Plant functional groups, grasses versus forbs, differ in their impact on soil carbon dynamics with nitrogen fertilization

European Journal of Soil Biology 75 (2016) 79e87

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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Plant functional groups, grasses versus forbs, differ in their impact on soil carbon dynamics with nitrogen fertilization Jin Hua Li a, *, Ji Zhang a, Wen Jin Li a, Dang Hui Xu a, Johannes M.H. Knops b, Guo Zhen Du a a b

State Key Laboratory of Grassland Agro-Ecosystems, School of Life Sciences, Lanzhou University, Lanzhou 730000, PR China School of Biological Sciences, University of Nebraska, 348 Manter Hall, Lincoln, NE 68588-0118, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 October 2015 Received in revised form 22 March 2016 Accepted 26 March 2016

Nitrogen (N) addition in N-limited grasslands often increases aboveground productivity, decreases species richness and leads to changes in species composition. In contrast to these consistant results in aboveground vegetation parameters, there is no consistant pattern in how N fertilization affects soil organic carbon (SOC) dynamics. Our objectives were to test how plant functional group changes caused by N fertilization affect soil C dynamics and determine if different plant functional groups respond similarly. We conducted a two-factorial experiment to examine soil C dynamics with N fertilization and soil inoculation with field microbial communities in a greenhouse pot experiment. We used six plant species (two grasses and four forbs) that are dominant within sub-alpine meadows on the east part of the Qinghai-Tibetan plateau. For both grasses and forbs, N fertilization and soil inoculation, alone or in combination, decreased SOC by 4e10% and increased soil microbial biomass. For grasses, N fertilization combined with inoculation caused much lower SOC content and higher soil microbial biomass carbon (MBC) as compared to inoculation alone. In contrast to forbs, grass aboveground biomass was significantly negatively correlated with SOC change and positively correlated with MBC change. Nitrogen fertilization combined with inoculation significantly increased basal respiration and cumulative C mineralization rates for both grasses and three of the four forbs as compared to inoculation alone. Grasses had higher basal respiration rates than forbs under these two treatments. Despite higher aboveground grass biomass, N-fertilization lowered the SOC pool by increasing soil MBC and basal respiration rates, thus increasing C decomposition. Overall, in these sub-alpine meadows, grasses and forbs impact on soil C dynamics differs and grasses, but not forbs, may reduce soil C sequestration in response to N fertilization. © 2016 Elsevier Masson SAS. All rights reserved.

Handling editor: C.C. Tebbe Keywords: Soil organic C Soil microbial respiration Soil microbial biomass C mineralization N fertilization Grass

1. Introduction Many terrestrial ecosystems are N-limited [1], and N addition can dramatically increase productivity, decrease species richness and lead to changes in plant species composition [2e4]. However, the effects of N addition on soil organic carbon (SOC) dynamics and soil C sequestration differ between sites [4e8]. Some studies have reported that N fertilization can increase plant productivity and soil C sequestration [8e10] because of decreased SOC decomposition [11e14], linked with a decrease in microbial biomass and activity. Other studies have shown that N fertilization-induced increases in

* Corresponding author. E-mail address: [email protected] (J.H. Li). http://dx.doi.org/10.1016/j.ejsobi.2016.03.011 1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.

plant productivity do not necessarily result in increased soil C accrual [5,8]. A negative effect of N fertilizers on soil C sequestration may result from increased SOC decomposition caused by a “priming effect”, i.e. accelerated SOC decomposition caused by increased microbial biomass and/or activity because N-fertilization stimulates plant growth and thereby increases rhizodepositions and other plant residue inputs [15,16]. Grasslands may differ in plant diversity and dominance of functional groups, and their subsequent impact on soil C sequestration among sites [4,17]. High plant species richness can increase soil C sequestration [17,18]. However, such increase can largely be attributed to the presence and abundance of specific species or functional groups [17,19], rather than to species richness per se. For instance, in tallgrass prairie grasslands in North America, C4 grasses and legumes increased soil C accumulation by 193% and 522%,

