Spatial niche differentiation and bacillin 20 mediated modulation shape fungal community diversity and network structure in roots and rhizospheres of corn and wheat

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Kabir, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8164785/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background The phytomicrobiome plays a crucial role in nutrient cycling, plant growth promotion, and stress resilience in cereals. However, how spatial niche differentiation (root vs. rhizosphere), host genotype and microbial biocontrol agents jointly shape fungal community structure and function remains poorly understood. Results Using ITS-based amplicon sequencing, we examined the effects of compartmental identity, host species ( Zea mays and Triticum aestivum ) and a Bacillus thuringiensis -derived bacteriocin (bacillin 20) on fungal community diversity, composition and co-occurrence networks. Alpha and beta diversity analyses revealed strong compartmental segregation, with rhizosphere communities exhibiting greater richness and taxonomic heterogeneity than roots, which harbored more even and host-filtered assemblages. Setophoma , Myrmecridium , Periconia , and Talaromyces as dominant root taxa, while Fusarium , Chaetomium , Alternaria , and Exophiala were enriched in rhizospheres. Bacillin 20 treatments, particularly at higher concentrations, enhanced the abundance of saprotrophic guilds and increased network complexity in rhizosphere communities. In contrast, root-associated networks were more compact and modular, reflecting stronger host-mediated ecological filtering. Functional annotation using FUNGuild revealed that saprotrophic fungi predominated in corn, whereas symbiotrophic guilds dominated wheat, highlighting host-specific functional partitioning. Conclusion Our findings demonstrated that spatial compartmentalization is the primary driver of fungal community assembly in cereals, with host genotype and Bacillus -mediated modulation exerting secondary but significant effects on composition and trophic function. Bacillin 20 acted as an ecological modulator, promoting saprotrophic activity and metabolic flexibility in rhizospheres while maintaining stable, symbiotic networks within roots. This study establishes a hierarchical model of cereal mycobiome organization, providing a framework for phytomicrobiome-informed biocontrol and sustainable crop management strategies. Bacillin 20 Rhizosphere Cereal crops Community assembly Network ecology FUNGuild Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction 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. Methods Extraction and purification of bacillin 20 Bacillin 20, a bacteriocin originally isolated from Bacillus thuringiensis NEB17, was extracted and purified following previously established protocols with minor modifications ( 16 , 18 ). Briefly, B. thuringiensis NEB17 was grown in King’s B medium for 48 h at 28 ± 2°C on a rotary shaker (150 rpm). Cultures were harvested at late-log phase (OD₆₀₀ ≈ 1.0), and 2 L of culture broth was extracted via butanol phase partitioning. The organic phase was concentrated by rotary evaporation under vacuum, resuspended in 18% acetonitrile, and subjected to high-performance liquid chromatography (HPLC; Waters Corp.) using a reverse-phase C18 column. Elution was performed with a linear acetonitrile water gradient containing 0.1% trifluoroacetic acid ( 19 – 21 ). Fractions corresponding to bacillin 20 peaks were pooled, lyophilized, and stored at -20°C until use. Field experimental design and bacillin 20 application 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. Sample collection and processing 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. DNA extraction and ITS amplicon sequencing 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). Bioinformatics and statistical analyses 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 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. Results Alpha diversity patterns of fungal communities in roots and rhizospheres of corn and wheat ITS-based amplicon sequencing revealed strong compartmental and host-specific structuring of fungal diversity (Fig. 1 ). Across both Zea mays (corn) and Triticum aestivum (wheat), rhizosphere samples consistently exhibited higher richness and diversity than root-associated fungal communities. Shannon and Simpson indices (Fig. 1 A-B) showed significantly greater fungal diversity and evenness in the rhizosphere ( p < 0.05), reflecting a broader and more heterogeneous assemblage of soil fungi. In contrast, the root endosphere displayed lower richness (Chao1, ACE, and Observed Richness; Fig. 1 C-E) but higher Pielou’s evenness (Fig. 1 F), suggesting a more selective colonization of endophytic taxa within plant tissues. These patterns mirror those observed for bacterial communities, underscoring compartment identity as the dominant ecological filter with the rhizosphere supporting a diverse, saprotroph-rich fungal pool, while roots maintain a restricted subset of symbiotic or host-adapted lineages. Beta diversity of fungal communities in roots and rhizospheres of corn and wheat 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. 