Spatio-temporal variations determine plant–microbe competition for inorganic nitrogen in an alpine meadow
Key Laboratory of Ecosystem Network Observation and Modelling, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, 11A Datun Road, Chaoyang District, Beijing 100101, China
Key Laboratory of Ecosystem Network Observation and Modelling, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, 11A Datun Road, Chaoyang District, Beijing 100101, China
1. Plant–microbe competition for available nitrogen (N) has been suggested to be an important mechanism controlling N limitation of plants in a variety of ecosystems. However, spatio-temporal patterns of competition between plants and microbes for soil N remain unclear.
2. Short-term 15N tracer experiments were conducted during a growing season (July, August and September) in an alpine meadow on the Tibetan Plateau to unravel spatio-temporal patterns of plant–microbe competition for NH4+ and NO3−.
3. Alpine plants were poorer competitors than soil microorganisms for inorganic N in July compared with August and September. Occupation of soil volume by roots and root density (high in August and September) played a greater role in plant–microbe competition than air temperature or precipitation (high in July).
4. In topsoils (0–5 cm, highest root density), alpine plants effectively competed with soil microorganisms for N and showed a preference for 15NO3−, while soil microorganisms that preferentially took up 15NH4+ out-competed plants below 5 cm soil depth (lower root density). Competition between plants and soil microorganisms for inorganic N strongly depended on root density (P < 0.0001, R2 = 0.93, exponential decay model).
5.Synthesis. Plant–microbe competition for inorganic N showed a clear spatio-temporal pattern in alpine meadows depending on (i) root density and therefore soil depth, (ii) inorganic N form, and (iii) different periods during the growing season. These findings have important implications for our understanding of above-ground–below-ground interactions and plant–microbial competition for available N.
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The Tibetan Plateau has been regarded as ‘the third pole of the Earth’ (Qiu 2008). The low temperature at this high altitude depresses soil organic matter decomposition, but does not affect N immobilization by microorganisms (Song et al. 2007). Thus, the inorganic N concentration in these meadows is low, and plant growth is strongly limited by available N (Zhou 2001). Although one of our previous studies showed that organic N may be a significant N source for alpine plants (Xu et al. 2006), inorganic N contributed more than 80% to plant N nutrition. Although concentrations of dissolved organic N (DON) were slightly higher than dissolved inorganic N (DIN) in the alpine meadow soils (Table 1), the largest fraction of DON is not directly available for microorganisms and roots (Blagodatskaya et al. 2009). We therefore focused only on inorganic N uptake by plants and microorganisms in this study. The inorganic N concentration in the topsoil also showed a clear seasonal pattern, increasing in early July and in mid-August, but decreasing in late July (Zhou 2001). Interactions between plant species can mediate the competition for inorganic N with soil microorganisms (Song et al. 2007), indicating strong competition for available N during the growing season in alpine meadows.
Table 1. Characteristics of the upper 10 cm of soils at the study site. Means ± 1 SE are shown (n = 6–8). Dissolved organic N (DON) measured as total dissolved N minus dissolved inorganic N (DIN). Data from Xu et al. (2006)
8.0 ± 0.1
Bulk density (g cm−3)
0.70 ± 0.05
12.8 ± 0.2
Soil organic C (%)
7.06 ± 0.37
Total soil N (%)
0.55 ± 0.03
Microbial biomass N (g N m−2)
6.5 ± 0.3
DON (g N m−2)
1.8 ± 0.1
DIN (g N m−2)
1.4 ± 0.4
We here performed a short-term 15N tracer experiment to investigate the temporal and spatial competition for NH4+ and NO3−. We tested the following three hypotheses, i.e. alpine plants compete more efficiently than soil microorganisms
1 for inorganic N in the topsoil (0–5 cm) compared with the soil layers from 5 to 15 cm (defined here as the subsoil), because root density is much higher in the topsoil than in the subsoil (Zhou 2001; Tao et al. 2006);
2 for inorganic N in the middle of the growing season compared with later stages because of higher growth rates and subsequent higher root production in the middle of the growing season related to high temperature and rainfall;
The experiment was conducted at the Haibei Alpine Meadow Ecosystem Station of the Chinese Academy of Sciences, Qinghai Province (37°36′60″ N, 101°19′14″ E, 3215 m a.s.l.). The area is located in the typical alpine meadow zone and climate. Average annual temperature and annual precipitation were −1.7 °C and 600 mm, respectively, during the past 25 years. Average July temperature and rainfall were c. 10.0 °C and 110 mm, respectively. The dominant plant species are Kobresia humilis Serg., Stipa aliena Keng., Poa sp., Festuca ovina Linn., Gentiana aristata Maxim, Gentiana straminea Maxim., Saussurea superba Anth., and Gueldenstaedtia diversifolia Maxim. (Zhou 2001). The soil is classified as Mat-Gryic Cambisol (Chinese Soil Taxonomy Research Group 1995), corresponding to Gelic Cambisol (WRB 1998). An overview over soil properties are presented in Table 1 (Xu et al. 2006).
