Cereal crops such as wheat (Triticum aestivum) and maize (Zea mays) form the cornerstone of global food security, collectively providing more than half of the caloric intake for the human population and serving as critical feed and bioenergy resources. As global agriculture faces mounting challenges from soil degradation, climate variability, and the need to reduce agrochemical dependence, the plant-associated microbiome has emerged as a key frontier for developing sustainable and resilient cropping systems (1, 2). Among plant microbiomes, the root-associated microbial community, encompassing the rhizosphere (the soil region influenced by roots) and the root endosphere (the internal root tissue), play central roles in nutrient acquisition, stress tolerance and disease suppression (3, 4).

The rhizosphere is a metabolically active hotspot where root exudates comprised of sugars, amino acids and secondary metabolites shape microbial recruitment and activity (5, 6). Plants release up to 40% of their photosynthetically fixed carbon into this zone, establishing gradients of nutrients and signaling molecules that support copiotroph, metabolically versatile microorganisms (7). By contrast, the root endosphere represents a highly selective environment, where only a limited subset of microbial taxa can penetrate plant tissues, tolerate host immune responses and establish mutualistic or commensal relationships (7). This compartmental dichotomy, which is an open, dynamic rhizosphere versus a controlled, selective endosphere, creates distinct ecological filters that shape microbial diversity and function. The rhizosphere, enriched by root exudates and soil interactions, harbors a metabolically versatile community dominated by saprotrophic and copiotroph taxa (3). In contrast, the endosphere is a host regulated, nutrient-limited niche where only specialized microbes capable of overcoming plant defenses and establishing stable associations can persist. These contrasting environments impose strong spatial and physiological constraints, leading to compartment-specific microbial assemblages and functional specialization that collectively sustain plant health and nutrient cycling within the root-soil interface (8).

While bacterial community assembly at the root-soil interface has been well studied, fungal communities remain comparatively underexplored, despite their central roles in nutrient mineralization, carbon turnover, plant growth promotion, and pathogen antagonism (9, 10). Recent multi-kingdom analyses suggest that fungi, like bacteria, are strongly influenced by spatial niche, plant genotype, and soil conditions, but may exhibit unique assembly dynamics due to their filamentous growth and saprotrophic-symbiotic versatility (11–13). Understanding the functional partitioning and interaction networks of fungal taxa within the root and rhizosphere compartments is therefore essential for unraveling the ecological foundations of plant-microbe symbioses in cereals.

In addition to intrinsic ecological processes, agronomic interventions such as microbial inoculants and biocontrol treatments can reshape plant microbiomes. Among these, Bacillus spp. are of particular interest due to their ability to produce antimicrobial peptides, phytohormones, and signaling molecules that modulate both plant physiology and microbial community dynamics (14, 15). Bacillin 20 has gained attention for its bioactivity and potential for microbiome modulation. It was initially characterized as thuricin 17, later redefined based on revised insights into its composition and structure, is a peptide produced by Bacillus thuringiensis NEB 17 and belongs to the bacteriocin family of ribosomally synthesized peptides that inhibit related microbial strains (16). Bacillus thuringiensis-derived bacillin 20 has been shown to enhance plant growth, induce stress tolerance, and modify soil microbial interactions in oilseed and legume systems. Although bacteriocins are classically viewed as narrow-spectrum antibacterial agents, their deployment in the rhizosphere has ripple effects on the broader microbial community by altering bacterial competition, modifying resource availability, and indirectly influencing fungi through cross-kingdom interactions (17). However, its broader ecological impacts on fungal community composition, diversity, and co-occurrence networks within cereal root compartments remain largely unknown. Here, we investigate how spatial niche (root vs. rhizosphere), host species (wheat vs. corn), and bacillin 20 treatment collectively shape fungal community structure and function in cereal crops. Using ITS-based amplicon sequencing coupled with diversity, indicator, and network analyses, we hypothesized that compartmental identity (root vs. rhizosphere) acts as the primary ecological filter shaping fungal community diversity, composition, and network organization in cereals, and that host genotype and bacillin 20 further modulate this assembly process by influencing the abundance and functional roles of key fungal taxa. By integrating taxonomic and network perspectives, this study provides new insights into the hierarchical organization and ecological specialization of fungal communities in cereals and establishes a framework for microbiome-informed biocontrol strategies aimed at improving crop productivity and resilience.

Field experiments were conducted at McGill University’s Emile A. Lods Research Centre (45°28′ N, 73°45′ W) using corn (Zea mays) hybrid Pioneer 3921 and wheat (Triticum aestivum) Chamran 2. Seeds of corn and wheat were collected from McGill University’s Emile A. Lods Research Centre. Bacillin 20 was applied at 10⁻⁹ M and 10⁻¹¹ M, concentrations previously shown to enhance germination, growth, and stress tolerance in crops such as soybean, and canola, and also Arabidopsis (17, 19, 21). Five treatment regimes were established: T1-control; T2- bacillin 20 (10⁻⁹ M) foliar spray along with herbicide; T3- bacillin 20 (10⁻¹¹ M) foliar spray along with herbicide; T4 -herbicide spray at first stage + foliar spray of bacillin 20 (10⁻⁹ M) at 6 leaf stage; T5- herbicide spray at first stage + foliar spray of bacillin 20 (10⁻¹¹ M) at 6 leaf stage. The experiment followed a randomized complete block design with four replicates per treatment. Each 5 m plot (14 rows) was established on clay loam soil, with 19 cm between rows and 80 cm between blocks.