2 A), the first two axes explained 41.85 and 15.91% of total variation, whereas in wheat (Fig. 2 B), 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. Genus-level fungal community composition across hosts and treatments Genus-level composition varied markedly across hosts, compartments, and bacillin 20 treatments (Fig. 3 ). In both crops, rhizosphere microbiomes exhibited greater taxonomic heterogeneity and functional turnover than roots, consistent with more dynamic soil environments. In corn, roots were dominated by Myrmecridium , Setophoma , Talaromyces , and Calophoma , which together represented over 60% of total abundance across treatments. Rhizospheres contained a broader array of genera, including Fusarium , Chaetomium , Alternaria , and Exophiala taxa. Notably, under bacillin 20 treatments T4 and T5, Fusarium and Chaetomium increased in abundance, suggesting treatment-induced enrichment of specific fungal guilds (PERMANOVA, R = 0.402, p < 0.001). In wheat, root communities were dominated by Setophoma , Myrmecridium , Periconia and Neosetophoma , while rhizospheres were enriched in Alternaria , Fusarium , Cordyceps and Chaetomium . Rhizosphere assemblages displayed higher variability across treatments, indicative of greater environmental responsiveness and functional plasticity. Statistical analyses confirmed significant differences between compartments (PERMANOVA, R = 0.532, p < 0.001) and among treatments (ANOVA, p < 0.05). Together, these results show that spatial niche and host identity jointly structure fungal community assembly, with rhizospheres favoring metabolically versatile taxa involved in nutrient cycling, while roots select for a smaller consortium of endophytic and symbiotic fungi. Core fungal microbiome in roots and rhizospheres of corn and wheat Analysis of the core fungal microbiome revealed both conserved and host-specific assemblages (Fig. 4 ). Across Zea mays and Triticum aestivum , core root communities were dominated by Setophoma , Myrmecridium , Periconia , Talaromyces , Fusarium and Neosetophoma , whereas rhizospheres were enriched in Solicoccozyma , Exophiala , Alternaria and Chaetomium . These dominant members of Ascomycota and Basidiomycota indicated functional partitioning between compartments. In corn, Myrmecridium and Fusarium comprised over half of the core root mycobiome, while Fusarium, Talaromyces and Cladosporiunm were more abundant in rhizospheres. In wheat, Setophoma , Periconia , Neostophoma and Fusarium dominated the root core, whereas only three genera – Holtermannia, Fusidium and Zygotorulaspora were present in rhizospheres. The overlap between compartments suggests a conserved core of multifunctional fungi, while host-specific enrichments highlight selective filtering and ecological adaptation. Collectively, these patterns indicate that root-associated taxa are specialized for endophytic lifestyles, whereas rhizosphere taxa perform saprotrophic and decomposer functions, contributing to soil carbon and nutrient turnover. Indicator fungal taxa distinguishing root and rhizosphere compartments Indicator species analysis identified distinct compartment-enriched fungal taxa in both crops (Fig. 5 ). In corn (Fig. 5 A), rhizosphere communities were enriched in Chaetomium , Alternaria , Solicoccozyma and Fusarium , representing saprotrophic decomposers thriving in nutrient-rich soil habitats. Conversely, roots were dominated by Setophoma , Myrmecridium , Talaromyces and Neosetophoma , taxa typical of endophytic or weakly pathogenic fungi adapted to plant tissues. In wheat (Fig. 5 B), rhizosphere-enriched taxa included Exophiala , Cordyceps and Alternaria , while roots favored Setophoma , Periconia and Talaromyces . These associations illustrate strong spatial niche filtering, with rhizospheres harboring functionally diverse decomposers and roots supporting specialized symbiotic or