Fertilization Promotes the Recovery of Plant Productivity but Decreases Biodiversity in a Khorchin Degraded Grassland

Abstract

Fertilization is a critical measure for vegetation restoration and ecological reconstruction in degraded grasslands. However, little is known about the long-term effects of different combinations of nitrogen (N), phosphorus (P), potassium (K) on plant and microbial communities in degraded grasslands. This study conducted a four-year (2017–2020) N, P, K addition experiment in the Khorchin Grassland, a degraded typical grassland located in Zhalute Banner, Tongliao City, Inner Mongolia, to investigate the effects of fertilization treatment on plant functional groups and microbial communities after grazing exclusion. Our results showed that the addition of P, NP, and NPK compound fertilizers significantly increased aboveground biomass of the plant community, which is mainly related to the improvement of nutrient availability to promote the growth of specific plant functional groups, especially annual and biennial plants and perennial bunchgrasses. However, the addition of N, P, and NP fertilizers significantly reduced the species diversity of the plant community. At the same time, the addition of N, P, and NP fertilizers and the application of N and NP significantly reduced fungal species diversity but had no significant effect on soil bacteria. Our study provides new insights into the relationships between different types of fertilization and plant community productivity and biodiversity in degraded grasslands over four years of fertilization, which is critical for evaluating the effect of fertilization on the restoration of degraded grassland.

1. Introduction

The primary production and ecological processes of natural grasslands are primarily co-limited by nitrogen (N), phosphorus (P), and potassium (K), although other nutrients, such as sulfur, can also be important limiting factors depending on specific conditions [1,2]. In grassland management, fertilization is commonly used to enhance plant productivity. However, this management practice may have uncertain impacts on critical components of the grassland ecosystem, including vegetation and microbial communities. Studies have shown that the addition of exogenous nutrients has become an effective strategy to enhance grassland productivity and promote vegetation restoration. However, prolonged and excessive fertilization often leads to a reduction in plant diversity, changes in plant species composition, and soil acidification, which threaten the stability of the ecosystem [3].

Global climate change (particularly manifesting as altered precipitation patterns and increased frequency of extreme events in the region), combined with inappropriate grassland cultivation and overgrazing, has contributed to large-scale grassland degradation [4]. It has been reported that the Khorchin Grassland in Inner Mongolia has experienced varying degrees of degradation, with the community species composition having shifted to semi-xerophytic sand plants and xerophytic meadow plants. Due to harsh climatic conditions, heavy sandy soils prone to wind erosion, and nutrient-poor soils, the restoration of degraded grasslands in this region is relatively challenging [5,6]. Grassland restoration is a long-term and complex process, primarily focusing on two key elements: plant and microbial communities [5,6,7]. At present, grazing exclusion is widely regarded as one of the simple and effective restoration methods, which has become a common management strategy in recent decades [8]. However, the restoration of grasslands through fencing alone is relatively slow [8,9]. Therefore, taking other measures to accelerate the restoration of degraded grassland, such as fertilization, has become the focus of current research.

It is generally believed that nitrogen (N) and phosphorus (P) are the key elements influencing grassland plant growth and soil nutrient cycling [10]. Nitrogen enrichment can affect soil organisms and nitrogen cycling by directly altering soil N availability and soil pH. Plant-related inputs are common limiting factors of soil organisms and affect a wide range of soil processes [10]. The plant diversity of grassland communities has often decreased with the addition of nitrogen [11,12]. This could be caused by the increased biomass of plant communities, which is accompanied by intensified light competition [13]. Grassland vegetation degradation is typically accompanied by soil degradation [14]. Studies have shown that grassland degradation leads to a decline in soil nutrient content, such as nitrogen and phosphorus. Degraded grasslands are more constrained by nitrogen and phosphorus levels than natural grasslands [15,16]. Therefore, appropriate fertilization is an essential management practice for maintaining soil nutrient balance and promoting the restoration of degraded grasslands. Fertilization with different nitrogen forms can influence plant community structure, diversity, cover, height, biomass, soil properties (e.g., acidification risks from ammonium-based N fertilizers), and other factors [16,17]. It rapidly replenishes the nutrients required for forage growth that are deficient in the soil. However, excessive nitrogen addition can lead to nutrient imbalances in both soil and plants, resulting in soil acidification and toxic effects on plant growth [16,17]. Previous studies have shown that fast-growing plant species tend to dominate the plant community following fertilization, while the abundance of slow-growing perennial species decreases within the community [18,19]. A meta-analysis revealed that species with large rhizomes benefit the most under nutrient addition conditions [20].

