Effects of warming on soil fungal community and its function in a temperate steppe

Abstract

Background

The potential effects of global warming on soil fungal communities and their functions remain uncertain. To address this issue, we investigated the effects of 3-year simulated field warming on the community and function of fungi in a temperate steppe of Inner Mongolia, northern China.

Methods

The diversity and structure of the fungal community were measured by high-throughput sequencing. The functionality of fungal communities was identified by comparison with the ITS reference database.

Results

Our results showed that warming did not affect the diversity of fungi, but significantly increased the complexity of the fungal community, with fungal taxa more closely associating with each other. We observed that plant pathogens and arbuscular mycorrhizal fungi were the most abundant functional groups. Meanwhile, warming significantly decreased the relative abundance of animal pathogens.

Conclusions

Warming significantly increased the complexity of the fungal community, with soil pH being the main factor affecting soil fungal function. Our findings emphasize that the response of the fungal community and its functional groups to warming has significant implications for ecosystem biogeochemical cycling.

Background

Global warming greatly impacts the biogeochemical cycles of terrestrial ecosystems and aggravates the loss of biodiversity (Hartmann et al. 2013). As important decomposers, soil microorganisms play a crucial role in driving nutrient cycling and have complex effects on plant growth and belowground carbon allocation, fixation and release (Cheng et al. 2017; Gao et al. 2021; Xue et al. 2016; Zou et al. 2022). Consequently, to mitigate the negative effects of warming on continental ecosystems, it is imperative to explore the response of soil microbial structure and functions to warming (Habtewold et al. 2021; Nottingham et al. 2022; Sun et al. 2023; Yu et al. 2018). However, the impact of warming on the interactions between microbial communities and specific functional groups is poorly understanded.

Soil fungi serve as the primary drivers of soil organic matter decomposition and mineralization (Bowles et al. 2014). Therefore, it is very important to investigate the response of soil fungal community structure and function to warming. Previous studies have shown that fungi are more tolerant than bacteria to arid environments, primarily due to their ability to form mycelium network that would be capable of transporting water and nutrients (Kaisermann et al. 2015; Khalvati et al. 2005; Yuste et al. 2011). Warming can directly impact soil temperature and moisture, and further influence plant community composition and above- and belowground biomass (Weltzin et al. 2003). Thus, the change of quality and quantity of plant litter and root exudation can subsequently affect the soil microbial community (Chen et al. 2021). Moreover, many studies have also found that fungi, more than bacteria, rely more on plants as habitat and the source of substrates (Peay et al. 2013; van der Linde et al. 2018; Wang et al. 2024). For example, warming had a strong environmental filtering effect on arbuscular mycorrhizal fungal community by decreasing plant carbon distribution in grasslands on the eastern Tibetan Plateau (Xu et al. 2022). Thus, the changes of plant carbon substrates induced by warming may result in changes in the fungal communities.

Studies on the impacts of climate warming on soil fungal communities have mainly focused on the change of α- and β-diversity and the abundance of fungal taxa (Nottingham et al. 2022; Wang et al. 2024; Zhou et al. 2021). Nottingham et al. (2022) showed that short-term (2-year) warming reduced the fungal diversity due to loss of rare taxa and increased the relative abundance of Glomerales-arbuscular mycorrhizae and Ascomycota, which included thermotolerant saprophytic and pathogenic species.

Che et al. (2019) found that long-term (6-year) warming significantly increased the proportion of Dothideomycetes (a potentially oligotrophic class of fungi) and decreased the proportion of active plant symbiotic lineages (e.g., Glomerales). Thus, warming shifted in fungal community composition in the direction of heat-resistant and oligotrophic species. A key question is whether the process of changing the microbial community structure will also change the interactions between fungal groups. Zhou et al. (2021) found that 5-year experimental warming significantly affected the diversity and composition of the fungal community, with the fungal network structure showing high resistance and stability to warming. In the ecologically fragile Loess Plateau, warming increased fungal network stability and reduced bacterial network stability, implying that warming enhances the competitive advantage of fungi over bacteria (Wang et al. 2024). However, in a Tibetan alpine meadow ecosystem, 6 years of field warming significantly attenuated the interactions among fungal taxa (Che et al. 2019). This suggests that the fungal communities of different ecosystems may respond differently. Therefore, it might be important to understand the fungal interactions among different fungal groups induced by changes in soil nutrients and plant biomass due to warming.

