Precipitation and soil microbial community influence plant response to warming and N addition in a desert steppe

doi:10.21203/rs.3.rs-1337752/v1

Aims

Global environmental changes are known to affect terrestrial ecosystems. However, how plants and soil microorganisms respond to warming and nitrogen deposition in a desert steppe with strong seasonal drought remains largely unexplored.

Methods

Based on a 13-year manipulative field experiment, we investigated the effects of warming and nitrogen addition on soil microbial communities and plant net photosynthetic rates during two hydrologically contrasting months (6 mm in June and 106 mm in July) in a desert steppe in Inner Mongolia.

Results

We found that in the wet month, warming and nitrogen (N) addition significantly increased soil microbial biomass. Warming and N addition significantly increased soil inorganic N and increased leaf N concentration, thus promoting the net photosynthetic rate of Stipa breviflora. Moreover, warming and N addition significantly shifted soil microbial composition with an increase in soil bacterial phospholipid fatty acids (PLFAs) and reduced fungal PLFAs. The increased soil inorganic N indirectly increased leaf N and plant photosynthesis by soil microbial composition. These changes were not significant in the dry month.

Conclusions

Our study indicates that warming and N addition can promote plant photosynthesis by increasing soil N availability and changing the structure of the soil microbial community. These changes only occurred when there was sufficient precipitation. These results highlight the crucial role of soil water availability and the soil microbial community in influencing plant responses to global change drivers (e.g., warming and N addition).

desert steppe

global warming

nitrogen deposition

plant-soil-microorganism

drought

wet

The Earth’s surface temperature has risen by 0.85 ℃ between 1880 and 2012. If the temperature continues to rise at the current rate, global warming will reach 1.5 ℃ in the mid-21st century (IPCC, 2018). Such unprecedented changes in temperature are predicted to affect soil microbial communities and plant growth with the potential feedbacks to climate (Gong, Zhang & Guo, 2019; Wu et al., 2020). Since the industrial revolution, human beings have increased nitrogen (N) deposition from the atmosphere by applying chemical fertilizers and burning fossil fuels (Galloway et al., 2008), a trend which is likely to double or even more from their current values (10 kg N ha⁻¹ yr⁻¹) by 2050 (Galloway et al., 2008; Phoenix et al., 2012), As N is one of the key limiting nutrients for terrestrial plant growth (LeBauer & Treseder, 2008), increased N deposition could have widespread ecological consequences, including effects on species composition, biodiversity and ecosystem functions (e.g., plant growth and carbon storage) (Isbell et al., 2013).

Soil microbial communities, as the catalyst of soil nutrient cycling and transformation, are an important component of terrestrial ecosystems (Liang et al., 2011; Lucas et al., 2007; Stroud, Paton & Semple, 2007). Soil microorganisms not only play an important driving role in soil processes, but also are sensitive biological indicators of climate change (Delgado-Baquerizo et al., 2017; Schindlbacher et al., 2011). Although many studies have shown that warming and nitrogen may have impacts on soil microbial communities, the existing reports on the effects of warming and N deposition on soil microorganisms are not consistent, with the reported increase (Gong, Zhang & Guo, 2019) or decrease (Alvarez, Santanatoglia & Garcia, 1995; Fu et al., 2012) in soil microbial biomass. Moreover, it has been also been reported that warming changes the structure of soil microbial communities. A few studies have shown that warming reduced the fungi to bacteria (F/B) ratio because of depletion of carbon substrate and increase in soil nutrient with a lower soil C/N ratio (Deng et al., 2018; Melillo et al., 2017; Zhang et al., 2005), but it remains largely unclear how changes in soil nutrients and soil microbial composition affect plant growth. The increase soil nutrient could directly increase plant growth by increasing nutrient uptake by roots, but soil microorganisms also compete for soil nutrients. Thus, it remains largely unknown whether the effects of increased soil nutrients on plant growth are regulated by soil microbial communities.

The effects of N addition on increased soil nutrient availability are clearly shown (Lu et al., 2011; Ren et al., 2018), but the response of soil microbial communities are ecosystem specific, For example, studies have reported increase of soil microbial biomass in desert (Liu & Greaver, 2010) and meadow steppe (Gong, Zhang & Guo, 2019) and decrease in forests (Liu & Greaver, 2010) and semi-arid grassland (Li et al., 2010). Most studies suggest that N addition would also significantly change the structure of the soil microbial community (Clegg, Lovell & Hobbs, 2003; Griepentrog et al., 2015), with either an increase in the F/B ratio (Chen et al., 2019) or non-significant change (Huang, Li & Su, 2015). Moreover, some researchers have suggested that warming and N addition have additive effects on soil microorganisms (Adair et al., 2019; Gutknecht, Field & Balser, 2012). However, it is less clear whether N addition directly influences plant growth by increasing soil nutrient availability or indirectly by the shift in soil microbial composition.