host-interactive taxa. Network structure of fungal communities in roots and rhizospheres Co-occurrence network analysis revealed striking contrasts in network topology between root and rhizosphere fungal communities in both hosts (Fig. S1 ). Rhizosphere networks exhibited higher connectivity, modularity, and inter-phyla associations, indicating greater ecological complexity and cooperative interactions among fungal taxa. In contrast, root networks were more compact, with fewer but stronger connections, suggesting host-mediated ecological filtering and niche specialization. In corn, root networks (Fig. S1 A) were dominated by Ascomycota taxa such as Talaromyces , Schizothecium , and Clohesyomyces , whereas the rhizosphere (Fig. S1 B) displayed increased connectivity among Ascomycota and Basidiomycota taxa, notably Fusidium , Exophiala , and Pullularia . Similarly, in wheat, root networks (Fig. S1 C) were largely formed by Ascomycota and Basidiomycota , while rhizosphere networks (Fig. S1 D) included additional nodes from Mortierellomycota and Chytridiomycota , reflecting expanded functional guild diversity. These results suggest that rhizosphere fungal communities form dense, metabolically flexible interaction networks, whereas root assemblages are highly specialized and structurally constrained, consistent with their adaptation to host-controlled environments. Functional guild composition of fungal communities in corn and wheat FUNGuild analysis revealed clear differences in the trophic composition of fungal communities between roots and rhizospheres, as well as between host species (Fig. S2). Across all samples, fungi were assigned to three major trophic guilds, such as, saprotrophs, pathotrophs and symbiotrophs along with mixed guilds (pathotroph-saprotroph, saprotroph-symbiotroph and pathotroph-symbiotroph). In Zea mays (corn) (Fig. S2A), saprotrophic fungi dominated the overall community, accounting for more than 50% of total relative abundance, followed by mixed trophic modes (pathotroph-saprotroph-symbiotroph and pathotroph-symbiotroph) and a smaller fraction of pathotrophs. The rhizosphere exhibited a higher proportion of saprotrophs and mixed guilds, suggesting active decomposition and nutrient cycling processes, while roots harbored a relatively higher abundance of pathotroph-symbiotroph fungi, indicating selective recruitment of functionally flexible taxa within plant tissues. In Triticum aestivum (wheat) (Fig. S2B), symbiotrophic fungi, including arbuscular mycorrhizal and mutualistic taxa, represented the most abundant functional group, particularly in the rhizosphere, accounting for up to 70% of the total community. Root-associated communities, by contrast, contained greater proportions of pathotrophs and pathotroph–saprotroph guilds, reflecting niche specialization under host control. Overall, the dominance of saprotrophic guilds in corn and symbiotrophic guilds in wheat underscores host- and compartment-specific functional partitioning of fungal communities, consistent with differences in root architecture, exudation patterns and microhabitat conditions. These results demonstrate that spatial niche differentiation and host genotype jointly influence not only taxonomic composition but also the ecological function of cereal-associated mycobiomes. Discussion 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. Spatial niche filtering dominates fungal community assembly The strong separation of fungal communities between root and rhizosphere compartments across both hosts underscores the primary role of spatial niche differentiation in microbiome assembly. Rhizospheres supported higher diversity and richness, dominated by saprotrophic and decomposer fungi, while root endospheres exhibited reduced diversity but higher evenness—an indicator of selective colonization and host control. These findings are consistent with previous reports showing that compartmental filtering exerts a stronger influence on microbial structure than soil type or host genotype ( 2 , 8 , 17 ). Enrichment of Fusarium , Chaetomium , and Alternaria in rhizospheres reflects the copiotrophic and decomposer capacities of these taxa, which thrive in exudate-rich, nutrient-dense microenvironments and contribute to carbon and nitrogen cycling ( 5 , 6 ). In contrast, root-enriched taxa such as Setophoma , Talaromyces , and Myrmecridium exemplify endophytic specialization, capable of surviving in low-nutrient conditions and evading plant immune defenses. This compartment-driven ecological partitioning aligns with models