Warming can alter the community structure of soil fungi, as well as their functional groups (Xiong et al. 2014). The functional groups of fungi are generally divided into ectomycorrhizal fungi (ECM), endomycorrhizal fungi (ERM) and arbuscular mycorrhizal (AM) fungi (Michaelson et al. 2008; Väre et al. 1992). Che et al. (2019) found that 6-year warming significantly decreased the proportions of saprophytic fungi, suggesting that warming weakens the ability of soil fungi to decompose organic matter. Arbuscular mycorrhizal fungi mainly provide nitrogen, phosphorus and other nutrients for plants (Ezawa and Saito 2018; Hodge and Fitter 2010; Kong et al. 2022). It was reported that warming significantly reduced the proportions of arbuscular mycorrhizal fungi (Che et al. 2019), indicating that warming adjusts the nutrient acquisition strategies of plants. Changes in plant aboveground and belowground processes induced by warming would directly and indirectly change soil fungal functions. The response of these fungal functional groups to warming would affect the soil nutrient cycle (Treseder et al. 2016; Trumbore et al. 1996). Therefore, it is very important to study the response of functional groups to warming.

To explore how warming and warming duration impact the complexity and function of the fungal community network structure, we conducted a 3-year field warming experiment in a temperate steppe of Inner Mongolia. The objectives of this study were: (1) to assess the effects of warming on the fungal diversity and community composition; (2) to examine the effects of warming on the network structure of soil fungal communities; (3) to explore the effects of warming on the change of fungal functional groups. We hypothesize that, firstly, warming would change plant biomass and thus change the fungal diversity and community composition, and secondly, warming would increase the plant aboveground biomass and further increase the quantity and quality of soil organic matter, and thus increase the decomposition of plant residues by saprophytic fungi among fungal functional groups.

Methods

Site description

The study area was located at the Hulun Lake Reserve of Inner Mongolia (117.05° E, 48.75° N), China. The climate of this study site was a typical temperate continental semi-arid climate, with long-term annual mean temperature and annual mean precipitation of −0.45 °C and 283 mm, respectively. The total precipitation was 211.3, 203.6 and 299.4 mm in 2018, 2019 and 2020, respectively, and mainly fell during the growing season, from June to September. The vegetation was dominated by Stipa krylovii. The soil is classified as chestnut based on the FAO soil classification.

Experimental design

In June, 2018, we built open-top-chambers (OTCs) to simulate climate warming. The experiment was set up as a paired design with the treatment of warming and control. Each treatment assigned 5 blocks and the distance between plots was 5 m. Each block included a 5 m × 5 m region, in which a 1.5 m × 1.5 m quadrat was arranged as control, and a 1.5 m × 1 m (diameter × height) chamber was constructed to simulate warming.

Soil temperature was estimated at 0.5-h intervals using an electronic temperature recorder (DS1921G Thermochron iButton datalogger) buried at depth of 0–10 cm from May 31 to October 9 in 2018, 2019 and 2020. The OTCs significantly increased soil temperature, on average by 0.95 and 1.21 °C during the growing seasons of 2019 and 2020, respectively (Additional file 1: Fig. S1). The soil temperature data for 2018 were unfortunately lost during the course of the experiment. More details about the construction and installation of the OTCs can be found in Yu et al. (2021).

Field sampling and lab analysis

In August 2019 and 2020, the aboveground living biomass was measured by harvesting all living plants within a 0.4 m × 0.2 m quadrat. The belowground biomass (0–10 cm) was collected from a soil core (10 cm in diameter). Both above- and below-ground biomasses were over-dried (60 °C) to a constant weight. Five soil cores (0–10 cm, 3 cm in diameter) were randomly sampled and pooled into one composite sample for each plot. The visible roots and other plant residues were removed and soil samples were sieved through 2 mm mesh, then divided into two parts. The first part was store at 4 °C for measuring soil organic carbon (SOC), total nitrogen (TN), soil pH and soil moisture. The second part was stored at  −80 °C for high-throughput sequencing of the soil fungi.