Fungi and bacteria are the two main groups in soil microbial communities [21,22]. Soil microbial diversity and composition are highly influenced by soil physicochemical properties, particularly soil pH and nutrient levels [23,24]. While extensive research exists globally on fertilization effects in grasslands, studies remain limited for degraded sandy grasslands like the Khorchin ecosystem regarding simultaneous impacts of nutrient inputs (N, P, NP, and NPK additions) on coupled plant-microbial community dynamics. Understanding the response of degraded grassland plants and soil microbial communities to different nutrient additions is essential for evaluating the effectiveness of nutrient inputs in grassland restoration. This knowledge is of significant practical importance for determining appropriate fertilization strategies for the restoration of degraded grasslands.

The Khorchin Grassland is located at the intersection of the Northeast Plain and the Inner Mongolia Plateau, the semi-humid and semi-arid zones, and the agricultural and pastoral transition areas. It is ecologically fragile and plays a critical role as an important ecological barrier in northern China. For a long time, factors such as climate change, inappropriate grassland cultivation, and overgrazing have led to varying degrees of degradation in the Khorchin Grassland, posing a serious threat to the ecological health of the grassland. This study selected a moderately degraded grassland in the Khorchin region and established a nutrient addition experiment within grazing-exclusion plots. This study addresses two key questions: (1) What are the effects of different fertilization treatments on the recovery of plant productivity? (2) What are the effects of different fertilization treatments on the composition and structure of plant and soil microbial communities? This study evaluates the responses of plant and microbial communities to different nutrient additions in a degraded grassland, aiming to explore effective measures for improving forage yield and quality. The findings provide scientific guidance for the restoration and management of degraded grasslands in the Khorchin region of Inner Mongolia.

2. Materials and Methods

2.1. Study Region

The degraded grassland experimental area (119°13′48″–121°56′05″ E, 43°50′13″–45°35′31″ N) is located in Zhalute Banner, Tongliao City, at the northwestern edge of the Khorchin Grassland in Inner Mongolia. The region has a temperate continental monsoon climate, characterized by distinct seasons. The average annual temperature in Zhalute Banner over the past 30 years (1990–2020) was 6.6 °C. During the experiment period (2017–2020), the average annual temperature was 6.7 °C. The average annual temperature in the first year of the trial was 5.8 °C, while for the second to fourth years, the average temperatures were 6.67 °C, 7.39 °C and 6.95 °C, respectively. Over the past 30 years, the average annual precipitation was 346.86 mm, the annual total was 2882.7 h of sunshine, and the sunshine percentage was 65%. During 2017–2020, the average annual precipitation was 362.27 mm. The average annual precipitation in the first year of the trial was 336.26 mm, and for the second to fourth years, the average annual precipitation figures were 346.40 mm, 359.15 mm, and 407.28 mm, respectively (data from China Meteorological Data Service Center: https://sci.cma.cn (accessed on 1 January 2021)). The average annual evaporation was 1957 mm, and the average relative humidity was 49%. The soil at the experimental site was classified as chestnut soil (FAO Soil Classification Systems, China soil map based harmonized world soil database (HWSD (v1.1) (2009), National Tibetan Plateau /Third Pole Environment Data Center). The primary characteristics of Chestnut soil are a chestnut-colored upper profile and a lower horizon with filiform, mottled, or reticulate calcic layers. The vegetation is typical grassland, characterized by a relatively uniform canopy without shrubs and a total cover of approximately 30–40%. The dominant species in the vegetation community are Stipa grandis, Cleistogenes chinensis, and Leymus chinensis, which are characteristic of an ecosystem type widely distributed across the Eurasian steppe.