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respectively [17]. Within alpine meadows, litter quality (e.g. N content and C:N) rather than litter quantity has been shown to drive soil C sequestration [20]. Plant species identity also can affect the soil microbial community size, composition and activity which in turn can influence grassland functions such as soil C storage [19,21]. Sub-alpine meadow is one of the dominant grassland types present in the Qinghai-Tibetan Plateau and because of its high SOC content may play an important role in global warming [22,23]. Nitrogen fertilization may decrease SOC [4,24]. Nitrogen fertilization at rates of 5, 10 and 15 g m
2 yr
1 decreased surface layer soil C accumulation by altering vegetation composition, i.e. increased grass dominance and decreased legume and forb biomass [4,25], which also caused a lower plant tissue C:N ratio [4]. However, little is known about how these plant functional groups influence soil C dynamics in response to N fertilization. We conducted a short term experiment with six plant species that are common in sub-alpine meadows to examine how different plant species from different functional groups affect soil C dynamics with N fertilization. We used sterilized soil inoculated with field microbial communities to examine the potential of plant C exudates to “prime” microbial growth and/or activity and determine soil C sequestration. We hypothesized, based on previous fertilization studies [5,8,15,16], that N fertilization and soil inoculation, alone or in combination, increase microbial biomass carbon (MBC), microbial respiration and C mineralization rates and as a consequence decrease SOC. And we hypothesized that functional groups differ in their effect on soil microbial biomass and activity, and thereby affect soil C storage and sequestration in sub-alpine meadows [19,21]. 2. Material and methods 2.1. Site description Soil for both the pot experiment and inoculum was collected from the surface layer 15 cm of a sub-alpine meadow with high plant diversity (plant species richness of 40e50 per 0.25 m2) [25] at the Research Station of the Alpine Meadow Ecosystem of Lanzhou University, located in Hezuo, Gansu, eastern Qinghai-Tibetan Plateau of China (N34 550 , E102 530 , 2900 m above sea level). Hezuo has a 30-year mean annual precipitation of 550 mm, with 85% of the precipitation occurring during the growing season from June to September (Institute of Hezuo Meteorology). The mean annual temperature is 2.4  C, ranging from
8.3  C during DecembereFebruary to 11.9  C during JuneeAugust periods. The soils in this area are chestnut soils or Haplic Calcisols according to the FAO classification or sub-alpine meadow soils according to the Chinese Soil Classification System [26]. The soil texture is 20% sand, 60% silt and 20% clay. The sub-alpine meadow is dominated by perennial sedge Kobresia humilis Serg (Cyperaeae). Common species include grasses, such as Stipa aliena, Elymus nutans Griseb, Festuca sinensis Keng ex S. L. Lu, forbs, such as Saussurea superba Anth., Gentiana lawrencei Burk. var farreri T.N.Ho, Gentiana straminea Maxim., Potentilla nivea Linn., Potentilla fragarioides L., Scirpus distigmaticus Tang et Wang; and Cyperaeae sedges such as Kobresia pygmaea C.B. Clarke in Hook, and Carex sp. 2.2. Preparation of soils and inoculum To examine the potential of plant C to “prime” microorganisms and their subsequent effect on soil C sequestration, soils collected from the field were sterilized and then inoculated with the soil microorganisms from the same sub-alpine meadow. Soils taken