Water is a limiting factor in the desert steppe of northern China. It has been found that different hydrological conditions affect response of plants to global change drivers. For instance, Ren et al. (2018) found that plant nutrient resorption efficiency had different responses to warming and N addition under different water availability conditions, and Yang et al. (2011) reported that precipitation enhancement changed the structure and composition of the plant community (Yang et al., 2011a). These effects may further affect the soil microbial community (Zhang et al., 2015a). Water may co-limit the soil microbial community along with other factors such as temperature and soil fertility. Warming has been found to interact with fluctuation in water availability and to affect the soil microbial community in arid and semi-arid ecosystems (Allison & Treseder, 2008; Liu, Zhang & Wan, 2009; Nielsen & Ball, 2015). In addition, it has been demonstrated that the effect of N on soil microbial composition may also strongly depend on water status (Bi et al., 2012; Herman et al., 1993; Huang, Li & Su, 2015). Therefore, the effects of global change such as warming, increasing N deposition and changing in rainfall patterns on plants and soil microorganisms in the desert steppe are still unclear.

To investigate how soil microorganisms and plant growth change in the context of global change (i.e., warming and N deposition), and whether and how these changes vary with variation in water availability, we conducted an experiment using a 13-year warming and N addition experiment platform in a Stipa breviflora desert steppe in northern China. In 2018, we sampled in two hydrologically contrasting months (i.e., dry in June and wet in July) to examine the characteristics of soil microorganisms and the dominant plant (S. breviflora) in response to warming and N deposition. We aimed to answer the following questions: (1) How do warming and N addition affect plant net photosynthesis? (2) How do warming and N addition affect soil microbial biomass and community? (3) Is the effect of warming and N addition on plant and soil microorganism additive and regulated by hydrology?

2.1｜Study site and experimental design

The study was conducted at the site of a long-term warming and N addition experiment in a desert steppe at Siziwang Banner, Inner Mongolia, northern China (41°46′43.6″N, 111°53′41.7‍″‍E, at 1,456 m a.s.l.). Long-term mean annual precipitation in this area is 229 mm, 78% of which falls in June to September. Mean annual temperature was 3.9 ℃ in 2018, with monthly mean temperature of -18.2 ℃ in January and 21.2 ℃ in July. The soil in the study site has a sandy loam texture and is classified as Kastanozem based on the FAO (Food and Agriculture Organization of the United Nations) classification system. The perennial grass, S. breviflora was the dominant species in our study site, and made up approximately 40% of aboveground plant biomass. The growing season in this desert grassland runs from early April to late September.

Since May 2006, the experiment used a split-plot design with warming as the main plot and N addition as the subplot. Six pairs of 3 m × 4 m main plots were established and the distance between two main plots in each pair was 3 m. One main plot in each pair was assigned to the ambient treatment (W0, no warming) and the other to the warming treatment (W1). Each main plot was divided into two 3 m × 2 m subplots, with one of the subplots randomly assigned to no N addition (N0, ambient N addition rate) and the other to N addition (N1). Thus, there were four treatments: no warming and no N addition as the control (C), warming without N addition (W), N addition without warming (N) and combined warming and N addition (WN). Each treatment had six replicates, so there were 24 subplots in total. The warming plots were heated continuously for 24 h a day using MSR-2420 infrared radiators (Kalglo Electronics Inc. Bethlehem, PA, USA). The infrared radiators were positioned 2.25 m above the ground in each warming plot and set to a radiation output of about 2000 W. In each no-warming plot, a dummy radiator of the same shape and size was positioned at the same height to simulate the shading and other effects of the radiators. The surface soil temperature in the warming plots was 1.3 ℃ higher than in the control (Wang et al., 2015b). Before a rainfall event in late June every year, NH₄NO₃ was dissolved in water and evenly sprayed into the N addition plot. At the same time, the same amount of water was sprayed into the no-N addition plot. The N addition rate was 10 g N m^-2 yr ^-1, which is equal to the current yearly N deposition for Inner Mongolian grassland ecosystems (Bai et al., 2010).