2.2. Design of Experiments

Experimental plots were established in a moderately degraded grassland (classified according to national standard GB 19377-2003 Parameters for degradation, sandification, and salification of rangelands) with flat terrain and evenly distributed vegetation [17]. In early June 2017, enclosure treatments were applied to the plots by setting up fences to prevent grazing. A randomized block design was employed, with treatments including nitrogen (N) fertilization, phosphorus (P) fertilization, a combination of nitrogen and phosphorus (NP) fertilization, and a combination of nitrogen, phosphorus, and potassium (NPK) fertilization. A control (CK) group was established with no fertilization. Each treatment had three 30 × 5 m plots, and the five treatment groups included a total of 15 plots. A 2 m buffer zone was established between adjacent plots. The fertilizers used in the experiment were as follows: urea (N ≥ 46%), superphosphate (P₂O₅ ≥ 44%), and potassium oxide (K₂O ≥ 51%). Treatments consisted of four nutrient addition regimes: (1) control (CK): No fertilization; (2) N: urea 180 kg/ha; (3) P: superphosphate 180 kg/ha; (4) NP: urea 90 kg/ha + superphosphate 90 kg/ha; and (5) NPK: urea 90 kg/ha + superphosphate 90 kg/ha + potassium oxide 40 kg/ha. Based on weather forecasts, artificial fertilization was conducted in each plot prior to rainfall.

2.3. Sample Collection and Testing

During the peak growing seasons (June–August) of 2020, three 1 × 1 m quadrats were randomly established within each plot, resulting in a total of 45 quadrats. Plants were classified into four functional groups: annual and biennial plants (AB), perennial forbs (PF), perennial bunchgrasses (PB), and perennial leguminous plants (PL). The height, cover, and density of plants within each quadrat were measured, with clumping grasses recorded by the number of tussocks and other grasses recorded by individual plants. Plant natural height was measured using a tape measure, and vegetation cover was estimated visually. Density is the number of individuals/the square area. The aboveground biomass of all plant species within each quadrat was collected by species, and the samples were brought to the laboratory and oven-dried at 70 °C for 48 h to determine the biomass of different plant species. We used the method of single diagonal sampling to collect three soil samples of 0 to 20 cm from each square with a soil drill of 2.5 cm in diameter [25]. Soil samples were collected from each plot by drilling three holes using the single-diagonal method. The samples were then pooled, homogenized, and transferred into liquid nitrogen containers for transport to the laboratory for soil microbial analysis. High-throughput sequencing was used to classify and quantify the species of bacteria and fungi in the soil microbial community [26].

DNA was extracted from 0.25 g soil microbial samples using the Power Soil^® DNA lsolation Kit (Aide Technology, Beijing, China), and steps refer to the instructions. The full-length primer sequences of the bacterial 16S rRNA V1~V9 region are as follows: 27F (5′-AGRGTTTGATYNTGGCTCAG-3′); 1492R (5′-TASGG HTACCTT-GTTASGACTT-3′). The full-length ITS rRNA primer sequence of the fungus is as follows: ITS1 (5′-CTTGGTCATTTAGAGGAAGTAA-3′); ITS4 (5′-TCCTCCGCTTATTGATATGC-3′).

PCR amplification system: KOD FX Neo Buffer (2×) 10 µL, dNTP (2 mmol/L) 4 µL, KOD FX Neo (TOYOBO company, Osaka, Japan) 0.4 µL, 5 ng DNA template, 1 µL of forward primer (10 µmol/L), 1 µL of reverse primer (10 µmol/L), 10 µL supplemented with ddH2O. PCR reaction program: 95 °C pre-denaturation for 5 min; denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, 72 °C for 1 min, 35 cycles; extending at 72 °C for 5 min. The PCR products were detected by 2% agarose gel electrophoresis, and the concentration was determined by using a Nano Drop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

The above PCR products were purified and quantified to construct the SMRT Bell sequencing library. After passing the quality inspection, PacBio Sequel was used for sequencing. The original data were corrected by SMRT Link and version 8.0 software to obtain CCS (circular consensus sequencing) sequence. The CCS sequence of each sample was identified according to the barcode, and the chimera sequence was removed using Usearch (V10.0) software to obtain a high-quality CCS sequence. The forward and reverse primers were identified by cut adapt v2.7 (error rate 0.2). The CCS sequences without primers were discarded, and the CCS length was filtered. The sequences with 97% similarity level were clustered by the RDP classifier Bayesian algorithm, and the OTUs (operational taxonomic units) were filtered by 0.005% of all sequences as the threshold. The number of OTUs in soil samples was obtained, and the OTU-Venn diagram was drawn. Based on the Silva and UNITE databases, the OTUs of bacteria and fungi were classified and annotated, and the species of each OTU’s representative sequence was annotated. The information and richness of bacteria and fungi were obtained.

