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.