Briefly, soil pH was estimated using a 1:2.5 soil to water ratio by pH probe (PHS-3CU, Shanghai Yueping Scientific Instrument (Suzhou) Manufacturing Co., Ltd). Soil water content was measured gravimetrically at 105 °C for 12 h. TN and SOC were measured by elemental analyzer (Vario EL III, Elemental Analysis System GmbH, Germany).

DNA extraction, PCR amplification and sequencing

Soil DNA was extracted from 0.5 g fresh soil using PowerSoil DNA Isolation Kit (MO BIO). After the DNA quality test, primers ITS3 (5′-GCATCGATGAAGAACGCAGC-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) were used to amplify the ITS2 region. The PCR amplification conditions included: denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 45 s for 31 cycles, and a final extension at 72 °C for 10 min. The PCR products were purified by Omega kit following the manufacturer’s protocol, and then the purified PCR products were paired-end sequenced using the Illumina HiSeq 2500 platform.

The raw data were analyzed using an in-house Galaxy Pipeline (https://dmap.denglab.org.cn/) (Feng et al. 2017). The low-quality sequences were discarded through Trimmomatic-0.33. We combined the forward and reverse sequences by Flash software in the Mothur platform. Then, the high-quality sequences were clustered into operational taxonomic units (OTUs) at 97% identity via UPARSE method (Edgar 2013). The fungal taxonomy was assigned by blasting the ITS reference database (Schoch et al. 2012). We collected a total of 101,701 high-quality sequences. We resampled to 55,540 sequences per sample to account for sequencing depth before further analysis.

Data analysis

The α-diversity and relative abundance of the fungal community were calculated using the Galaxy Pipeline platform (https://dmap.denglab.org.cn/) (Louca et al. 2016). One-way ANOVA was used to test the effects of warming and warming duration on the soil fungal diversity index. We used the database of FUNGuild to predict the soil fungal community function. Repeated measures ANOVA was used to test the effects of warming, warming duration and their interaction (warming × warming duration) on soil physicochemical properties, aboveground and belowground biomass, and fungal diversity index and function. We used Spearman correlation matrices to construct the fungal network based on the package of “psych” in R Studio software (4.3.3). Only OTUs with relative abundance > 0.01% and statistically significant correlations (absolute correlation coefficient > 0.6, p-value < 0.05) were used to construct the network. Then, we loaded the correlation table into Gephi software (0.9.2) to calculate the network topological property and visualize the co-occurrence networks. We used Mantel test to verify the soil biogeochemical effects on the soil fungal communities. Additionally, we constructed a partial least squares path model (PLS-PM) using the package “plspm” to assess how experimental warming affected plant biomass, soil nutrients and pH, and their feedbacks to soil fungal diversity, communities and function (R Development Core Team 2016).

Results

Soil fungal diversity and community structure

Warming had no significant effects on soil fungal Shannon diversity and Inv-Simpson indices in 2019 and 2020 (Fig. 1). At the phylum level, the fungal community was dominated by Ascomycota, Basidiomycota and Glomeromycota (Fig. 2).

Fig. 1

The effects of warming on fungal Shannon Index (A) and Inv-Simpson Index (B) in 2019 and 2020. Values are means ± SE (n = 5). Different letters indicate the significant differences (p < 0.05) according to the Duncan’s post-hoc test

Fig. 2

The effects of warming on the relative abundance of dominant fungal taxa in 2019 and 2020

Soil fungal function prediction and complex networks

The effect of warming on the fungal functional groups was mainly concentrated in plant pathogen, arbuscular mycorrhizal fungi, and endophyte (Fig. 3). Warming significantly decreased the relative abundance of animal pathogens (p < 0.05, Fig. 3). Warming duration had significant effects on animal pathogens and plant pathogens (p < 0.05, Additional file 1: Table S2). There was a significant interaction between warming and warming duration on animal pathogens (p < 0.05, Additional file 1: Table S2).