2.4. Data Analysis

The Shannon diversity indicator of each plot was calculated:

$$H=-\sum _{i=1}^s{(P}_{i}ln{P}_i)$$

where S represents the total number of plant species in the quadrat, and P_i is the ratio of the number of individuals in the group i to the total number of individuals in the community [8].

All data used in this study met the Shapiro–Wilk test of normality and the Levene’s test of homogeneity of variance. One-way ANOVA analysis with Tukey’s HSD test was conducted to evaluate the effects of different fertilization treatments on aboveground biomass, diversity, height, density, and cover of plant and soil microbial community. To analyze the differences in the soil microbial composition among different fertilization treatments, the nonmetric multidimensional scaling (NMDS) based on Bray–Curtis distance was run, using the Envfit function from the Vegan package in R (R Core Development Team). The goodness-of-fit for the data points was estimated by the stress value, where the stress value < 0.2 indicates that the result is acceptable.

3. Results

3.1. Effect of Different Fertilization Treatments on the Plant Community

Compared with the CK group, the addition of N, P, NP, and NPK significantly increased the aboveground biomass of the plant community (Figure 1a) while decreasing species diversity (Figure 1b). The effects of different fertilization treatments on plant functional groups were different. After four years of fertilization, the height of AB in the P addition group was significantly higher than that in the CK, N, and NP addition groups, but not significantly different from that in the NPK addition group (Figure 2a). There was no significant difference in height and aboveground biomass of PF in CK, N, P, NP and NPK addition groups (Figure 2b,n). The density of AB in the P addition group was significantly higher than that in the other four groups (Figure 2e). The cover of AB in the N, P, NP, and NPK addition groups was significantly higher than that in the CK group (Figure 2i). The aboveground biomass of AB in the P and NPK addition groups was significantly higher than in the CK group (Figure 2m). The density of PF in the CK group was significantly higher than that in the other four groups (Figure 2f). The cover of PF in the N, P, and NPK groups was significantly lower than that in the CK group (Figure 2j). The height of PB in the NP group was significantly higher than that in the other four groups (Figure 2c). The density of PB in the P, NP, and NPK groups was significantly higher than that in the CK group (Figure 2g). There was no significant difference in cover of PB in CK, N, P, NP and NPK addition groups (Figure 2k). The aboveground biomass of PL in the N group was significantly higher than that in the CK group (Figure 2p). There was no significant difference in height, density and cover of PL in CK, N, P, NP and NPK addition groups (Figure 2d,h,l).

3.2. Effect of Different Fertilization Treatments on the Microbial Community

The effects of different fertilization treatments on the community structure of fungi and bacteria were different (Figure 3). The application of N, P, and NP significantly changed the composition of the fungal community, while there was no significant difference between the NPK treatment and the control (Figure 3a). Fertilization had little effect on bacterial community composition, and there was no significant difference under different fertilization treatments (Figure 3b). In addition, the application of N and NP significantly reduced fungal species diversity (Figure 4a) but had no significant effect on bacterial richness (Figure 4b).