describing microhabitat filtering and host selection as fundamental drivers of microbial diversification at the root-soil interface ( 4 , 8 ). Host genotype modulates core and indicator taxa composition While spatial niche effects dominated, host genotype further refined fungal community structure and functional guild composition. Wheat mycobiomes displayed greater compositional variability and responsiveness to bacillin 20 treatments than those of corn, reflecting higher functional plasticity and environmental sensitivity. Similar host-dependent patterns have been reported in barley ( 35 ), rice ( 36 ) and maize ( 37 ), where variations in root exudate profiles and secondary metabolite composition influence fungal recruitment and persistence. The persistence of Setophoma , Periconia and Talaromyces as core taxa across both hosts indicated an evolutionarily conserved endophytic backbone within cereal crops. In contrast, rhizosphere-specific enrichment of Exophiala , Chaetomium and Cordyceps illustrated the functional divergence toward saprotrophic and decomposer roles. Together, these findings supported a model in which cereal microbiomes comprise a stable, multifunctional core complemented by host-specific taxa that respond dynamically to environmental and physiological contexts ( 3 , 4 ). Biocontrol-mediated modulation of fungal guilds and functional traits Bacillus -derived peptide bacillin 20 significantly altered fungal community composition and functional guild distribution, primarily within the rhizosphere. Treatments T4 and T5 enriched saprotrophic and mixed trophic guilds ( Fusarium , Chaetomium ), consistent with Bacillus -mediated enhancement of organic matter decomposition and nutrient turnover. These results parallel studies in canola ( 17 ), wheat and maize systems ( 9 , 12 ) where Bacillus inoculants stimulated soil nutrient cycling. The limited structural change observed within root-associated communities highlights the buffering capacity of host filtering, which stabilizes the endophytic microbiome against external interventions. Nonetheless, bacillin 20 induced functional differentiation, favoring biosynthetic and redox-active taxa in roots, consistent with reports that Bacillus -derived bacillin 20 influence fungal metabolism and antioxidant responses without disrupting symbiotic stability ( 15 , 38 ). These findings underscore the potential of Bacillus -based treatments as ecological modulators rather than community disruptors, capable of enhancing fungal functionality in a compartment-dependent manner. Network organization reflects ecological specialization Network analyses revealed a clear dichotomy between rhizosphere and root fungal interactions. Rhizosphere networks were highly connected and modular, involving cross-phyla associations among Ascomycota, Basidiomycota, Mortierellomycota, and Chytridiomycota, consistent with the cooperative, resource-sharing nature of soil fungal consortia ( 8 , 39 – 41 ). In contrast, root networks were more compact, dominated by tightly linked Ascomycota taxa, reflecting ecological constraint and host-mediated filtering. This compartmental divergence illustrates a trade-off between metabolic versatility and symbiotic stability. The rhizosphere network’s complexity confers adaptability to environmental fluctuations, whereas the root’s constrained structure ensures stability and efficient host-microbe cooperation ( 3 ). Similar patterns in rice and maize demonstrate that high network modularity enhances functional robustness in open environments, while tightly linked endophyte networks underpin nutrient exchange and host defense ( 42 , 43 ). Ecological and practical implications Together, our results establish a mechanistic framework in which spatial compartmentalization defines the foundation of cereal mycobiomes, host genotype refines community composition, and biocontrol treatments act as functional modulators within this hierarchy. The rhizosphere emerges as a metabolically dynamic and interaction-rich hub supporting nutrient turnover, while the root endosphere functions as a selective, host-regulated niche harboring symbiotic and defensive lineages. From an applied perspective, this compartmental understanding underscores the potential for targeted microbiome engineering. Rhizosphere-directed Bacillus -based formulations may enhance soil nutrient cycling and suppress pathogens, whereas root-focused inoculants or breeding strategies could reinforce beneficial symbioses and stress tolerance. Integration of multi-omics