from the top 15 cm (Ah horizon) of the sub-alpine meadow were sieved to 6 mm to remove stones and large plant particles, homogenized, and sterilized by g-irradiation (25 kGy). Soil MBC was measured before and after sterilization. Soil MBC after sterilization was 0.138 g kg
1, which was less than 5% of the mass prior to sterilization. The SOC content averaged 30.72 g kg
1. The soil sample used as inoculum was collected from the top 15 cm of the same sub-alpine meadow 3 days before the inoculation treatments. The microbial inoculum was made by adding 15 L of sterile MilliQ-filtered water to 15 kg fresh soil [27]. These mixtures were left for 5 h to enable large particles to sink, after which the supernatant was sieved through a 75 mm mesh, followed by two sievings through a 45 mm mesh and two sievings through a 30 mm mesh. This method omitted the micro-arthropods and nematodes but allowed most other microorganisms in the suspension to pass through [28]. 2.3. Pot experiment The pot experiment was carried out in a greenhouse with an average 16/8 h day/night cycle and a light:dark temperature regime of 20/12  C. Temperature conditions were regulated by computerized control of heating, vents, screens and a mobile air conditioning unit. The light regime was minimally 16 h/d of light, and natural daylight was supplemented with metal halide lamps (225 mmol m
2 s
1 photosynthetically active radiation) to ensure optimum light supply. Before transplanting seedling, 11 cm high pots with an inner diameter of 9e13 cm (base to top), sterilized with alcohol, were filled with 900 g g-irradiation sterilized soil and cultured for thirty days in the greenhouse. Soils were watered to field capacity every six days to ensure them not too loose. Seeds of six individual species (grasses Festuca sinensis Keng ex S. L. Lu, Elymus nutans Griseb, and forbs Lamium amplexicaule Linn., Aconitum gymnandrum Maxim, Elsholtzia densa Benth., and Potentilla fragarioides L.), collected at the field site, were sterilized with a 0.1% chloride solution for 3 min and sown on glass beads, moistened with demineralized water. For germination seeds were placed in a cabinet with a 16:8 h light:dark photo regime and an 18:10  C temperature regime. Prior to this procedure, a seed germination pre-experiment was carried out. Based on the differences among the species, sowing started at different times to ensure all species germinated at the same time and were of a comparable ontogenetic state at the start of the experiment. One week after germination, the seedlings were transplanted. Six seedlings of the same species were planted per pot. The pots were randomly divided among four treatments (four replicates per treatment): N fertilization and soil inoculation (N-M), N fertilization alone (N-O), soil inoculation alone (O-M), and a control with neither N fertilization nor soil inoculation (O-O). All pots were randomly placed on trolleys in a greenhouse. The positions of the trolleys were changed three times a week to minimize effects of microclimate differences within the greenhouse. Once planted, the N-M and O-M pots were injected with a total of 145 ml microbial inoculum originating from the field soil community or, as a control (N-O and O-O), 145 ml of sterile MilliQfiltered water at the first, second, and third day after transplanting. A week later, the N-addition treatments started: pots were watered 5 days a week for 9 weeks, each time with 25 ml of either sterile MilliQ-filtered water (no N addition; O-M and O-O pots) or NH4NO3 solution (N addition; N-M and N-O pots), at a rate of 2.1 g N m
2 yr
1. The approximate growing season in the Tibetan Plateau is about 5 months, and the N addition rate is comparable to the fertilization rate in previous field N fertilization experiments in the Tibetan Plateau [4,25]. We harvested the aboveground biomass and roots of all plants 10 weeks after planting. Aboveground

J.H. Li et al. / European Journal of Soil Biology 75 (2016) 79e87

biomass was oven-dried at 70  C, and weighed on a per pot basis. The roots were sieved, washed, oven-dried at 70  C, and weighed also on a per pot basis.

all treatments and for each plant species.

2.4. Soil sampling and analysis

3.1. Effect of N fertilization and inoculation on SOC and MBC changes

From each pot, 300 g of soil was collected after plant harvesting, sieved to 2-mm to remove roots, homogenized and divided into two portions. One portion was air dried and sieved to analyze SOC. The other portion was used immediately for analysis of MBC and C mineralization rate. Soil organic C was estimated using the dichromate oxidation method [29] on 0.2 g dry soil for each soil sample. Soil MBC was determined using the chloroform fumigation extraction method [30]. Three 25 g field-moist sub-samples were fumigated with alcohol-free CHCl3 for 24 h at 25  C. The samples were then extracted for MBC by adding 100 ml of 0.5 M K2SO4, shaked for 60 min and filtered through Whatman No. 2 paper. Three 25 g nonfumigated soil sub-samples were processed in the same manner. The carbon concentration in the extract was determined using the same method as for SOC, and MBC was calculated as the difference in C concentrations between fumigated and non-fumigated samples divided by an efficiency factor of 0.38 [30]. Soil organic C and MBC changes were calculated as the differences in their concentrations prior and after the pot experiment. Soil incubation for C mineralization was carried out in the laboratory for 28 days. The dry weight equivalent of 30 g of the sieved field-moist soil samples were weighed into 250 ml Schott jars. Small beakers filled with 15 ml of 1 M NaOH were placed at the soil surface in jars to trap the evolved carbon dioxide (CO2). The jars were fastened airtight and incubated for 28 days at 25  C. The moisture content of the samples was periodically adjusted to 30% of water holding capacity, as this amount is generally considered to mimic the moisture content of field conditions [31]. Constant soil water content was maintained by weighing each sample once a week and adding water to the soil. The CO2 evolved from the soil was measured at 1, 7, 14, 21, and 28 day after incubation. At each measurement day, the small beaker with NaOH solution was removed and replaced by a new one with fresh NaOH. The CO2 absorbed in the NaOH solution was titrated with 0.1 M HCl after the addition of BaCl2. A NaOH solution without soil, incubated as above, was also titrated. Basal respiration was calculated as the rate of C mineralization after the first day (24 h) of incubation. The cumulative C mineralization was calculated as the sum of CO2 released during the 28 days of incubation. 2.5. Statistical analysis The data were analyzed using SPSS 16.0 and graphs were plotted using Sigma Plot 10.0. Three-way ANOVA was used to test the effects of plant species, N fertilization and inoculation on plant aboveground and root biomass (Table A1, Fig. A1), SOC, soil MBC, and C mineralization rates, and the least significant difference (LSD) test was used to separate means at P ¼ 0.05. When the 3-way interaction was significant, the 3-way analysis was divided into 2way analyses of N fertilization and inoculation for each species. The per family rate error critical value was used to tell which were statistically significant in the 2-way analyses. Because of the variation in SOC after different treatments (Fig. 1a), C mineralization rate was expressed per unit of SOC present within each treatment. A regression analysis was used to analyze the relationship between plant biomass and soil MBC and SOC changes for all treatments and for each plant species. A regression analysis was also used to analyze the relationship between the cumulative C mineralization rates at the end of incubation (28th day) and soil MBC and SOC for

