Shannon‒Wiener diversity index (H): (4)
Pielou evenness index (E): (5)
where Hri represents the relative height,Cri indicates the relative coverage,Dri depicts the relative density,Bri is the relative biomass, S represents the number of species in the community, and IVi is a representation of the importance value of species i.
2.4. Soil properties analysis
The pH value was determined by the acidometer method (water‒soil mass ratio of 2.5:1; PHS-3G digital pH meter, Shanghai) (Cao et al., 2017). The soil water content (SWC) was determined gravimetrically by drying the soil samples (105 °C, 24 h). The bulk density (BD) was measured gravimetrically after oven-drying (105 °C, 24 h). The soil organic carbon (SOC) was determined using the dichromate oxidation method (Walkley and Black, 1934). Total phosphorus (TP) was measured with the Mo-Sb colorimetric method using a spectrophotometer (Lambda25 UV-visspectrometer, United States). Kjeldahl nitrogen (KN) was determined using the Kjeldahl method.
2.5. DNA extraction and Illumina NovaSeq sequencing
Total genomic DNA from samples was extracted using the CTAB method. DNA concentration and purity were monitored on 1 % agarose gels. According to the concentration, DNA was diluted to 1 ng/µL using sterile water.
ITS rRNA genes of distinct regions(ITS1-1F) were amplified using specific primers (ITS1-1F-F (5’-CTTGGTCATTTAGAGGAAGTAA-3’), and ITS1-1F-R (5’-GCTGCGTTCTTCATCGATGC-3’) with the barcodes (Bokulich and Mills, 2013; Hugerth et al., 2014). All PCRs reactions were carried out with 15 µL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs), 2 µM of forward and reverse primers, and approximately 10 ng of template DNA. Thermal cycling consisted of initial denaturation at 98 ℃ for 1 min, followed by 30 cycles of denaturation at 98 ℃ for 10 s, annealing at 50 ℃ for 30 s, and elongation at 72 ℃ for 30 s, and finally 72 ℃ for 5 min. The same volume of 1X TAE buffer was mixed with the PCR products, and electrophoresis was performed on a 2% agarose gel for detection. PCR products were mixed in equidensity ratios. Then, the mixture of PCR products was purified with a Qiagen Gel Extraction Kit (Qiagen, Germany).
Sequencing libraries were generated, using a TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer’s recommendations and index codes were added. The library quality was assessed on a Qubit@ 2.0 Fluorometer (Thermo Scientific). Finally, the library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated.
2.6. Statistics and analysis
All data are shown as the mean and standard error. Diversity metrics were calculated using the core-diversity plugin within QIIME2. Feature level alpha diversity indices, such as the Simpson index, Chao1 richness index, and Shannon diversity index were calculated to estimate the fungal diversity. Data on soil physicochemical properties and microbial diversity indices were analyzed by independent T-test, one-way ANOVA, multifactor ANOVA and Pearson’s correlation analysis using SPSS 26.0. Bar graphs were generated in OriginPro 2021 (Originlab Corporation, USA). Principal coordinate analysis (PCoA) using version R4.2.1, and Bray-Curtis distances were used to visualize the effects of grazing exclusion and grassland type on fungal β-diversity. PERMANOVA (permutational multivariate analysis of variance) was performed in the ’vegan’ package of R using the ANOSIM function to test for the significance of differences in community and plotted using the ’ggplot2’ package in R 4.2.1. Finally, we implemented structural equation modeling (SEM) based on the lavaan package (Rosseel., 2011) to assess the effects of grazing exclusion and grassland type on fungal diversity through changes in plant and soil abiotic variables, the statistical analyses of which were carried out using R version 4.2.1.
Results
3.1. Soil physicochemical properties as affected by grazing exclusion and grassland type
In the 0-5 cm soil layer (Table 1), grazing exclusion significantly affected the physicochemical parameters of all three studied grassland types. In the temperate desert, grazing exclusion resulted in a 139.34% increase in SOC and a 36.36% increase in TP. In the temperate steppe, SWC and SOC increased, while BD was reduced by 16.67%. In the mountain meadow, KN increased. Grazing exclusion significantly increased the differences in SWC, SOC, KN and N:P among the three grassland types, while the final pH, BD, TP, C:N and C:P values were consistent with the results before exclusion. In the 5-10 cm soil layer, grazing exclusion significantly increased TP in temperate deserts by 34.1% while significantly decreasing BD by 9.8% and N:P by 47.1%. In temperate steppes, none of the effects of grazing exclusion on the measured soil physicochemical properties were significant. In mountain meadows, grazing exclusion resulted in a significant increase in SWC and SOC. After grazing exclusion, C:N was significantly higher in temperate deserts than in temperate steppe and mountain meadows, while SOC and BD were not significantly different between temperate steppe and mountain meadows.
Table 1 Soil physicochemical properties as affected by grazing exclusion and grassland type.