approaches-metagenomics, transcriptomics, and metabolomics will be essential to link community composition with function and to mechanistically resolve how Bacillus-derived metabolites orchestrate microbial interactions within plant-associated ecosystems. Limitations and future directions While this study provides a detailed view of fungal community structuring in cereal roots and rhizospheres, several limitations warrant mention. Amplicon sequencing, while effective for taxonomic profiling, does not capture functional gene expression or metabolite dynamics. Combining shotgun metagenomics and metatranscriptomics would provide deeper insights into the functional mechanisms of bacillin 20 mediated modulation. Additionally, the co-occurrence networks presented here are correlative, and future validation through synthetic community experiments and perturbation assays is necessary to confirm interaction types (mutualistic, competitive, or neutral). Multi-site, multi-year field trials encompassing diverse soil types and genotypes would help determine the broader ecological generality of these findings. Future research should also link microbial shifts with plant physiological and immune responses to better understand how microbiome restructuring translates into crop performance and resilience. Conclusions 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. Abbreviations ASV Amplicon Sequence Variant LEfSe Linear discriminant analysis HPLC High-performance liquid chromatography RPM revolutions per minute Declarations Data Availability The datasets used in this study are available online with the accession number PRJNA1366560 in the NCBI SRA database. Conflict of Interest The authors declare that there is no conflict of interest and any commercial or financial relationships. Ethics approval and consent to participate This research does not include or report the results of a clinical trial; therefore, trial registration and ethical approval was not required. Clinical trial number Not applicable Consent for publication Not applicable Competing interests There are no competing interests among authors. Funding This research project was funded by CXC Montreal in Quebec, Canada. Authors contributions BA: conceptualization, experimentation, processing sequencing data, statistical analysis and draft the manuscript. MN: experimentation and sampling. RE and JL: analysis of sequencing data. AK: draft and revision of the manuscript. MH: conceptualization, and manuscript revision. DS: conceptualization, fund acquisition, supervision, and manuscript drafting. All authors contributed to the article and approved the submitted version. Acknowledgements Authors are grateful to the molecular biology laboratory facilities at the Institut de Recherche en biologie vegetale (IRBV), Montreal. References Kumar R, Farda B, Mignini A, Djebaili R, Koolman L, Paul A, et al. 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Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 13 Jan, 2026 Reviews received at journal 27 Dec, 2025 Reviewers agreed at journal 22 Dec, 2025 Reviewers agreed at journal 16 Dec, 2025 Reviewers invited by journal 16 Dec, 2025 Editor assigned by journal 27 Nov, 2025 Submission checks completed at journal 26 Nov, 2025 First submitted to journal 26 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8164785","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":561442183,"identity":"53fba878-8028-4a17-bfe9-2b9f55a2184d","order_by":0,"name":"Bulbul Ahmed","email":"","orcid":"","institution":"Macdonald Campus of McGill University","correspondingAuthor":false,"prefix":"","firstName":"Bulbul","middleName":"","lastName":"Ahmed","suffix":""},{"id":561442187,"identity":"5669ed47-13f9-47d5-a524-e316a926fb7b","order_by":1,"name":"Mahtab Nazari","email":"","orcid":"","institution":"Macdonald Campus of McGill University","correspondingAuthor":false,"prefix":"","firstName":"Mahtab","middleName":"","lastName":"Nazari","suffix":""},{"id":561442189,"identity":"1ce2a0b6-59a9-4a49-8234-d67c0d63b83f","order_by":2,"name":"Rachid Elfermi","email":"","orcid":"","institution":"University Mohamed VI Polytechnic","correspondingAuthor":false,"prefix":"","firstName":"Rachid","middleName":"","lastName":"Elfermi","suffix":""},{"id":561442190,"identity":"1b498da4-d3f1-4f71-980f-bedaddf0055a","order_by":3,"name":"Jean Legeay","email":"","orcid":"","institution":"University Mohamed VI Polytechnic","correspondingAuthor":false,"prefix":"","firstName":"Jean","middleName":"","lastName":"Legeay","suffix":""},{"id":561442191,"identity":"9c4cb15b-710d-4163-aa0c-a7c5968613f8","order_by":4,"name":"Ahmad H. 