2.2｜Field sampling and biogeochemistry measurements

We studied the changes in plant photosynthesis rate and soil microorganisms under warming and N addition during months with different hydrological conditions in 2018. Total precipitation in June was only 6.3 mm, but total precipitation in July was 106.1 mm (Figure S1). Thus, this provided a unique opportunity to investigate how the response of plant productivity and soil microorganisms to warming and N addition depends on soil water availability. We collected 0-10 cm mineral soil samples by taking three 3 cm diameter cores in each plot on June 15, June 30, July 14 and July 30 in 2018. We then mixed the three soil samples from each plot into one. The mixed soil sample was sieved (2 mm) to remove impurities such as stones and roots. Soil inorganic N was measured by a continuous flow spectrophotometer (FIAstar 5000 Analyzer; Foss Tecator, Hillerod, Denmark). Soil temperature and moisture in the 0-‍10 cm layer were measured by soil temperature and water content sensors (ECH₂O-TE/TM, Decagon Devices Inc., USA).

2.3｜Plant leaf N concentration and photosynthesis

Three S. brevifloraplants in each plot were randomly selected to measure photosynthesis rate using a Li-6400 Portable Photosynthesis System (Li-Cor, Inc, Lincoln, NE, USA) at the end of June and July 2018. In addition, we randomly collected some leaves of these plants, dried them at 65℃ for 48 h, weighed and ground them to determine N concentration using the Auto-Kjeldahl method (Kjektec System 1026 distilling unit; Kjeltec Systems, Sweden).

2.4｜Soil microorganisms

Soil microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) were determined using the chloroform-fumigation-extraction (CFE) method (Vance, Brookes & Jenkinson, 1987). Soil microbial community structure was assessed using phospholipid fatty acids (PLFA) analysis (Bossio et al., 1998). We used the sum abundance of the following FAs: i14:0, i15:0, i16:0, i17:0, a15:0, a17:0, 16:1ω7c, 17:1ω8c, 18:1ω9c, 18:1ω7c, cy17:0 and cy19:0 to predict bacterial biomass, and fungal biomass was predicted from the abundance of the fungi-specific FA 18:2ω6,9 (Frostegard, Tunlid & Baath, 2011). F/B ratio was calculated from fungi FAs and bacteria FAs.

We used a DNeasy PowerSoil kit (QIAGEN GmbH, Hilden, Germany) to extract total genomic DNA from a 0.5 g soil sample. The final DNA concentration and purification were determined with a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and DNA quality was checked by 1% agarose gel electrophoresis. Bacteria 16S rRNA genes were amplified with PCR primers 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). Fungal internal transcribed spacer (ITS) rRNA genes were amplified with PCR primers ITS1 (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS2 (5’- GCTGCGTTCTTCATCGATGC-3’) (Fierer et al., 2012; Leff et al., 2015).

We mixed the same volume of 1X loading buffer (containing SYB green) with PCR products and applied electrophoresis on 2% agarose gel for detection. PCR products were mixed in equidensity ratios. Then, the mixed PCR products were purified with Qiagen Gel Extraction Kit (Qiagen, Germany). Purified amplicons were in equimolar concentration and were paired-end sequenced for 16s rRNA or ITS rRNA gene sequencing on an Illumina HiSeq 2500 platform (Illumina, San Diego, USA) following the manufacturer's recommendations. Operational taxonomic units (OTUs) were clustered with 97% similarity cut-off using UPARSE (version 7.0.1001, http://drive5.com/uparse/) (Edgar, 2013). For each representative sequence, the GreenGene Database (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi) (DeSantis et al., 2006) was used based on the RDP classifier (Version 2.2, http://sourceforge.net/projects/rdp-classifier/) (Wang et al., 2007) algorithm to annotate taxonomic information.

2.5｜Statistical analyses

We used repeated-measures split-plot ANOVA to examine the effects of warming and N addition, sampling date and their interaction on soil physical and chemical properties, soil microorganisms and plant photosynthetic rate and leaf N concentration. All statistical analyses were performed in the nlme package of R (Version 4.0.3, R Core Team, 2020). The code used in R was “lme(Y ~ W*N*D, random = ~1|Rep/Plot, correlation = corAR1(form = ~1|Rep/Plot))”, where Y is the response variable (such as soil MBC, MBN), W is binary (0 = no warming, 1 = warming), N is binary (0 = ambient N, 1 = N addition), D denotes the sampling date(e.g., 15-Jun, 30-Jun, 14-Jul and 30-Jul), and Rep is an integer (1 to 6 for replicates from north to south). The denominator degrees of freedom for main effects and interactions in these models reflect sample sizes for the plots to which warming and N addition were randomly allocated. In addition, in order to study the responses of each factor to warming and N addition at each sampling date, we used repeated-measures split-plot two-way ANOVA for each factor at each sampling date (Table S1).