81

3. Results

Nitrogen fertilization, inoculation, and plant species had significant 3-way interactions on SOC and MBC changes (Table 1). The 2-way analyses at each species level showed that N fertilization or inoculation alone had significant impacts on SOC and MBC changes, except for F. sinensis and E. densa for which N fertilization alone had no significant impact on MBC (Table 2; Fig. 1). The interaction of N fertilization and inoculation had significant effects on SOC change for both grasses and forbs (except L. amplexicaule), and significant effects on MBC change for grasses and two forbs of L. amplexicaule and A. gymnandrum (Fig. 1a; Table 2). Soil organic C under N fertilization and soil inoculation, alone or in combination, decreased by 4e10% for most plant species as compared to the SOC concentration prior to the pot experiment. Grasses had higher SOC decrease under N-M treatment than O-M treatment. However, there was no consistent pattern for forbs. SOC increased for forbs of A. gymnandrum under N-M and O-M treatments, P. fragarioides under NeO treatment (Fig. 1a). Soil MBC increased compared to the value prior to pot experiment for both grasses and forbs (Figs. 1b, Fig. A2). There was no significant difference in soil MBC change among plant species under the O-O treatment. Nitrogen fertilization combined with inoculation (N-M) had much higher soil MBC than inoculation alone (O-M) for grasses and one forb, P. fragarioides whereas lower MBC for forb of A. gymnandrum. In general, plant aboveground biomass was negatively correlated with SOC change whereas positively correlated with MBC change (Fig. A3). However, on species level, relationships between plant aboveground biomass and SOC change or MBC change were significant only for grasses F. sinensis and E. nutans (Fig. 2). 3.2. Effect of N fertilization and inoculation on C mineralization rate Nitrogen fertilization, inoculation, and plant species had significant 3-way interactions on soil C mineralization rates (Table 1). Two-way ANOVA analyses at species level showed N fertilization or inoculation individually had significant effects on both basal respiration rates and C cumulative mineralization rates for four of the six species (except one grass and one forb, E. nutans and E. densa) (Table 2). The interaction of N fertilization and inoculation had significant effects on basal respiration rates (Fig. 3; Table 2) and cumulative C mineralization rates (Fig. 3; Table 2) for both grasses and forbs (except for E. densa). Soil organic C mineralization rates decreased during the incubation period. However, the differences were statistically insignificant among treatments over the incubation between 7 days and 28 days. Grasses had higher basal respiration rates than forbs under N-M and O-M treatments. N fertilization combined with inoculation treatment led to the highest C mineralization rates for both grasses and forbs (except A. gymnandrum), whereas the N-O treatment led to the lowest C mineralization rates for grass of E. nutans, and forbs of L. amplexicaule, and P. fragarioides (Fig. 3). The cumulative C mineralization rates for all treatments at the end of the 28 days incubation ranged from 61 to 114 mg kg
1 SOC (Fig. 4). Nitrogen fertilization combined with inoculation (N-M) had much higher cumulative C mineralization rates than N-O treatment for both grasses and forbs whereas higher cumulative C mineralization rates than O-M treatment for grasses and forbs (except E. densa) (Fig. 4).