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17:09:25","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127650,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8164785/v1/549f0edd9e253d8ba6f55641.html"},{"id":98626134,"identity":"39476010-5eeb-4934-96d4-54f513a75273","added_by":"auto","created_at":"2025-12-19 17:09:33","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":73835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlpha diversity of fungal communities in root and rhizosphere compartments of corn and wheat across Bacillin 20 treatments. \u003c/strong\u003eBoxplots showing alpha diversity indices of fungal communities based on ITS amplicon sequencing under five Bacillin 20 treatments (T1-T5). Diversity metrics include (A) Shannon diversity, (B) Simpson’s evenness, (C) Chao1 richness, (D) ACE, (E) Observed richness, and (F) Pielou’s evenness. Each panel compares the diversity between root and rhizosphere compartments for\u0026nbsp;\u003cem\u003eZea mays\u003c/em\u003e\u0026nbsp;(corn) and\u0026nbsp;\u003cem\u003eTriticum aestivum\u003c/em\u003e\u0026nbsp;(wheat). T1- control; T2 – bacillin 20 at 10\u003csup\u003e-9\u003c/sup\u003e M (seed treatment); T3 – bacillin 20 at 10\u003csup\u003e-11\u003c/sup\u003e M seed treatment; T4 - bacillin 10\u003csup\u003e-9\u003c/sup\u003e (foliar treatment at 6 leaf stage); T5 – bacillin 20 at 10\u003csup\u003e-11\u003c/sup\u003e M (foliar treatment at 6 leaf stage). Statistical significance among treatments and between compartments was determined by ANOVA followed by Tukey’s HSD test (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"groupimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8164785/v1/3d9897d611b947afb94b850d.jpeg"},{"id":98625112,"identity":"6e5496dd-dc64-4fca-8421-37057b712db9","added_by":"auto","created_at":"2025-12-19 17:08:56","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53182,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBeta diversity of fungal communities in root and rhizosphere compartments of corn and wheat under Bacillin 20 treatments. \u003c/strong\u003ePrincipal coordinates analysis (PCoA) based on Bray-Curtis dissimilarities of ITS amplicon data showing differences in fungal community composition between root and rhizosphere compartments across bacillin 20 treatments (T1-T5). T1- control; T2 – bacillin 20 at 10\u003csup\u003e-9\u003c/sup\u003e M (seed treatment); T3 – bacillin 20 at 10\u003csup\u003e-11\u003c/sup\u003e M seed treatment; T4 - bacillin 10\u003csup\u003e-9\u003c/sup\u003e (foliar treatment at 6 leaf stage); T5 – bacillin 20 at 10\u003csup\u003e-11\u003c/sup\u003e M (foliar treatment at 6 leaf stage). Each point represents one sample, colored by compartment (root or rhizosphere) and shaped by treatment. (A)\u0026nbsp;\u003cem\u003eZea mays\u003c/em\u003e\u0026nbsp;(corn) and (B)\u0026nbsp;\u003cem\u003eTriticum aestivum\u003c/em\u003e\u0026nbsp;(wheat) show clear separation between root and rhizosphere communities. Ellipses represent 95% confidence intervals for each compartment. PERMANOVA (Adonis) confirmed significant compartmental effects on community composition (corn: R = 0.402,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e\u0026nbsp;= 0.001; wheat: R = 0.532,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e\u0026nbsp;= 0.001).\u003c/p\u003e","description":"","filename":"groupimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8164785/v1/b55442f29f1067729882146d.jpeg"},{"id":98521952,"identity":"16952349-3bd1-4020-91bc-5fc3676ee596","added_by":"auto","created_at":"2025-12-18 13:52:15","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":276834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenus-level composition of fungal communities in root and rhizosphere compartments of corn and wheat across Bacillin 20 treatments. \u003c/strong\u003eStacked bar plots showing the relative abundance of dominant fungal genera identified from ITS amplicon sequencing across five bacillin 20 treatments (T1-T5). Each bar represents the mean relative abundance of fungal genera in root and rhizosphere compartments of\u0026nbsp;\u003cem\u003eZea mays\u003c/em\u003e\u0026nbsp;(corn) and\u0026nbsp;\u003cem\u003eTriticum aestivum\u003c/em\u003e\u0026nbsp;(wheat). Colors correspond to major genera as indicated in the legend. T1- control; T2 – bacillin 20 at 10\u003csup\u003e-9\u003c/sup\u003e M (seed treatment); T3 – bacillin 20 at 10\u003csup\u003e-11\u003c/sup\u003e M seed treatment; T4 - bacillin 10\u003csup\u003e-9\u003c/sup\u003e (foliar treatment at 6 leaf stage); T5 – bacillin 20 at 10\u003csup\u003e-11\u003c/sup\u003e M (foliar treatment at 6 leaf stage).