Fig. 3

The effects of warming on the relative abundance of fungal functional groups. Values are means ± SE (n = 5). Different letters indicate significant differences (p < 0.05) according to the Duncan’s post-hoc test

The network structure of the soil fungal communities showed significantly different patterns under control and warming treatments (Fig. 4; Table S3). In 2019, the control network included 91 nodes and 478 links, and consisted of 292 positive correlations and 186 negative correlations. Meanwhile, in the warming network, there were 91 nodes and 451 links (330 positive correlations and 121 negative correlations). Warming decreased the fungal network modularity and clustering index. In 2020, the control network consisted of 145 nodes and 1086 links, which included 684 positive correlations and 402 negative correlations. A greater number of nodes (154) and links (811 positive correlations and 537 negative correlations) were detected in warming fungal network. Thus, warming decreased network modularity.

Fig. 4

Patterns of fungal interactions under the control (A) and warming (B) treatments. A red edge indicates a positive correlation, and a green edge indicates a negative correlation

The mechanisms affecting fungal community structure

According to Mantel test, the aboveground biomass and soil moisture were the main factors affecting the fungal community under warming (p < 0.05, Additional file 1: Table S4). We utilized a partial least squares path model to investigate the warming-induced changes in plant biomass–soil pH–soil nutrients and soil fungal community structure and function (Fig. 5). The model revealed that warming had positive effects on plant biomass and soil pH, but negative effects on soil nutrients. The plant biomass had negative effects on network properties and fungal community structure, but no significant effects on fungal diversity and function. Soil nutrients also had no significant effects on fungal diversity, community structure and function. The soil pH had direct negative effects on fungal function. However, the fungal network properties and community structure had no significant effects on fungal function.

Fig. 5

Partial least squares path models (PLS-PM) of warming effects on plant biomass–soil nutrients–soil pH–fungal communities and function system. Red solid lines indicate positive relationships (p < 0.05), blue solid lines indicate negative relationships (p < 0.05) and gray light lines indicate no significance (p > 0.05). The numbers represent the total effects between factors. Biomass includes the aboveground and belowground biomass. Soil nutrients includes TN (soil total nitrogen), SOC (Soil organic carbon), C:N (ratio of soil organic carbon to soil total nitrogen), and soil pH (soil acidity). The fungal network properties include nodes, links, modularity and clustering. The fungal communities were calculated based on Bray-Curtis distance. The fungal diversity is the Shannon diversity. The fungal functions include animal pathogen, arbuscular mycorrhizal, ectomycorrhizal, endophyte, fungal parasite, plant pathogen, and soil saprotroph

Discussion

Warming affected soil fungal community structure

In our study, warming duration had no significant effects on fungal diversity. This was consistent with previous studies that showed short-term (15-month) and long-term (6-year) warming did not alter the diversity of fungi (Che et al. 2019; Xiong et al. 2014), indicating that the fungal diversity may not be sensitive to warming. Yuste et al. (2011) also found that fungal communities were resistant to change in a 10-year precipitation exclusion experiment in a water-limited Mediterranean ecosystem, implying that fungal communities could resist the arid environment. However, Wu et al. (2022) showed that long-term climate warming reduced fungal biodiversity in tall-grass prairie, which was driven by soil microclimate and geochemistry. Therefore, the effects of warming on fungal diversity vary between ecosystem types, and it takes a long time for fungal diversity to respond to warming.

In contrast, in our study we found that warming significantly altered the relative abundance of fungal communities, with a significant increase in the relative abundance of Ascomycota. The Ascomycota are an important group of saprophytic fungi and saprophytic fungi have been recognized as critical decomposers of soil organic matter (Xiong et al. 2014). It reflects that warming increases the ability of saprophytic fungi to degrade soil organic matter. This may be because warming significantly increased plant aboveground biomass and litter in 2020, which in turn increased the relative abundance of Ascomycota.