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Fig. 1. Effects of nitrogen fertilization and soil inoculation on SOC and MBC changes, which were calculated as the differences in their concentrations prior and after the pot experiment (mean ± SE, n ¼ 4) after planting with 6 different plant species. Different letters above/below bars for the same panel indicate significant differences between treatments at P ¼ 0.05. Abbreviations: SOC soil organic C, MBC microbial biomass C, N-M nitrogen fertilization and soil inoculation, N-O nitrogen fertilization alone, O-M soil inoculation alone, O-O neither nitrogen fertilization nor soil inoculation.

Table 1 Three-way ANOVAs examining the impact of N fertilization (N), inoculation (M) and plant species (S) effects and their interactions on soil organic C (SOC) and microbial biomass C (MBC) changes, and C mineralization rates. SOC and MBC changes were calculated as the differences in their concentrations prior and after the pot experiment. Source

Overall R2 N M S N *M N* S M* S N* M* S

df

1,69 1,69 5,69 1,69 5,69 5,69 5,69

SOC change

MBC change

C mineralization rates Day1

Day7

Day14

Day21

Day28

Cumulative

F

F

F

F

F

F

F

F

0.98 0.34 ns 59.09*** 220.86*** 24.94*** 209.11*** 209.74*** 101.06***

0.95 148.60*** 475.23*** 35.63*** 10.81** 27.85*** 35.07*** 8.86***

0.98 0.82ns 1566.80*** 160.79*** 348.30*** 38.18*** 134.64*** 26.44***

0.97 146.60*** 764.61*** 80.42*** 180.56*** 18.44*** 40.77*** 20.85***

0.86 4.01* 65.18*** 32.08*** 3.91 ns 13.42*** 16.46*** 4.07**

0.87 0.02 ns 11.73** 4.93** 22.55*** 22.22*** 48.65*** 5.23***

0.88 0.13 ns 39.39*** 20.16*** 95.51*** 52.30*** 4.27** 9.84***

0.96 37.12*** 696.4*** 68.08*** 15.23*** 4.29** 54.49*** 7.72***

*P < 0.05; **P < 0.01; ***P < 0.001; ns P > 0.05.

Table 2 Two-way ANOVA examining the impact of N fertilization (N) and inoculation (M) effects and their interactions on soil organic C (SOC) and microbial biomass C (MBC) changes, basal respiration rates, and C cumulative mineralization for six plant species. SOC and MBC changes were calculated as the differences in their concentrations prior and after the pot experiment. Functional groups

Species

Source

df

SOC change

MBC change

Basal respiration rates

Cumulative mineralization rates

Grasses

Festuca sinensis

N M N *M N M N *M N M N *M N M N *M N M N *M N M N *M

1,12 1,12 1,12 1,12 1,12 1,12 1,12 1,12 1,12 1,12 1,12 1,12 1,12 1,12 1,12 1,12 1,12 1,12

30.51*** 27.32*** 11.2** 298.11*** 224.63*** 95.52** 349.93*** 2.71ns 2.51ns 192.98*** 800.89*** 189.36** 18.10** 255.06*** 13.54** 65.42*** 14.98*** 231.51**

0.35ns 307.81*** 411.03*** 145.86*** 431.80*** 10.08* 13.69** 22.33** 18.75** 8.58* 47.50*** 34.53** 0.16ns 364.77*** 0.52ns 169.70*** 5.49* 0.43ns

46.04*** 224.68*** 73.93** 0.14ns 565.46*** 31.39** 94.08*** 117.12*** 95.27** 8.30* 31.61*** 66.86** 0.69ns 474.09*** 0.95ns 97.94*** 1312.04*** 230.47**

13.33** 340.20*** 13.45** 3.97ns 332.95*** 4.29* 70.55*** 1237.36*** 6.10* 5.93* 7.06* 24.12** 2.60ns 33.59*** 3.85ns 116.13*** 271.99*** 21.35**

Elymus nutans

Forbs

Lamium amplexicaule

Aconitum gymnandrum

Elsholtzia densa

Potentilla fragarioides

*P < 0.05; **P < 0.01; ***P < 0.001; ns P > 0.05.