We used a structural equation model (SEM) to investigate the direct and indirect effects of warming and N addition on soil microbial structure (F/B ratio) and the net photosynthetic rate of S. breviflora. The SEM analysis was carrierd out using the lavaan package in R (Oberski, 2014). We first considered a model including all pathways and sequentially eliminated non-significant pathways until we obtained the final model. The path coefficients were obtained using maximum likelihood estimation. We used the chi-square (χ²), P value, comparative fit index (CFI) and root mean square error of approximation (RMSEA) to evaluate the quality of fit of model. When 0 ≤ χ² ≤ 2df, 0.05 ＜ P ≤ 1, 0 ≤ RMSEA ≤ 0.05 and 0,97 ≤ CFI ≤ 1.00, the model has a good fit.

3.1｜Changes in soil pH, moisture and inorganic N

Soil temperature in the 0 ~ 10 cm soil layer was increased significantly by warming in 2018. On average, soil temperature in the 0 ~ 10 cm soil layer of the warming plots was increased by 1.01 ℃ (W0: 6.21 ± 0.24 ℃ [mean± SE, same below], W1: 7.22 ± 0.22℃; P < 0.001; Figure S2; Table S1), but it was not affected by N addition (N0: 6.76 ± 0.23℃, N1: 6.77 ± 0.23℃; P = 0.49; Table S1). There was no significant interaction between warming and N addition on soil temperature. Neither warming nor N addition had significant effect on soil moisture (Figure S3, Table 1). Soil pH did not change significantly under warming, but N addition affected soil pH significantly, and there was a significant interaction between warming and N addition (P < 0.01; Table S1). Additionally, compared with the control plots, N addition decreased soil pH by 0.08%, while warming + N addition significantly decreased it by 3.6% (P < 0.01, Figure S4). Warming and N addition had additive negative effects on soil pH (P < 0.01; Figure S4). There was no significant interaction between warming and N addition. In the dry month (i.e., June), neither warming nor N addition had a significant effect on soil moisture (June 15: C: 4.43% ± 0.16%, N: 4.93% ± 0.12%, W: 4.95% ± 0.20%; June 30: 3.91% ± 0.14%, N: 4.35% ± 0.10%, W: 4.37% ± 0.17%).

Similarly, soil inorganic N was significantly increased by warming in the wet month, but it was not affected in the dry month. N addition also increased soil inorganic N significantly, while no significant effects were identified in the dry month (P < 0.001; Table 1). Specifically, compared with control, warming significantly increased soil inorganic N by 40.5% and 40.8% on July 14 and July 30, respectively. Meanwhile, N addition significantly increased soil inorganic N by 88.9% and 89.2% on July 14 and July 30, respectively. Warming + N addition significantly increased soil inorganic N by 110.5% and 110.6% on July 14 and July 30, respectively (P < 0.001; Figure S5a; Table S2). In addition, we measured soil NH₄⁺ and NO₃^- content. Similar to the results for inorganic N, warming significantly increased NO₃^- by 45.0 % on July 14 and July 30, but it had no significant effect on NH₄⁺ (P < 0.05; Figure S5b, c; Table S3). N addition significantly increased NO₃^- by 94.2% on July 14 and July 30, and significantly increased NH₄⁺ by 67.8% on July 14 and July 30 (P < 0.001; Figure S5b, c; Table S3). No interactive effect of warming and N addition was found on them.

3.2｜Leaf N concentration and photosynthesis of Stipa breviflora in response to warming and N addition

In the relatively wet month, compared with the control plots (2.36% ± 0.06%), leaf N concentration of S.breviflora was significantly increased by warming (2.69% ± 0.07%), N addition (2.74% ± 0.04%) and warming + N addition (2.76% ± 0.03%) by 14.0%, 15.9 % and 16.9%, respectively (P < 0.001; Figure 1a). However, the significant increase in leaf N concentration did not occur in the dry month. Results from repeated-measures split-plot ANOVA showed that there was a significant interaction between warming and N addition on leaf N concentration (P < 0.01; Table 2).

Similar to the effects of warming and N addition on leaf N concentration, in the wet month, warming, N addition and warming + N addition significantly increased the net photosynthetic rate of S.breviflora from 6.18 ± 0.30 μmol m^-2 s^-1to 9.03 ± 0.51 μmol m^-2 s^-1, 9.24 ± 0.84 μmol m^-2 s^-‍1and 9.35 ± 0.60 μmol m^-2 s^-1, respectively. These changes corresponded to 46.1%, 49.5% and 51.2% increase in net photosynthetic rate by warming, N addition and warming + N addition (P < 0.01; Figure 1b). The results of repeated-measures split-plot ANOVA showed that there was a significant interaction between warming and N addition (P < 0.05; Table 2). However, these effects were not detected in the dry month (C: 1.74 ± 0.23 μmol m^-2 s^-1, W: 1.81 ± 0.17 μmol m^-2 s^-1, N: 2.10 ± 0.19 μmol m^-2 s^-1, WN: 1.79 ± 0.19 μmol m^-2 s^-1).