\u003c/p\u003e","description":"","filename":"floatimage39.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8164785/v1/24fff2aff16cc89492c52545.jpeg"},{"id":98626254,"identity":"92f898f3-a107-498a-9a2f-83675c221004","added_by":"auto","created_at":"2025-12-19 17:09:39","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":278396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCore fungal microbiome in root and rhizosphere compartments of corn and wheat. \u003c/strong\u003eStacked bar plots showing the relative abundance of core fungal genera detected in the roots and rhizospheres of \u003cem\u003eZea mays\u003c/em\u003e (corn) and \u003cem\u003eTriticum aestivum\u003c/em\u003e (wheat). Colors represent individual fungal genera that were present in ≥80% of samples within each compartment.\u003c/p\u003e","description":"","filename":"floatimage391.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8164785/v1/5b9266a73c123d12c2091ef2.jpeg"},{"id":98624457,"identity":"d384afde-1f4a-4fbb-8d5c-35e07d11d856","added_by":"auto","created_at":"2025-12-19 17:08:26","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIndicator fungal taxa distinguishing root and rhizosphere compartments in corn and wheat. \u003c/strong\u003eBubble plots showing fungal taxa significantly enriched in the root and rhizosphere compartments of (A) \u003cem\u003eZea mays\u003c/em\u003e (corn) and (B) \u003cem\u003eTriticum aestivum\u003c/em\u003e (wheat) based on indicator species analysis. Bubble size represents the mean relative abundance of each taxon, and color denotes the compartment in which it is enriched (pink: rhizosphere; blue: root).\u003c/p\u003e","description":"","filename":"groupimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8164785/v1/e15e52e1af43266be6c721e9.jpeg"},{"id":98631771,"identity":"5f96c7e3-103d-4af5-9ee0-1fe3539acde6","added_by":"auto","created_at":"2025-12-19 17:20:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2129070,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8164785/v1/c7a87bd5-004e-4bf9-a3cb-2ee64b92756c.pdf"},{"id":98521970,"identity":"2c50f7ae-0506-429e-ae62-af5dd454e22b","added_by":"auto","created_at":"2025-12-18 13:52:16","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":7532572,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8164785/v1/bf5c693bd3e9ef23845a16bc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Spatial niche differentiation and bacillin 20 mediated modulation shape fungal community diversity and network structure in roots and rhizospheres of corn and wheat","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCereal crops such as wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) and maize (\u003cem\u003eZea mays\u003c/em\u003e) 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 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe rhizosphere is a metabolically active hotspot where root exudates comprised of sugars, amino acids and secondary metabolites shape microbial recruitment and activity (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). 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 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). 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 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). 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 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile 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 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). 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 (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eIn addition to intrinsic ecological processes, agronomic interventions such as microbial inoculants and biocontrol treatments can reshape plant microbiomes. Among these, \u003cem\u003eBacillus\u003c/em\u003e 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 (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). 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 \u003cem\u003eBacillus thuringiensis\u003c/em\u003e NEB 17 and belongs to the bacteriocin family of ribosomally synthesized peptides that inhibit related microbial strains (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). \u003cem\u003eBacillus thuringiensis\u003c/em\u003e-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 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). 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.