J.H. Li et al. / European Journal of Soil Biology 75 (2016) 79e87

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Fig. 2. Relationships between SOC changes or MBC changes and plant aboveground biomass. Soil organic C changes or MBC changes were calculated as the differences in their concentrations prior and after the pot experiment (n ¼ 4) after planting with 2 grasses species. Abbreviations: SOC soil organic C, MBC microbial biomass C, N-M nitrogen fertilization and soil inoculation, N-O nitrogen fertilization alone, O-M soil inoculation alone, O-O neither nitrogen fertilization nor soil inoculation.

3.3. Relationships between soil properties and C mineralization parameters In general, cumulative C mineralization rate was negatively correlated with SOC whereas positively correlated with soil MBC (Fig. A4). Relationships between cumulative C mineralization rates and SOC or MBC were similar for the two grasses. However, there were some differences among the forbs (Table 3). In particular, the relationship between cumulative C mineralization rates and SOC for L. amplexicaule was insignificantly positive. The relationship between cumulative C mineralization rates and MBC for P. fragarioides was insignificantly positive while significantly negative for A. gymnandrum. 4. Discussion 4.1. Soil microbial biomass and C mineralization rates We found that soil MBC increased under N-M compared to O-M treatment as predicted which was consistent with studies by Chu et al. [16] and Reid et al. [8]. The increase in soil MBC with N fertilization and inoculation combination was likely promoted by plant growth (Fig. A3, Fig. 2) or the alleviation of microbial Nlimitation [32]. The increased soil MBC under O-O treatment for all plant species indicated that there was potential of plant C to “prime” microbial growth. The similar MBC changes for O-O and NO treatments of F. sinensis and E. densa suggested that microbial growth induced by these two species was not N limited. The increase in MBC was not consistent with a meta-analysis of ecosystem studies which has shown that microbial biomass on average declined by 15% with N fertilization because N addition

negatively affected microbial growth [33]. However, experimental studies examining MBC after N addition are mixed, some showing a decrease [13,14,34], others showing an increase [8,15,16]. Inoculum from field soil has been shown to contain beneficial microorganisms [35,36]. These microorganisms have indirect positive effects on plants by affecting the population density, dynamics and metabolic activities of soil-borne pathogens, or direct positive effects on plants exerted by rhizosphere microorganisms through a phytostimulation and a biofertilization of plants [35,36]. In our study, inoculation with field microbial community might have simply increased the number of viable cells in the soil, which could increase microbial growth and MBC. Inoculation might also favor beneficial microorganisms and microbial community functional diversity, promoting plant growth and resulting in improved MBC. However, their effect on plant growth and soil MBC was reported to be species-specific [37], which was in accordance to our study. Our study revealed similar patterns of reduced basal respiration rates and cumulative C mineralization rates (Fig. 3; Fig. 4) under N fertilization alone compared O-O treatment for most plant species, whereas the inverse was observed for MBC (Fig. 1b). The increase in microbial biomass (Fig. 1b) but decrease in basal respiration rates after N fertilization alone compared to O-O treatment for one grass and three forbs (Fig. 3) and the identical or reduced cumulative C mineralization for all grasses and two forbs (Fig. 3) suggested increasing microbial C use efficiency (CUE) through N fertilization as predicted by Schimel and Weintraub [38]. This might be due to a mechanism that adding N alleviates N limitation, which allows microorganisms to divert C from overflow metabolism into producing microbial biomass [38]. The absence of changes in the soil MBC and C mineralization rate with N fertilization alone compared to O-O treatment for

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Fig. 3. Effects of nitrogen fertilization and soil inoculation on SOC mineralization rates (mean ± SE, n ¼ 4) after planting with 6 different plant species. Abbreviations: SOC soil organic C, N-M nitrogen fertilization and soil inoculation, N-O nitrogen fertilization alone, O-M soil inoculation alone, O-O neither nitrogen fertilization nor soil inoculation.