A 25 × 25 m area, uniform in cover and species composition, was selected in a K. humilis meadow in 2004. In July, August and September, 90 circular microplots (diameter 10 cm) were randomly set up. These microplots were equally divided into two groups: 15NH4+ (injected with 15NH4NO3) and 15NO3− (injected with NH415NO3). Each group included three soil depth treatments (0–5, 5–10 and 10–15 cm) and three sampling times (4, 24 and 48 h after 15N addition), with five replicates per treatment. 15N tracers (98.2%15N enrichment for 15NO3− and 98.4% enrichment for 15NH4+) were dissolved in H2O and injected at 2.5 cm depth for the 0–5 cm soil depth treatment, at 7.5 cm for the 5–10 cm soil depth, and at 12.5 cm for the 10–15 cm soil depth. Each microplot was injected with 7 mL 15N solution, and the added amount corresponded to 0.32 g N m−2. In total, a four-factor design was constructed: the first factor was N form (15N in NO3− or NH4+); the second factor was season (July, August or September); the third factor was sampling time after 15N injection (4, 24 or 48 h); and the fourth factor was soil depth of 15N injection (2.5, 7.5 or 12.5 cm).
Sampling and analyses
Four, 24 and 48 h after 15N tracer injection, above-ground plant parts were collected using scissors. Soil samples were collected down to 15 cm depth and cooled immediately to 4 °C. Roots were carefully separated from soils and rinsed first with tap water, then for 30 min with 0.5 mmol L−1 CaCl2 solution and again with distilled water. Above-ground plant parts and roots were dried at 75 °C for 48 h, weighed for dry mass and ground to a fine powder using a ball mill (MM2, Fa. Retsch, Haan, Germany) for measuring N content, and 15N:14N ratios. Fresh soil was sieved to 2 mm and stored at −20 °C for later measurements. Aliquots (2 mg) of plant materials were weighed into tin capsules to analyse total N, C and 15N:14N ratios by continuous-flow gas isotope ratio mass spectrometry (MAT253, Finnigan MAT, Bremen, Germany), coupled by ConFlo III device (Finnigan MAT, Bremen, Germany) to an elemental analyser (EA 1112, CE Instruments, Milan, Italy).
15N incorporation into microbial biomass was determined by chloroform fumigation (Brookes et al. 1985). Twenty grams of fresh soil were fumigated with chloroform for 24 h and then immediately extracted with 60 mL 0.5 m K2SO4. An additional soil sample was extracted without fumigation.