3.3｜Soil microbial biomass in response to warming and N addition

The results from a mixed model with a repeated-measure split-plot design showed that warming and N addition had no significant effect on soil MBC (Table 1, MBC). N addition had a significant effect on soil MBN, but warming had no significant effect on it (P < 0.001 for N; Table 1, MBN). Soil MBC/MBN ratio was significantly affected by warming rather than N addition (P < 0.05 for W; Table 1, MBC/MBN). Therefore, we analyzed the effects of warming and N addition at each sampling date on soil MBC and MBN. On June 15 and June 30, neither warming nor N addition significantly affected soil MBC and MBN. However, on July 14, warming, N addition and warming + N addition significantly enhanced soil MBC by 10.4%, 12.4% and 24.4%, respectively (C: 106.7 ± 5.77 μg g^-1, W: 117.83 ± 4.47 μg g^-1, N: 19.97 ± 5.77 μg g^-1, WN: 132.77 ± 5.32μg g^-1; P < 0.05; Figure 2a). On July 30, warming, N addition and warming + N addition significantly enhanced it by 21.9%, 22.7% and 24.6%, respectively (C: 127.5 ± 6.14 μg g^-1, W: 155.40 ± 7.65 μg g^-1, N: 156.48 ± 7.81 μg g^-1, WN: 158.92 ± 6.71 μg g^-1; P < 0.05; Figure 2a). Similar to MBC, on July 14, soil MBN was significantly increased by 56.8%, 59.5% and 65.3% by warming, N addition and warming + N addition, respectively (C: 9.46 ± 0.99 μg g^-1, W: 14.84 ± 1.00 μg g^-1, N: 15.09 ± 1.52 μg g^-1, WN: 15.65 ± 1.85 μg g^-‍1; P < 0.05; Figure 2b). On July 30, warming, N addition and warming + N addition significantly increased soil MBN by 40.3%, 50.7% and 80.8%, respectively (C: 10.53 ± 1.02 μg g^-1, W: 14.78 ± 1.05 μg g^-1, N: 15.87 ± 1.76 μg g^-1, WN: 19.04 ± 1.20 μg g^-1; P < 0.01; Figure 2b). Due to a greater increase in MBN than MBC, warming significantly decreased MBC/MBN by 31.4% on July 14 and by 14.5% on July 30. N addition significantly decrease MBC/MBN by 30.1% on July 14 and by 17.4% on July 30 (P < 0.001; Figure 2c; Table S2). The results from repeated-measures split-plot ANOVA showed that warming and N addition had significant interaction on soil MBN and MBC/MBN on July 14, but no significant interaction was observed on MBC (P < 0.001; Table S2). However, on July 30, warming and N addition had significant interaction on MBC (P < 0.001 Table S2).

3.4｜Soil microbial community structure in response to warming and N addition

The results from a mixed model with a repeated-measure split-plot design showed that both warming and N addition significantly affected soil microbial FAs. There was a significant interaction between warming and N addition. Moreover, the FAs were significantly different at different sampling dates (P < 0.001; Table 1). Thus, we also analyzed the responses of soil microbial FAs at different sampling dates to warming and N addition. On June 15 and June 30, N addition had no significant effect on soil microbial FAs. On June 30, warming significantly decreased bacterial FAs and total FAs by 2.9% and 2.2%, respectively. Warming + N addition significantly decreased bacterial FAs and total FAs by 4.9% and 3.3%, respectively (P < 0.001; Figure 3, red lines; Table S2). On July 14, warming and N addition significantly increased bacterial FAs but decreased fungal FAs, resulting in decreases in the F/B ratio by 31.0% and 32.5%, respectively (P < 0.001; Figure3, blue lines; Table S2). Similarly, warming and N addition significantly decreased F/B ratio by 44.9% and 47.6% respectively on July 30 (P < 0.001; Figure 3, green lines; Table S2). In addition, warming + N addition significantly decreased F/B by 32.8% on July 14 and by 47.5% on July 30 (P < 0.001; Figure 3, blue and green lines; Table S2).

Moreover, in order to study the effect of warming on soil microbial community composition, we also carried out high-throughput sequencing analysis on soil samples from the control and warming plots. For soil bacteria, the relative abundance of Proteobacteria, Actinobacteria and Acidobacteria accounted more than half of the bacterial community. Warming did not change the relative abundance of soil bacteria at the phylum level of (Figure 4a). For soil fungi, Ascomycota and Basidiomycota were the main dominant community of fungi. Warming increased the relative abundance of Ascomycota and Basidiomycota by 12.2% and 174.0% (Figure 4b).