F. sinensis might result from reduced endophyte richness and fungal dominance due to increased N availability [39], as well as depressed microbial growth and metabolism [13,14,34]. F. sinensis is a graminoid species adapted to nutrient-poor soil [40,41], which harbor endophytic fungi that may facilitate resource acquisition in nutrient-poor environments [42]. Studies have shown that soil N enrichment (by chronic and/or high N inputs) was linked to a decline in soil lignin-degrading enzyme activity and/or fungal population densities, which were primarily responsible for lignin degradation [43,44], and that a decrease in mineralization of SOC resulted from modification of the structure and composition of soil microbial communities by increased N availability [45]. The low basal respiration for the forbs L. amplexicaule and E. densa and the high cumulative C mineralization rates with N fertilization alone might be due to the stimulation of carbondegrading enzyme activities by higher N availability [46,47], or through shifts in microbial enzymes [44]. In our study, we did not measure soil microbial community composition or enzyme activities; however, in a recent field study at the same site we found that N fertilization increased microbial functional diversity and modified microbial carbon utilization patterns and metabolism, resulting in a change in C mineralization rates [4]. The higher soil MBC, basal respiration and cumulative C mineralization rates for most plant species when N fertilization and

soil inoculation were combined, compared to soil inoculation alone, were consistent with our hypothesis. However, the findings of higher MBC under N fertilization and inoculation in combination than in treatment with inoculation alone were not consistent with other grasslands experiments showing that N addition decreased soil MBC and microbial respiration [13,14,34]. Our contrasting results can be because of a variety of reasons, such as differences in N fertilization rate, plant diversity and vegetation type, soil organic matter content or initial grasslands soil N content [48,49]. In our study, the higher soil MBC and C mineralization rates under N fertilization and soil inoculation in combination could reveal a greater microbial growth after inoculation and increased microbial demand for N. Nitrogen fertilization intensified the effects of inoculation alone on soil microbial biomass, microbial respiration and C mineralization rates. Our recent field study also showed that continuous N fertilization at 5 and 10 g m
2 yr
1 increased soil surface MBC and microbial functional diversity [4]. These results suggested that N fertilization might stimulate soil microbial biomass and microbial mineralization in such sub-alpine meadows with high SOC content raging from 30.7 to 33.8 g kg
1 [4]. 4.2. Soil organic C Some studies have reported that inorganic fertilizer application

J.H. Li et al. / European Journal of Soil Biology 75 (2016) 79e87

Fig. 4. Effects of nitrogen fertilization and soil inoculation on cumulative SOC mineralization rates (mean ± SE, n ¼ 4) after 28 days of incubation. Different letters above bars for the same panel indicate significant differences between treatments at P ¼ 0.05. Abbreviations: SOC soil organic C, N-M nitrogen fertilization and soil inoculation, N-O nitrogen fertilization alone, O-M soil inoculation alone, O-O neither nitrogen fertilization nor soil inoculation.

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We previously also found at the same site, in the field a decrease in plant C:N ratios with 6-year N fertilization, caused by dominance changes among the functional groups grasses and forbs [4]. However, our experiment lasted only about 2 months and there was little litter incorporated into the soil. The high C losses might be partially ascribed to low CUE, indicated by higher basal respiration and cumulative C mineralization rates under N-M treatment than O-M treatment (Figs. 3 and 4). Low CUE resulted in large C losses (through respiration and exudation) and reduced SOC concentration and potential C storage, as reviewed by Manzoni et al. [51]. Experimental conditions, such as the higher temperature than outside in the field, or other factors, such as leaching, could also be responsible for the high C losses. By using 13C or 14C isotope to measure CO2 fluxes, further studies could determine how much the mineralization or leaching contributed to C losses. In total, SOC were much lower under N fertilization and inoculation in combination (which was the simulation of N fertilization in the field study) than that under inoculation alone for both the grasses F. sinensis and E. nutans. With N fertilization, these two grasses, especially E. nutans, become dominant at our study site. Thus, sub-alpine meadow SOC may decrease in response to N fertilization, because of the increased grass biomass and increased basal respiration. However, other studies have found different impact of grasses on SOC. For instance, Soudzilovskaia et al. [52] found an increase of SOC due to an increase of grass aboveground

Table 3 Linear regressions between cumulative C mineralization rates and SOC or MBC. Abbreviations: SOC soil organic C, MBC microbial biomass C. Functional groups

Plant species

Cumulative C mineralization rates and SOC Model

Grasses Forbs

Festuca sinensis Elymus nutans Lamium amplexicaule Aconitum gymnandrum Elsholtzia densa Potentilla fragarioides