Calculation and statistics
15N atom% excess (APE) was calculated as the atom%15N difference between treated and control plants. 15N recovery by plants was calculated by multiplying the N content in the pool by its mass per square meter and APE, divided by total added 15N per square meter. 15N recovery by microbial biomass was calculated as the difference in 15N mass between fumigated and non-fumigated soil samples, divided by total added 15N per square meter (Zogg et al. 2000). In this study we did not apply a KEN factor to correct microbial 15N uptake for incomplete extraction of microbial N in the chloroform-fumigation samples for two main reasons. First, Jenkinson (1988) suggested that the correction factor should not be used when N immobilization controlling microbial activity is variable. In the alpine meadows N immobilization was the dominant process of soil N cycling, exceeding N mineralization by far (Song et al. 2007), and N immobilization varied between seasons. Secondly, as has been argued before (Hofmockel, Schlesinger & Jackson 2007), a recovery coefficient should not be applied in such studies due to the uncertainties associated with temporal variations in the extractability of N and the variability in incorporation efficiency into the cytoplasmic (soluble) vs. structural (insoluble) components (Bremer & van Kessel 1990). Therefore, the results given here represent a conservative estimate of the microbial biomass pool and isotope content. To consider a possible bias and for purpose of comparison with the conservative estimate, we also calculated the plant–microbe competition using the KEN factor of 0.54 (Brookes et al. 1985) to correct microbial 15N uptake for incomplete extraction (see Discussion section; termed ‘speculative estimate’, Fig. 4). The competition between plants and soil microorganisms for N was referred to as the ratio of 15N recovery by microbial biomass to 15N recovery by plants.
The standard errors of means are presented in figures and tables as a variability parameter. Multivariate anova was calculated to estimate the effects of N form, season, sampling time, soil injection depth, and their interactions on 15N recovery by microbial biomass, 15N recovery by plants and the ratio of 15N recovery by microbial biomass to 15N recovery by plants using the spss 16.0 software package (SPSS Inc., Chicago, IL, USA). The contribution of the factors and their interactions to the total variance was calculated by dividing the respective type III sum of squares by the total sum of type III sum of squares from multifactorial anova. All differences were tested at P < 0.05. Results of regression analysis of root biomass (or microbial biomass) vs. ratios of 15N recovery by microbial biomass to 15N recovery by plants were calculated by the spss 16.0 software package.
Spatio-temporal patterns of 15N recovery by microbial biomass from 15NH4+ and 15NO3−
There was a spatio-temporal effect on 15N uptake by microorganisms from 15NH4+ and 15NO3− in alpine meadows on the Tibetan Plateau (Table 2, Fig. 1 and also see Fig. S1 in Supporting Information): 15N recovery by microbial biomass showed a different pattern with increasing soil depth of 15N injection at different times during the growing season. In July, 15N recovery by microbial biomass from 15NH4+ and 15NO3− showed similar, but lower values in the two upper soil layers (0–5 cm and 5–10 cm depth), but higher values at 10–15 cm depth (Fig. 1; P < 0.05 for 15NH4+ and P < 0.05 for 15NO3−). In August, 15N recovery from 15NO3− remained constant with increasing soil depth (P = 0.19), whereas recovery from 15NH4+ declined with increasing soil depth of 15N injection (P < 0.005). Recovery from 15NH4+ and 15NO3− differed significantly in the two top soil layers (Fig. 1; P < 0.05). In September, recovery by microbial biomass from 15NO3− did not significantly change with increasing injection depth (P = 0.56), while recovery from 15NH4+ exhibited a pattern similar to that shown in July (Fig. 1; P < 0.05). In topsoil, recovery from 15NH4+ was significantly higher than from 15NO3− (Fig. 1; P < 0.05).
Table 2. Multifactorial analysis of variance for the effects of 15N form added, sampling time, season, soil injection depth and their interactions on 15N recovery by microbial biomass and by plants. The competition for 15N between plants and soil microorganisms is presented as ratio of 15N recovery by microbial biomass to 15N recovery by plants. P values for significant effects and interactions are in bold
Source of variation
15N recovery by microbial biomass
15N recovery by plants
Ratio of 15N recovery by microbial biomass to 15N recovery by plants
Time × Season
Time × Depth
Time × Forms
Season × Depth
Season × Forms
Depth × Forms
Time × Season × Depth
Time × Season × Forms
Time × Depth × Forms
Season × Depth × Forms
Time × Season × Depth × Forms
Spatio-temporal patterns of 15N recovery by plants from 15NH4+ and 15NO3−
15N uptake by plants from 15NH4 and 15NO3− also showed a clear spatio-temporal pattern (Figs. 2 and S2; Table 2). While 15N recovery decreased with increasing soil depth (P < 0.05), the decrease differed during the growing season (Table 2). In July, 15N uptake was significantly higher at 0–5 cm depth compared with deeper soil layers (P < 0.001), and was significantly higher for 15NH4+ versus 15NO3− (Fig. 2; P < 0.05). In August these values decreased with increasing soil depth (Fig. 2; P < 0.005). Throughout the growing season, uptake from 15NO3− was higher than from 15NH4+ (Figs 2 and S2, Table 2). 15N recovery in plants increased significantly with time after tracer injection, from 4 to 48 h after labelling (Table 2).
