The agents of Paenibacillus polymyxa enhance maize resistance to Fusarium stalk rot by regulating endophytic bacteria and soil microbial communities

Collect and mix PCR products of identical components. Recover target bands through 2% agarose gel electrophoresis and purify using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). Verify purified products via 2% agarose gel electrophoresis and quantify using a Quantus™ Fluorometer (Promega, USA). Construct libraries with the NEXTflex™ Rapid DNA-Seq Kit (Bioo Scientific, USA), including: (1) adapter ligation; (2) removal of self-ligated adapters using magnetic beads; (3) PCR amplification for template enrichment; (4) magnetic bead purification of PCR products to obtain final libraries. Perform paired-end sequencing on the Illumina MiSeq PE300 platform (Illumina Inc., USA; Shanghai Majorbio Bio-pharm Technology Co., Ltd.).
Control raw sequencing data quality with fastp (v0.20.0). Assemble sequences using FLASH (v1.2.7). Cluster OTUs at 97% similarity and remove chimeras using UPARSE (v7.1). Annotate taxonomic classifications with the RDP Classifier (v2.2) against the SILVA 16S rRNA database (v138) at 70% confidence threshold. Input microbiome data as matrices and calculate Alpha diversity indices for bacterial and fungal communities at the OTU level. Analyze differential species across treatments using Venn diagrams to identify core OTUs. Measure Beta diversity variations between groups via Unifrac (weighted/unweighted, sorted by distance metrics appropriate for this study) and Bray-Curtis distances, testing for significant differences. Validate group separation significance through Partial Least Squares Discriminant Analysis (PLS-DA). Perform multi-group comparisons of intergroup species differences using the Kruskal-Wallis rank-sum test. Analyze correlations among environmental factors, samples, and microbiota using RDA/CCA methods. Construct co-occurrence networks to examine evolutionary relationships between species. Predict bacterial and fungal functions using FAPROTAX and PICRUSt2, respectively. Conduct all analyses on the Majorbio I-Sanger Cloud Platform (www.i-sanger.com).
According to the above operation, the relationship between the number of otu and the species of each sample was obtained, and these relationships were analyzed and predicted.