3.5｜Pathways of warming and N addition effects on plant photosynthesis

The SEM showed that warming and N addition increased plant photosynthesis by changing the community structure (F/B ratio) of soil microorganisms. Warming directly changed the soil F/B ratio, and then increased leaf N concentration and plant photosynthesis. Warming and N addition indirectly changed the soil F/B ratio by increasing soil inorganic N, and then leaf N concentration (Figure 5). These results thus highlight the crucial role of soil microbial community in the response of plant photosynthesis and growth to global change drivers (i.e., warming and N addition).

4.1 Effects of warming and N addition on soil microorganisms and plant photosynthesis

Previous reports about the effects of warming and N addition on soil microbial biomass have been inconsistent. Some studies have shown that climate warming decreased soil microbial biomass (Fu et al., 2012) or had no significant effect on it (Papatheodorou, Stamou & Giannotaki, 2004). Gong et al. (2019) reported that warming increased soil MBC and MBN based on a 4-year experiment conducted in a meadow steppe. Through a meta-analysis, Xu et al. (2017) found that MBC and MBN showed positive responses to warming, while the MBC/MBN ratio showed a negative response. These findings are consistent with the results from the wet month in our experiment, which showed a decreased MBC/MBN ratio. On the one hand, N addition is thought to increase soil microbial biomass (Allen & Schlesinger, 2004; Nieminen & Setala, 2001). For instance, a recent study in a meadow steppe in northern China showed that N addition significantly enhanced soil MBC and MBN (Gong, Zhang & Guo, 2019), which is consistent with the result from the wet month in our experiment. On the other hand, N addition has been found to decrease or have no effect on soil microbial biomass. For example, a study on semi-arid, sandy grassland showed that soil MBC and MBN decreased significantly after five consecutive years of N addition (Li et al., 2010). No impact of N addition on soil MBC and MBN was observed in the study of a semi-arid desert (Huang, Li & Su, 2015). In this study, both soil MBC and MBN increased under N addition in the wet month, but these effects were not observed in the dry month of the experiment. The reason for this difference may be that the impact of atmospheric N deposition on soil microbial biomass is ecosystem specific and soil water dependent. For example, Johnson et al. (1998) found that N addition increased soil MBC in a healthland, decreased it in an acid grassland, and had no effect on it in a calcareous grassland. In addition, a meta-analysis of global data also showed that the responses of soil MBC to N addition varied between ecosystems (Liu & Greaver, 2010).

Similarly, microbial PLFAs were strongly affected by warming in the wet month. Warming, N addition and warming + N addition all increased bacterial FAs and decreased fungal FAs, and this led to a reduction in the F/B ratio. This is presumably because of decreased soil C substrates or increased soil nutrients with a lower soil C/N ratio, which is consistent with other studies showing that warming reduces the F/B ratio (Deng et al., 2018; Melillo et al., 2017). It is generally believed that long-term N input has a greater negative impact on fungi than on bacteria, resulting in the decrease in the F/B ratio (Frey et al., 2004; Leff et al., 2015). This may be because bacteria need more N to accumulate per unit of biomass C, and a lower C:N ratio may increase the relative abundance of bacteria (Elliott et al., 1983; Fierer et al., 2009). In our study, N addition increased soil inorganic N, which may lead to the decrease in the soil C/N ratio and therefore the F/B ratio.

Warming and N addition are usually considered to increase plant photosynthetic rate (Wu et al., 2011; Zhang et al., 2018). Nitrogen is an important element in the synthesis of chlorophyll and photosynthesis protein (Ordonez et al., 2009). As one of the most important indexes of leaf traits, leaf N content is closely related to the photosynthesis of plants (Wright et al., 2004). In our study, warming and N addition increased plant net photosynthetic rate in the wet month, which may be related to the nutrient supply of plants. Warming and N increased the leaf N concentration of S.breviflora by changing the structure of the soil microbial community. On the one hand, warming directly reduced the F/B ratio. On the other hand, warming and N addition decreased the F/B ratio by increasing soil inorganic N (Figure5). Warming and N addition may increase the abundance of some bacteria related to nutrient cycling and plant N absorption (Tao, Kelly & Radutoiu, 2019), hence leading to increased leaf N concentration and promotion of photosynthesis. Interestingly, the increase in soil inorganic N did not directly increase leaf N concentration, thus suggestion the potential role of competition between plants and soil microorganisms in soil nutrient uptake. Further studies are needed to clarify the roles of bacterial community change and the mechanisms affecting plant leaf N concentration, with a focus on plant vs. soil microbial competition for soil nutrients and the role of the soil microbial community in nutrient cycling.