Spatio-temporal changes in ratios of 15N recovery by microbial biomass to 15N recovery by plants
Multifactorial anova indicated significant effects of soil depth, 15N form added, season and sampling time on the 15N recovery ratio between microbial biomass and plants (Figs 3 and S3, Table 2). Besides these direct effects, the interactions between the four factors also significantly affected these ratios (Table 2). In July, recovery ratios were lower in the topsoil but higher at both 5–10 cm to 10–15 cm depth (Fig. 3; P < 0.05). The values at both subsoil layers were similar for 15NH4+ and 15NO3−, but significantly higher for the former than for the latter in the same soil layer (Fig. 3; P < 0.05). In August, the ratios increased with soil depth (P < 0.005) and were significantly higher for 15NH4+ than 15NO3− at both 0–5 cm and 10–15 cm (Fig. 3; P < 0.05). In September, ratios were similar to those in August (Fig. 3; P < 0.05). At all stages of the growing season, recovery ratios were higher for 15NH4+ than for 15NO3− (Fig. 3; P < 0.05). In both August and September, the values were around 1 in topsoils (Fig. 3). The recovery ratios decreased with time passed after 15N injection (4, 24 and 48 h), due to increasing 15N recoveries in plants, while time did not affect microbial 15N recovery.
Temporal frameworks are important to better understand relationships between above- and below-ground communities (Paterson 2003; Bardgett et al. 2005). Plant–microbe competition for inorganic N in an N-limited alpine meadow on the Tibetan Plateau showed that spatio-temporal variations are important for a better understanding of plant–soil interactions in alpine meadows.
15NO3− and 15NH4+ uptake by alpine plants significantly declined with increasing soil depth (Figs 2 and S2). In contrast, there was no clear trend for microbial uptake within the soil profile, although we found a clear seasonal pattern (Figs 1 and S1).
The first hypothesis that alpine plants compete more effectively with soil microorganisms for inorganic N in the topsoil but not in the subsoil was not fully supported by our results. Our conservative estimate showed that alpine plants took up a similar amount of 15N as the microbial biomass, (e.g. an equal amount of NO3− 4 h and 24 h after 15N injection in August and 48 h after 15N injection from July to September). Even more 15N was immobilized by microbial biomass especially from NH4+ (Figs 3 and S3). Nonetheless, alpine plants acquired more inorganic N from the topsoil than from the subsoil (Figs 2 and S2). This was related to higher root biomass in the topsoil, providing a spatial advantage for uptake of available soil N by roots over microorganisms. The distribution of roots and soil microorganisms as well as the mobility of the different N forms are important factors controlling competition for inorganic N between plants and microorganisms (Jackson, Schimel & Firestone 1989). In alpine Kobresia meadows, more roots were found in the topsoil compared with the subsoil (Zhou 2001; Tao et al. 2006). The ratio of root-to-soil volume (root volume did not include rhizosphere volume) was estimated to be around 0.62 in the top 0–10 cm soil layer, declining to about 0.26 in the 10–20 cm soil layer in the same meadow type close to our research site (G. Cao, unpublished data). We further found strong evidence that plant–microbe competition for available N strongly shifted in favour of plants as root biomass increased (Fig. 4), i.e. alpine plants out-competed soil microorganisms when root biomass exceeded 4.4 kg m−2. When a correction factor (KEN) of 0.54 (Brookes et al. 1985) was used to correct for incomplete extraction, alpine plants acquired more inorganic N than soil microorganisms with root biomass greater than 7.9 kg m−2. In contrast, microbial biomass showed a weak correlation with ratios of 15N recovery by microbial biomass to 15N recovery by plants (y = 0.18x1.03, R2 = 0.11, P < 0.001), but no correlation with root biomass (data not shown). This indicates that roots modify microbial uptake of inorganic N and their competition for inorganic N with plants. Soil depth as a proxy for root density therefore has been identified as a main factor defining plant–microbe competition for N uptake (Fig. 5).