Based on results of the Mantel test, we discovered that the main factors influencing fungal community structure under warming were belowground biomass and soil moisture. Because warming decreased soil moisture and the availability of soil nutrients, the fungal community would respond accordingly, especially the active fungal groups (Barnard et al. 2015; Fierer 2017) and the oligotrophic fungal communities (Che et al. 2019). This supports that water availability has been recognized as an important factor affecting fungal community structure (Barnard et al. 2015; Fierer 2017; Placella et al. 2012). It has been reported that plant regulation of microbial community composition by affecting the quantity and quality of plant litter further affects the availability of microbial resources (Bardgett and Wardle 2010; Zhang et al. 2017). In our study, the belowground biomass was also considered to be an important factor affecting the fungal community structure. Chen et al. (2021) postulated that the simultaneous interaction between the aboveground and belowground biomass was the main factor affecting the soil microbial community structure. Plant-driven changes in belowground biomass would directly affect the activity of the fungal groups and ultimately lead to interspecific competition and further alter the microbial community.

Warming affects the interaction of fungal taxa

Complex interactions between microbial groups play a significant role in maintaining the functioning of ecosystems (Barberán et al. 2012; Worrich et al. 2017). It is important to research the complex interrelationships between microbial taxa for predicting the response of ecosystem functional models to climate warming (Allison et al. 2010). In our study, analysis of the fungal community networks showed that warming increased positive interactions in 2019, reflecting that warming increases the cooperative behaviors between taxa. Warming significantly increased the complexity of the fungal network in 2020, indicating the facilitation of fungal interactions under warming. This may lead to an increase in grassland soil resilience to warming with greater stability of fungal communities (Treseder et al. 2016; Zhou et al. 2021).

Compared to the control in 2019 and 2020, the topological properties of the fungal networks showed that warming decreased the network structure modularity in 2019 and 2020, suggesting that warming reduces the ecological niches of fungal community coexistence. This is likely because warming acts as a deterministic filtering factor, selecting for certain fungal community, which could further result in divergent succession with reduced stochasticity. Previous work showed that the topological properties of fungal networks were closely related to soil nutrient availability and plant type (Chen et al. 2021). In our study, the changes in plant biomass caused by warming could be used as environmental filtering factors for explaining the changes in the topological properties of the fungal network. Therefore, warming could change plant biomass, resulting in changes to the fungal community structure and topological properties of the fungal network.

Warming alters the function of the fungal community

Soil microorganisms play an important role in biogeochemical cyclings, therefore it is important to study how their functions respond to climate warming (Zhou et al. 2020). In our study, warming did not affect the relative abundance of saprophytic fungi and ectomycorrhizal fungi, which may be due to the fact that the duration of warming here was shorter and the network structure of fungal community is resistant to drought (de Vries et al. 2018). We found that warming significantly decreased animal pathogenic fungi in 2019 and the main factor affecting fungal function was soil pH. Additionally, Labouyrie et al. (2024) showed soil pH is a driver of soil fungal richness in most climatic areas. Thus, in the future, we should pay more attention to the changes in the soil fungal community caused by soil pH under warming. There was high relative abundance among the plant pathogen and arbuscular mycorrhizal fungi functional groups. Plant pathogenic fungi have negative effects on plant growth, while arbuscular mycorrhizal fungi have positive effects on plant growth (Cozzolino et al. 2021). These two groups of fungi are antagonistic to each other, which may be due to the regulation of plant growth strategy, and further induce the change of fungal functional groups. Changes in fungal functional groups would affect the decomposition of soil organic matter (Pec et al. 2021), and have important impacts on the dynamic change of soil carbon under warming. Consequently, we need to pay attention to the long-term response of fungal functional groups to warming.

Conclusion

In general, our study revealed the response of fungal diversity and community composition to warming in the steppe of Inner Mongolia. Our results show that 3 years of warming did not affect fungal diversity but did significantly increase the complexity of the fungal community, reflecting that the fungal network structure has high resistance to warming. Warming significantly decreased the relative abundance of animal pathogen among the fungal functional groups, and the main factor affecting soil fungal function was found to be soil pH. To improve the accuracy of climate change models, further research should focus on how changes in soil pH affect the key fungal functional groups related to soil carbon concentrations under long-term experimental warming.