4.2｜The role of water availability in regulating plant and soil microorganism responses to warming and N addition

There was no significant effect on either leaf N concentration or plant photosynthesis from either warming or N addition in the dry month. However, warming and N addition and their interaction increased leaf N concentration and plant photosynthesis significantly in the following wet month. The reason may be due to the significant difference in water availability between these two months. In the arid desert steppe, water availability is the most important factor affecting plant functioning (Weltzin et al., 2003). A large number of studies have shown that precipitation and warming influence plant growth (Ganjurjav et al., 2020; Song, Niu & Wan, 2016; Wu et al., 2011; Zhao et al., 2019). Zhao et al. (2019) found that increasing precipitation by 15% would offset the negative effect of warming on plant biomass in an Tibetan alpine steppe. Ganjurjav et al. (2020) reported that warming and precipitation interact to advance plant spring phenology. It has also been reported that precipitation and N interact to affect plant growth (Kong et al., 2013; Wang et al., 2015a; Yang et al., 2011b; Zhang et al., 2015b). For instance, precipitation has been reported to alter the responses of plant biomass (Kong et al., 2013), community structure and composition to N addition (Yang et al., 2011b).

The activity of soil microorganisms is highly limited by water availability in water-limited ecosystems. It is considered that in the more arid soils, microbes are more sensitive to changes in water availability (Wang et al., 2018). Our result demonstrated that soil microbial biomass and community structure were stimulated by warming and N addition only when sufficient water was available in the desert steppe, as was the case in July, but not in June. In this semi-arid ecosystem, water seems to be the main limiting factor, while the effect of temperature was secondary and only effective when water conditions were sufficient. These results may indicate that the effect of warming on soil microorganisms depends on water availability (Liu, Zhang & Wan, 2009). Most microbial activities are usually inhibited under conditions of water stress, which may lead to the lack of response of microbial communities to warming. When water is abundant, the microbial population is more active than under drought conditions (Sheik et al., 2011). This may explain why warming only affected soil microbial biomass in the wet month of July, but not in the dry month of June in our study. Huang et al. (2015) reported that N addition did not significantly affect microbial biomass in interplant soils or the F/B ratio in a temperate desert. However, warming and N addition significantly increased soil microbial biomass in a meadow steppe (Gong, Zhang & Guo, 2019). These results may be due to the fact that water is an important limiting factor in desert, but not in meadow steppe. Most microbial functional groups respond to N addition positively only under sufficient water conditions (Zhang et al., 2015a). N addition limits the growth of microbes to a great extent, but its effect on microorganisms depends on variation in precipitation (Zhang et al., 2015a). Water is an important condition for nutrient diffusion and replenishment in soil. Abundant water availability could help to replenish N into soil solution and therefore increase available N for microbial use (Park, Kalbitz & Matzner, 2002). Consequently, N supplementation can promote the growth of microbes by reducing N restriction. These factors may explain why warming and N addition increased soil microbial biomass in the wet month in our experiment.

Generally, bacteria have a low carbon to nitrogen ratio (C/N) (3 ~ 5), while fungal biomass has a higher C/N (4 ~ 15) (Paul & Clark, 1996). Therefore, variations in MBC/MBN may reflect change in the proportions of fungi and bacteria to some extent (Sarathchandra et al., 1988). Variatio in MBC/MBN strongly determines the N and C utilization efficiency of microorganisms (Mooshammer et al., 2014). A study has shown that available C is more sufficient than available N under higher water content, and that soil microorganisms will preferentially fix available N. In this case, microorganisms may reduce C utilization efficiency and increase N utilization efficiency, resulting in a lower MBC/MBN (Chen et al., 2016). The F/B ratio may be related to ecosystem process such as decomposition, C sequestration, nutrient cycling and ecosystem self-regulation (Wang et al., 2019). There is a relationship between the dominance of fungi and bacteria and plant productivity (de Ruiter, Neutel & Moore, 2005; Ingham & Slaughter, 2004). An experiment with warming and mowing showed that warming changed microbial biomass and the F/B ratio only when plants were not mown (Zhang et al., 2005). Therefore, the effects of warming and N addition on the F/B ratio may be mediated by plant growth and strongly regulated by water status.

Overall, our results show that soil microorganisms and plant responses to warming and N addition are strongly regulated by seasonal precipitation in the water-limited desert steppe. Warming and N addition could stimulate plant photosynthesis and soil microorganisms only under high water availability conditions, while there was no change due to warming and N addition on plant and soil microorganisms under water stress. Warming and N addition directly increased soil inorganic N, but the effects of increased soil inorganic N on leaf N and plant photosynthesis were indirect by soil microbial composition. These results highlight the crucial role of soil microbial community in regulating the response of the plant community to global change drivers (i.e., warming and N addition).