Y Y Y Y Y Y

¼ ¼ ¼ ¼ ¼ ¼

684.39e20.31 X 213.38e4.08 X 27.10 þ 4.06 X 135.16e1.99 X 371.56e10.14 X 242.97e5.20 X

can cause a significant increases in SOC due to the positive effects of fertilizer on plant growth and, in turn, C inputs to the soil [50], combined with the negative effect of N fertilizer on litter decomposition and soil microbial respiration [12,34]. In southern Californian semiarid shrublands, chronic N inputs increased soil C storage [34] suggesting N fertilization could be used to sequester more atmospheric C in soils [50]. However, in our study, SOC decreased with N fertilization and inoculation combination treatments for five of the six species, in particular for the two grasses, and under inoculation alone for five species (Fig. 1a). Nitrogen fertilization and inoculation combination significantly increased plant aboveground biomass of grasses, instead of forbs, compared to treatment with soil inoculation alone (Fig. A1; Table A2). Yet, there was a general negative correlation between SOC change and plant aboveground biomass (Fig. A3a). The overall decrease of SOC contents under N fertilization and soil inoculation, alone or combined, was consistent with our hypothesis. This SOC reduction was partially ascribed to the increasing microbial biomass and C decomposition rates. The decreased SOC and basal respiration, and the restrained cumulative C mineralization rates under N fertilization alone relative to the O-O treatment were most likely resulting from the fact that the amount of C input to the soil by plant roots might be smaller than the amount of microbial C mineralization. The N fertilization-induced decrease in SOC concentration was likely caused by changes in plant biomass and in the plant C:N ratio.

Cumulative C mineralization rates and MBC

R2

p

Model

0.487 0.419 0.112 0.510 0.578 0.628

<0.01 <0.01 >0.05 <0.01 <0.01 <0.001

Y Y Y Y Y Y

¼ ¼ ¼ ¼ ¼ ¼

52.16 þ 21.37 X 72.26 þ 9.76 X 53.12 þ 17.16 X 86.83e7.68 X 66.99 þ 16.98 X 57.65 þ 4.48 X

R2

p

0.909 0.774 0.648 0.566 0.728 0.198

<0.001 <0.001 <0.001 <0.001 <0.001 >0.05

biomass and litter quality with N addition. Foranara and Tilman [17] also attributed the increase in soil C sequestration to the presence and abundance of the functional groups legumes and C4 grasses. One potential explanation of the differences between our study and these studies could be related to plant community composition and plant traits. Plant communities, in terms of both biomass and diversity, changed from forbs dominated to grasses and forbs dominated after N fertilization at our site [4,25]. We also recently found in a field study at the same site that grasses may reduce soil C sequestration in sub-alpine meadows on the Qinghai-Tibetan Plateau in response to N fertilization by altering the plant biomass composition and modifying the soil microbial communities C substrate utilization patterns [4]. These results were consistent with studies that have shown an increase in plant productivity and potential input of organic carbon into the soil after N fertilization [7,9], however, these changes did not result in increased soil C accrual [8]. Therefore, composition of plant functional groups and changes in their dominance in response to N addition may result in differential soil C sequestration. 5. Conclusions In this study, we demonstrated using a two factorial experiment with N fertilization and inoculation treatments for six plant species that grasses and forbs differed in their impacts on soil organic SOC dynamics. For both grasses, nitrogen fertilization decreased SOC

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but increased MBC. For five of the six species, fertilization increased basal respiration and cumulative C mineralization rates. Grasses had higher basal respiration rates than forbs with N fertilization. These findings combined with a field study suggested that N fertilization will lower SOC sequestration within sub-alpine meadows on the Qinghai-Tibetan Plateau by increasing grass dominance because of the grass induced increasing soil MBC and basal respiration rates, which increases soil C decomposition.

[21] [22]

[23] [24]

Acknowledgments

[25]

This research was supported by the Key Program of National Natural Science Foundation of China (No.41430749), the National Natural Science Foundation of China (No.31470480, No.30871823), and the Program for New Century Excellent Talents in University (NCET-11-0210). We are grateful to Prof. Nancy Collins Johnson for useful comments on the manuscript.

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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejsobi.2016.03.011.

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