Statistical and bioinformatics analyses

Statistical analysis was carried out using SPSS software (v.26.0.0.2). Analysis of variance (ANOVA) was employed to assess the significance of differences among various groups (*p<0.05, **p<0.01, and ***p<0.001 were considered to be significant). Bar graphs were plotted with Prism 9.5 (GraphPad Software, LLC). Alpha diversity for each sample was assessed based on four diversity indices: Chao1, Shannon, Simpson, and Ace. All sample indexes were calculated using QIIME (v.1.9.1) (Caporaso et al., 2010), and Kruskal-Wallis H test was used to evaluate the differences in alpha diversity index between different treatments. Rarefaction curves based on the Shannon and Ace indices were plotted for each sample, assessing the effectiveness of sequencing depth. Principal Coordinate Analysis (PCoA) based on the Bray-Curtis distance was performed using the R Package "vegan (v2.5-3)" to assess the similarity of microbial community structures among samples (Oksanen et al., 2016). Additionally, we performed a non-parametric PERMANOVA analysis to determine the significance of differences in microbial community structures between sample groups. Species composition circle plots, distribution histograms, heatmaps, and correlation heatmaps between species and environmental factors were generated with the ggplot2 (v.3.3.5) package. The correlations between soil factors and among soil factors and the root-associated microbial communities of the three alfalfa varieties were calculated by the "linkET" package in R, and generated a mantel test network graph. Visualize the shared and unique microbial taxa among different treatment samples based on a Venn diagram. Identify significantly different species between groups using the ANCOM differential analysis provided by QIIME2. Linear discriminant analysis effect size (Lefse), for which the logarithmic LDA score was set to 3.0 with statistical significance (p < 0.05), and the functional potential of a bacterial community predicted by PICRUSt2 were both performed on the Majorbio Cloud platform ⁴ .