4. Discussion

Our study showed that fertilization significantly increased the aboveground biomass of the community, regardless of the type of nutrient added, which also indicates that the degraded grassland is limited by nitrogen and phosphorus. The increase in aboveground biomass under different nutrient additions was mainly related to the response of different plant functional groups. N fertilizer addition unexpectedly increased perennial legume biomass (Figure 2p), which is different from previous studies, potentially due to suppressed N₂ fixation overriding initial growth inhibition [27]. Drought or low temperature may lead to the decrease of nitrogenase activity, and the growth of leguminous plants can be maintained by nitrogen application [28]. P fertilizer addition significantly increased aboveground biomass of annual and biennial plants. Generally, annual and biennial plants have higher phosphorus absorption efficiency and growth rates, and P fertilizer addition can directly alleviate the phosphorus limitation and increase their photosynthetic rate and resource competitiveness [29], which is also reflected in the increases in height, cover, and density. In addition, it has been suggested that P fertilizer addition may reduce plant dependence on arbuscular mycorrhizal fungi (AMF), and annual and biennial plants (non-mycorrhizal species) may benefit from this [30]. In our study, compared with the CK, we found that the effect of P fertilizer alone on characteristics of perennial leguminous and perennial bunchgrass plant communities was mostly not significant. It is well known that the rooting density of graminaceous species is very high, whereas several leguminous species have the ability to mobilize soil P by the excretion of carboxylates [31]. P availability may be strongly locally and temporarily improved in low-P soils by the release of carboxylates [31]. Therefore, the application of phosphate fertilizer alone may have little effect on gramineous and leguminous plants. The NP fertilizer addition significantly increased aboveground biomass of perennial bunchgrasses, mainly because perennial bunchgrasses usually have more developed root systems and can use resources more efficiently. The improvement of N and P availability can promote tillering ability and photosynthetic efficiency of perennial bunchgrasses [1]. Thus, their height and density increased significantly. We found that the addition of NPK is beneficial to the growth of both biennial and perennial grasses, increasing the cover of annual and biennial plants and the density of perennial bunchgrasses. This is related to the fact that improved nutrient availability enhances the ability of annual, biennial, and perennial bunchgrasses to compete for light [1]. In addition, previous studies have shown that the addition of K may alleviate the soil cation imbalance caused by phosphorus, enhance the disease resistance of perennial bunchgrasses, and enhance the resistance of annual grasses to drought stress [1,12,29]. Perennial forbs typically have high niche differentiation and resource conservation strategies, but in this study the response to fertilization was generally negative, which may be caused by increased competition for light after nutrient mitigation [32]. We found that the plant height of perennial forbs was lower in this study. The density and coverage of other functional groups increased after fertilization, while perennial forbs decreased, which also supported the intensification of light competition against perennial forbs.

Although fertilization increased plant productivity, it significantly decreased plant diversity. On the one hand, it is caused by the intensification of light competition among different plant functional groups [29]. On the other hand, it may also be affected by changes in soil properties. Previous studies have shown that long-term nitrogen fertilizer addition reduces soil pH, especially when nitrogen and phosphorus are applied simultaneously [28]. Nitrogen-induced H+ release, in conjunction with metal ion fixation of phosphorus, further reduces pH and leads to soil acidification, which can be an important cause of the decline in plant diversity [10]. When phosphate fertilizer is applied alone for a long time, it can increase the accumulation of heavy metals in soil and cause toxic effects on plants [15]. The application of NP may trigger relative deficiencies of other nutrients, such as potassium, so the addition of NPK may have relatively little effect on plant diversity.

We also found that four years of fertilization also had important effects on soil microbial community composition and structure. Fertilization changed the community composition and diversity of fungi but had relatively little effect on bacteria. Fungi are K-strategists, which grow slowly and adapt to resource-poor environments, while bacteria are mostly r-strategists, which respond quickly to resource changes and have strong adaptability to environmental changes [21]. Most studies have demonstrated that fungi are sensitive to soil pH, soil acidification leads to a decrease in fungal diversity, and bacteria have a wide range of adaptation to pH changes [33]. In addition, some fungi are closely related to host plants, such as AMF, and nutrient enrichment leads to a decrease in plant photosynthetic products transferred below ground, and a decrease in plant diversity may also contribute to the decrease in fungal diversity [22]. The decrease in plant and microbial diversity caused by fertilization is not conducive to the long-term stability of grassland communities. Some studies have shown that mowing can mitigate the adverse effects of nutrient addition on plant diversity by reducing the dominant position of grasses [34]. Compared with other fertilization treatments, NPK addition has fewer negative effects on plant and microbial communities and is also conducive to the recovery of community productivity, which may be a more appropriate fertilization strategy.

5. Conclusions

Our findings demonstrate that fertilization promoted the recovery of plant community productivity in general, mainly because the increase in nutrient availability promoted the growth of different plant functional groups, especially annual and biennial plants and perennial bunchgrasses. However, fertilization had an adverse effect on biodiversity, reducing plant and soil fungal diversity. Therefore, when applying fertilizer to restore degraded grassland, plant community productivity can be quickly restored through fertilization, but it is not conducive to the restoration of community stability. Other interference measures should be taken to promote the restoration of biodiversity. In conclusion, this study provides new insights into the relationships between different types of fertilization and plant community productivity and biodiversity in degraded grasslands over a four-year period, which is critical for evaluating the effect of fertilization on the restoration of degraded grasslands.