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.

收集并混合组分一致的聚合酶链式反应（Polymerase Chain Reaction，PCR）扩增产物。通过2%琼脂糖凝胶电泳回收目的条带，并使用AxyPrep DNA凝胶提取试剂盒（Axygen Biosciences，美国加利福尼亚州联合城）进行纯化。通过2%琼脂糖凝胶电泳验证纯化后的产物，并使用Quantus™荧光计（美国普洛麦格公司）进行定量。使用NEXTflex™快速DNA测序试剂盒（美国Bioo Scientific公司）构建文库，具体步骤包括：(1) 接头连接；(2) 利用磁珠去除自连接接头；(3) PCR扩增以富集模板；(4) 对PCR产物进行磁珠纯化，最终获得测序文库。在Illumina MiSeq PE300测序平台（美国Illumina公司；上海美吉生物医药科技有限公司）上进行双端测序。
使用fastp（v0.20.0）对原始测序数据进行质量控制。使用FLASH（v1.2.7）对测序序列进行拼接。以97%的相似性阈值对操作分类单元（Operational Taxonomic Unit，OTU）进行聚类，并使用UPARSE（v7.1）移除嵌合体序列。使用RDP分类器（v2.2），以70%的置信度阈值，比对SILVA 16S rRNA数据库（v138）进行物种分类注释。将微生物组数据以矩阵形式输入，并在OTU水平上计算细菌和真菌群落的α多样性指数。使用维恩图分析不同处理组间的差异物种，以识别核心OTU。通过加权/未加权Unifrac距离（选用适配本研究的距离度量方式）和Bray-Curtis距离分析组间β多样性差异，并检验其显著性。使用偏最小二乘判别分析（Partial Least Squares Discriminant Analysis，PLS-DA）验证组间分离的显著性。使用Kruskal-Wallis秩和检验对多组间的物种差异进行组间比较。使用冗余分析（Redundancy Analysis，RDA）与典范对应分析（Canonical Correspondence Analysis，CCA）方法分析环境因子、样本与微生物群落之间的相关性。构建共现网络以探究物种间的进化关联。分别使用FAPROTAX和PICRUSt2预测细菌和真菌的群落功能。所有分析均在美吉I-Sanger云平台（www.i-sanger.com）上完成。
基于上述实验流程，获得各样本中OTU数量与物种之间的关联，并对这些关联进行分析与预测。