol oxidase, peroxidase and organic matter dynamics of soil, Soil Biol. Biochem. 42 (3) (2012) 391–404. [14] X.Q. Zhou, C.R. Chen, Y.F. Wang, Z.H. Xu, J.C. Duan, Y.B. Hao, S. Smail, Soil extractable carbon and nitrogen, microbial biomass and microbial metabolic activity in response to warming and increased precipitation in a semiarid Inner Mongolian grassland, Geoderma 206 (2013) 24–31. [15] K.R. Saiya-Cork, R.L. Sinsabaugh, D.R. Zak, The effects of long term nitrogen deposition on extracellular enzyme activity in an Acer saccharum forest soil, Soil Biol. Biochem. 34 (9) (2002) 1309–1315.

[16] S. Hu, Y. Zhang, R.J. Shi, S.Q. Han, H. Li, H. Xu, Temporal variations of soil microbial biomass and enzyme activities during the secondary succession of primary broadleaved-Pinuskoraiensis forests in Changbai Mountains of Northeast China, Chin. J. Appl. Ecol. 24 (2) (2013) 366–372. [17] Y.L. Yang, Effects of Simulated Warming on Soil Microbial and Enzyme Activity in the Alpine Forests, Sichuang Agri. Univer., Ya'an, 2012. [18] C. Hamel, K. Hanson, F. Selles, A.F. Cruz, R. Lemkea, B. McConkeya, R. Zentnera, Seasonal and long-term resource-related variations in soil microbial communities in wheat-based rotations of the Canadian prairie, Soil Biol. Biochem. 38 (8) (2006) 2104–2116. [19] D. Johnson, J.R. Leake, J.A. Lee, C.D. Campbell, Changes in soil microbial biomass and microbial activities in response to 7 years simulated pollutant nitrogen deposition on a heathland and two grasslands, Environ. Pollut. 103 (2–3) (1998) 239–250. [20] Z.Y. Wang, F. Zhu, S.F. Su, M.M. Zhang, W.F. Duo, Q. Fu, Adcances in the effects of nitrogen deposition on soil microbiological characterisrics of forest ecosystem, J. Cent. S. Fore Tech. Univ. 31 (5) (2011) 202–206. [21] Y.C. Xu, Q.R. Shen, W. Ran, Effects of zero-tillage and application of manure on soil microbial biomass C, N, and P after sixteen years of cropping, Acta Pedol. Sin. 39 (1) (2002) 89–96. [22] W.Q. Yang, K.Y. Wang, Advances in forest soil enzymolog, Sci. Silvae Sin. 40 (2) (2004) 152–159. [23] J. Sardans, J. Peñuelas, M. Estiarte, Warming and drought alter soil phosphatase activity and soil P availability in a Mediterranean shrubland, Plant Soil 289 (1–2) (2006) 227–238. [24] H. Fang, J.M. Mo, Effects of nitrogen deposition on forest litter decomposition, Acta Ecol. Sin. 26 (9) (2006) 3127–3136. [25] J.L. Campbell, M.J. Mitchell, Groffman P.M. Mitchell, L.M. Christenson, J.P. Hardy, Winter in northeastern North America: a critical period for ecological processes, Front. Ecol. Environ. 3 (6) (2005) 314–322. [26] D.F. Cusack, M.S. Torn, W.H. McDowell, W.L. Silver, The response of heterotrophic activity and carbon cycling to nitrogen additions and warming in two tropical soils, Glob. Chang. Biol. 16 (9) (2010) 2555–2572. [27] X.Q. Zhou, C.R. Chen, Y.F. Wang, Z.H. Xu, H.Y. Han, L.H. Li, S.Q. Wan, Warming and increased precipitation have differential effects on soil extracellular enzyme activities in a temperate grassland, Sci. Total Environ. 444 (2013) 552–558. [28] P. Kardol, M.A. Cregger, C.E. Campany, A.T. Classen, Soil ecosystem functioning under climate change: plant species and community effects, Ecology 91 (3) (2010) 767–781. [29] D.A. Burns, The effects of atmospheric nitrogen deposition in the Rocky Mountains of Colorado and southern Wyoming, USA-a critical review, Environ. Pollut. 127 (2) (2004) 257–269. [30] J.E. Compton, L.S. Watrud, L.A. Porteous, S. DeGroodb, Response of soil microbial biomass and community composition to chronic nitrogen additions at Harvard forest, For. Ecol. Manag. 196 (1) (2004) 143–158. [31] J.D. Aber, A.H. Magill, Chronic nitrogen additions at the Harvard Forest (USA): the first 15 years of a nitrogen saturation experiment, For. Ecol. Manag. 196 (1) (2004) 1–5 (2004). [32] M.M. Carreiro, R.L. Sinsabaugh, D.A. Repert, D.F. Parkhurst, Microbial enzyme shifts explain litter decay responses to simulated nitrogen deposition, Ecology 81 (9) (2000) 2359–2365. [33] Y.T. Zhao, X.F. Li, S.J. Han, Y.L. Hu, Soil enzyme activities under two forest types as affected by different level of nitrogen deposition, Chin. J. Appl. Ecol. 19 (12) (2008) 2769–2773. [34] B. Berg, E. Matzner, Effect of N deposition on decomposition of plant litter and soil organic matter in forest systems, Environ. Res. 5 (1) (1997) 1–25. [35] J.L. DeForest, D.R. Zak, K.S. Pregitzer, A.J. Burton, Atmospheric nitrate deposition and the microbial degradation of cellobiose and vanillin in a northern hardwood forest, Soil Biol. Biochem. 36 (6) (2004) 965–971. [36] L.H. Tu, H.L. Hu, T.X. Hu, J. Zhang, Y.L. Xiao, S.H. Ge, H.R. Li, H.Z. Dai, Effects of simulated nitrogen deposition on soil enzyme activities in a Betula luminifera plantation in rainy area of West China, Chin. J. Appl. Ecol. 23 (8) (2012) 2129–2134. [37] S.W. Gong, T. Zhang, R. Guo, H.B. Gao, L.X. Shi, J.X. Guo, W. Sun, Response of soil enzyme activity to warming and nitrogen addition in a meadow steppe, Soil Res. 53 (3) (2015) 242–252. [38] Q. Zhang, W. Zhou, G.Q. Liang, J.W. Sun, X.B. Wang, P. He, Distribution of soil nutrients, extracellular enzyme activities and microbial communities across particle-size fractions in a long-term fertilizer experiment, Appl. Soil Ecol. 94 (2015) 59–71. [39] X.G. Song, T.X. Hu, J.R. Xian, C.L. Xiao, Soil enzyme activities and its response to stimulated nitrogen deposition in an evergreen broad-leaved forest, southern Sichuan, Acta Ecol. Sin. 29 (3) (2009) 1234–1240.