Funding

The National Natural Science Foundation of China (31760146), the Fok Ying Tung Education Foundation (171018), the local science and technology development fund guided by the central government (2020ZY0008), Applied Technology Research and Development Fund of Inner Mongolia (2021GG0088), the CAS "Light of West China" Program, the Innovative Team of Grassland Resources from the Ministry of Education of China (IRT_17R59), and the Natural Science Foundation of Inner Mongolia Autonomous Region of China (2021BS03006).

Authors’ contributions

H.R. and G.H. designed and supported the research. H.R. and Y.Z. conducted the research (sampling and laboratory analysis). Y.Z. and H.R. performed the data analyses. Y.Z., H.R., K.Y. and A.W. conceived this study and wrote the manuscript. All authors contributed to manuscript revisions and gave final approval for publication.

Conflicts of interest/Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics approval

Not applicable

Consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and material

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable

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TABLE 1 Results from mixed model with a repeated-measures split-plot design on the effects of warming and N addition on soil moisture, inorganic N, microbial biomass and microbial PLFAs.

	df₁	df₂	SM	Inorganic N	MBC	MBN	MBC/MBN	Total FAs	FAs(B)	FAs(F)	F/B
Warming (W)	1	15	0.01	9.32**	1.93	16.77	8.58*	24.92***	65.41***	22.45***	44.89***
Nitrogen (N)	1	15	0.55	60.06***	2.46	20.82***	3.32	41.10***	118.87***	24.40***	61.85***
Sampling date (D)	3	60		538.86***	147.85***	84.94***	5.35**	3678.77***	3919.04***	38.86***	124.27***
W × N	1	15	0.02	0.97	1.39	0.10	0.26	28.85***	79.56***	18.21***	50.276***
W × D	3	60		12.46***	4.57**	5.78**	0.43	53.74***	94.83***	6.79***	16.92***
N × D	3	60		82.83***	3.48*	10.08***	2.32	45.75***	101.46***	8.12***	19.10***
W × N × D	3	60		1.06	3.00*	5.76**	5.57**	16.75***	49.42***	8.24***	15.03***

Note. The F ratios are presented together with their levels of significance. *P < 0.05, **P < 0.01, ***P < 0.001. df₁ and df₂ denote the numerator and denominator degrees of freedom, respectively. SM represents soil moisture. MBC, MBN and MBC/MBN represent soil microbial biomass carbon, soil microbial biomass nitrogen and the ratio of MBC/MBN, respectively. Total FAs, FAs(B), FAs(F) and F/B represent total FAs, bacterial FAs, fungal FAs and the ratio of fungi/bacteria of soil samples, respectively.

TABLE 2 Results from mixed model with a repeated-measures split-plot design on the effects of warming and N addition on leaf N concentration (Leaf N) and net Photosynthetic rate (P_net) of Stipa breviflora in each month.

		df₁	df₁	Leaf N	P_net
Jun	Warming (W)	1	15	3.04	0.37
	Nitrogen (N)	1	15	3.96	0.77
	W × N	1	15	1.21	0.96
Jul	W	1	15	11.85**	7.63*
	N	1	15	18.34***	9.95**
	W × N	1	15	8.75**	6.59*

Note. The F ratios are presented together with their levels of significance. *P < 0.05, **P < 0.01, ***P < 0.001. df₁ and df₂ denote the numerator and denominator degrees of freedom, respectively.

Supplementaryfiles.docx

Precipitation and soil microbial community influence plant response to warming and N addition in a desert steppe

Status:

Version 1

Abstract

Aims

Methods

Results

Conclusions

Figures

1. Introduction

2. Material And Methods

2.1｜Study site and experimental design

2.2｜Field sampling and biogeochemistry measurements

2.3｜Plant leaf N concentration and photosynthesis

2.4｜Soil microorganisms

2.5｜Statistical analyses

3. Results

3.1｜Changes in soil pH, moisture and inorganic N

3.2｜Leaf N concentration and photosynthesis of Stipa breviflora in response to warming and N addition

3.3｜Soil microbial biomass in response to warming and N addition

3.4｜Soil microbial community structure in response to warming and N addition

3.5｜Pathways of warming and N addition effects on plant photosynthesis

4. Discussion

4.1 Effects of warming and N addition on soil microorganisms and plant photosynthesis

4.2｜The role of water availability in regulating plant and soil microorganism responses to warming and N addition

5. Conclusion

Declarations

Funding

Authors’ contributions

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