At the flowering stage, five healthy plants per replicate were randomly collected from each treatment. Entire root systems were excavated to a depth of ~ 15 cm. Fine roots were carefully separated, and adhering soil was retained as rhizosphere material. Approximately 0.5 g of root tissue and 1 g of rhizosphere soil per plant were pooled to generate composite samples (36 in total). All samples were transported on ice and stored at -20°C until DNA extraction. Nodulation parameters were not included in this analysis to avoid confounding effects of genotype–environment interactions. The study focused on characterizing fungal community structure and diversity rather than quantifying symbiotic nitrogen fixation.

We followed the previously established methods (17, 22, 23) for DNA extraction, PCR and sequencing. In brief, total genomic DNA was extracted from 100 mg of root tissue using the DNeasy Plant Mini Kit (Qiagen, Toronto, Canada) and from 250 mg of rhizosphere soil using the DNeasy PowerSoil Pro Kit (Qiagen). DNA concentration and purity were assessed using a NanoDrop spectrophotometer, and quality was verified by 1% agarose gel electrophoresis. For fungal community profiling, the internal transcribed spacer (ITS2) region was amplified using the primer pair CS1_ITS3_KYO2 (5′ACACTGACGACATGGTTCTACAGATGAAGAACGYAGYRAA-3′) and reverse primer CS2_ITS4 (5′TACGGTAGCAGAGACTTGGTCTTCCTCCGCTTATTGATATGC-3′) (17, 24). PCR reactions (25 µL) contained 1.5× Platinum™ Direct PCR Universal Master Mix (ThermoFisher), 0.25 µM of each primer, 1.5× Platinum™ GC Enhancer, and approximately 20 ng of DNA template. Amplicons were visualized, purified, and sequenced on an Illumina MiSeq platform (2 × 300 bp paired end reads) at the McGill Genome Québec Innovation Centre (Montreal, Canada).

Raw ITS reads were processed in R (v4.0.2) using the DADA2 pipeline (25). Sequences were quality-filtered (maxEE = c (2, 2); truncQ = 2), denoised, merged, and checked for chimeras. Amplicon sequence variants (ASVs) were inferred and assigned taxonomy using the UNITE v9.0 fungal reference database (26). ASVs identified as plant-derived or unclassified eukaryotes were removed. The dataset was rarefied to the minimum read depth using the vegan package (v2.5-6). Alpha diversity indices (Observed Richness, Chao1, Shannon, Simpson) were calculated using phyloseq, while beta diversity was assessed based on Bray-Curtis dissimilarities followed by principal coordinates analysis (PCoA). Significant differences in community structure were evaluated via PERMANOVA (adonis, 999 permutations) (27). ANOVA with Tukey’s HSD (agricolae v1.3-3) (28) was used for parametric data, and Wilcoxon rank-sum or Kruskal–Wallis with Dunn’s post hoc tests were applied for non-parametric comparisons.

Differentially abundant taxa were identified using LEfSe (Kruskal-Wallis p < 0.05; LDA > 3.0) (29) and edgeR (30). Indicator species were determined using indicspecies (31), and core taxa were defined as those present in ≥ 80% of samples within each compartment. Microbial co-occurrence networks were generated from filtered ASV tables (relative abundance > 0.01%; presence ≥ 20% of samples) using Spearman correlations (p > 0.6; FDR-adjusted p < 0.05) (32). Networks were visualized in igraph (33), and node roles were classified according to Zi–Pi analysis (34) into module hubs, connectors, and peripheral taxa.

Principal coordinates analysis (PCoA) of Bray-Curtis dissimilarities revealed clear segregation between root and rhizosphere fungal communities in both crops (Fig. 2). In corn (Fig. 2A), the first two axes explained 41.85 and 15.91% of total variation, whereas in wheat (Fig. 2B), they accounted for 57.64 and 10.82%, respectively. PERMANOVA analysis confirmed that compartment identity significantly influenced fungal composition (corn: R = 0.402, p = 0.001; wheat: R = 0.532, p = 0.001). Rhizosphere assemblages clustered more tightly, reflecting stable soil and exudate associated fungal consortia, whereas root-associated communities formed discrete clusters indicative of stronger host filtering and niche specialization. These findings demonstrate that spatial niche differentiation between root and rhizosphere compartments is the primary determinant of fungal community composition, consistent across both host species and treatment conditions.

This study provides an integrative view of how spatial niche (root vs. rhizosphere), host genotype (corn vs. wheat), and Bacillus-based bacteriocin (bacillin 20) collectively shape the structure and organization of fungal communities in corn and wheat microbiomes. Through ITS-based amplicon sequencing, we showed that compartment identity is the principal determinant of fungal diversity, composition, and network topology, surpassing host and treatment effects. The results demonstrated a hierarchical assembly process wherein the rhizosphere harbored diverse, interaction-rich fungal consortia, while the root maintained a more selective, host-filtered subset of functionally specialized taxa. These findings advance current understanding of compartmental microbiome ecology and highlight how biocontrol interventions interact with spatial filtering to modulate plant-associated fungal networks.

In summary, this study establishes that spatial compartmentalization is the dominant determinant of fungal community assembly in cereals, with host genotype and Bacillus-derived metabolites serving as modulatory factors. Rhizosphere mycobiomes exhibit high diversity and interaction complexity, while root mycobiomes are selective, structured, and functionally specialized. Bacillin 20 further enhanced compartmental functional divergence, promoting saprotrophic activity and nutrient cycling in rhizospheres and biosynthetic and redox-linked processes in roots. This work contributes to a unified ecological framework where spatial filtering, host adaptation, and microbial signaling converge to organize cereal root microbiomes, providing a foundation for developing microbiome-informed biocontrol and sustainable agriculture strategies.