Several studies suggested that plants acquire more of the N required for growth during the early growing season, while soil microorganisms immobilize more N late in the growing season after plant senescence (Jaeger et al. 1999; Bardgett et al. 2002). In this study, we were unable to observe such a pattern, because roots exploited more soil volume in both August and September. Our second hypothesis, i.e. that alpine plants compete more effectively with soil microorganisms for inorganic N in the middle versus the late growing season, was therefore not supported. Above-ground biomass in alpine meadows is known to increase fast in July because of higher temperature and precipitation (Zhou 2001), but our study showed that at this time of the year they were poorer competitors for inorganic N compared with microorganisms. Leaf senescence already starts in September in alpine meadows on the Tibetan Plateau, but the competitive strength of plants for inorganic N in September was similar to that in August (Fig. 3). This reflects differences in below-ground biomass during the growing season: Pu et al. (2005) showed that the below-ground biomass of alpine plants was low in July despite fast above-ground biomass accumulation, but that their below-ground biomass was high both in August and September. In this study, ratios of shoots to roots were higher in July (0.21) than in August (0.18) and September (0.19). This indicates more root accumulation during the late growing season, thereby effectively allowing roots to compete for available N with soil microorganisms. However, we did not apply a conversion factor (KEN) commonly used in the chloroform-fumigation extraction technique to account for incomplete extraction (Jenkinson, Brooks & Powlson 2004) and thus to correct microbial 15N uptake. The reason is that soluble 15N and insoluble 15N are in disequilibrium in short-term 15N uptake experiments, which could have underestimated microbial 15N uptake (Fig. 4).
In support of the third hypothesis, the ratios of 15N recovery by microbial biomass to 15N recovery by plants from NO3− were lower than from NH4+ (Figs 3 and S3). One explanation for this uptake pattern is that specific plant species preferentially take up NO3− while other species prefer NH4+. For example, shrubs preferentially acquired 15NH4+, while Carex species took up more 15NO3− than 15NH4+ in subarctic tundra ecosystems (Sorensen et al. 2008), while several other studies showed that certain plant species preferentially took up NO3− in alpine meadows (Miller, Bowman & Suding 2007; Song et al. 2007). We therefore suggest that the high mobility of NO3− in soils (Nye & Tinker 1977; Owen & Jones 2001; Miller & Cramer 2004) and the importance of NO3− in balancing cation uptake can help explain the high uptake of 15NO3− by plant roots.
Compared with previous studies, we investigated simultaneously spatio-temporal patterns of plant–microbe competition for NH4+ and NO3− in the relatively unexplored alpine meadows on the Tibetan Plateau using a short-term 15N experiment. Our results demonstrate that spatio-temporal variations determine plant–microbe competition for inorganic N in alpine meadows and that root biomass is a critical factor modifying plant–microbe competition for inorganic N (Fig. 5). Root biomass below the threshold of 4.4 kg m−2 indicates that microorganisms compete more effectively than alpine plants without using the KEN factor. Alpine plants showed a preference for NO3−, and the factor season influenced plant–microbe competition for inorganic N mainly through affecting the distribution of root biomass in alpine meadows. Overall, our findings have important implications for the understanding of above-ground–below-ground interactions and plant–microbial competition for available N.
We thank two anonymous referees who have improved the manuscript with their constructive comments. The research was supported by the National Natural Science Foundation of China (30870424), the National Basic Research Program of China (2005CB422005) and the Chinese Academy of Sciences Visiting Professorship for Senior International Scientists.