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExtraction and purification of bacillin 20\u003c/h2\u003e \u003cp\u003eBacillin 20, a bacteriocin originally isolated from \u003cem\u003eBacillus thuringiensis\u003c/em\u003e NEB17, was extracted and purified following previously established protocols with minor modifications (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Briefly, \u003cem\u003eB. thuringiensis\u003c/em\u003e NEB17 was grown in King\u0026rsquo;s B medium for 48 h at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C on a rotary shaker (150 rpm). Cultures were harvested at late-log phase (OD₆₀₀ \u0026asymp; 1.0), and 2 L of culture broth was extracted via butanol phase partitioning. The organic phase was concentrated by rotary evaporation under vacuum, resuspended in 18% acetonitrile, and subjected to high-performance liquid chromatography (HPLC; Waters Corp.) using a reverse-phase C18 column. Elution was performed with a linear acetonitrile water gradient containing 0.1% trifluoroacetic acid (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Fractions corresponding to bacillin 20 peaks were pooled, lyophilized, and stored at -20\u0026deg;C until use.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eField experimental design and bacillin 20 application\u003c/h3\u003e\n\u003cp\u003eField experiments were conducted at McGill University\u0026rsquo;s Emile A. Lods Research Centre (45\u0026deg;28\u0026prime; N, 73\u0026deg;45\u0026prime; W) using corn (\u003cem\u003eZea mays\u003c/em\u003e) hybrid Pioneer 3921 and wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) Chamran 2. Seeds of corn and wheat were collected from McGill University\u0026rsquo;s Emile A. Lods Research Centre. Bacillin 20 was applied at 10⁻⁹ M and 10⁻\u0026sup1;\u0026sup1; M, concentrations previously shown to enhance germination, growth, and stress tolerance in crops such as soybean, and canola, and also \u003cem\u003eArabidopsis\u003c/em\u003e (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Five treatment regimes were established: T1-control; T2- bacillin 20 (10⁻⁹ M) foliar spray along with herbicide; T3- bacillin 20 (10⁻\u0026sup1;\u0026sup1; M) foliar spray along with herbicide; T4 -herbicide spray at first stage\u0026thinsp;+\u0026thinsp;foliar spray of bacillin 20 (10⁻⁹ M) at 6 leaf stage; T5- herbicide spray at first stage\u0026thinsp;+\u0026thinsp;foliar spray of bacillin 20 (10⁻\u003csup\u003e11\u003c/sup\u003e 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.\u003c/p\u003e\n\u003ch3\u003eSample collection and processing\u003c/h3\u003e\n\u003cp\u003eAt the flowering stage, five healthy plants per replicate were randomly collected from each treatment. Entire root systems were excavated to a depth of ~\u0026thinsp;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\u0026deg;C until DNA extraction. Nodulation parameters were not included in this analysis to avoid confounding effects of genotype\u0026ndash;environment interactions. The study focused on characterizing fungal community structure and diversity rather than quantifying symbiotic nitrogen fixation.\u003c/p\u003e\n\u003ch3\u003eDNA extraction and ITS amplicon sequencing\u003c/h3\u003e\n\u003cp\u003eWe followed the previously established methods (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) 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\u0026prime;ACACTGACGACATGGTTCTACAGATGAAGAACGYAGYRAA-3\u0026prime;) and reverse primer CS2_ITS4 (5\u0026prime;TACGGTAGCAGAGACTTGGTCTTCCTCCGCTTATTGATATGC-3\u0026prime;) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). PCR reactions (25 \u0026micro;L) contained 1.5\u0026times; Platinum\u0026trade; Direct PCR Universal Master Mix (ThermoFisher), 0.25 \u0026micro;M of each primer, 1.5\u0026times; Platinum\u0026trade; GC Enhancer, and approximately 20 ng of DNA template. Amplicons were visualized, purified, and sequenced on an Illumina MiSeq platform (2 \u0026times; 300 bp paired end reads) at the McGill Genome Qu\u0026eacute;bec Innovation Centre (Montreal, Canada).\u003c/p\u003e\n\u003ch3\u003eBioinformatics and statistical analyses\u003c/h3\u003e\n\u003cp\u003eRaw ITS reads were processed in R (v4.0.2) using the DADA2 pipeline (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Sequences were quality-filtered (maxEE\u0026thinsp;=\u0026thinsp;c (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e); truncQ\u0026thinsp;=\u0026thinsp;2), denoised, merged, and checked for chimeras. Amplicon sequence variants (ASVs) were inferred and assigned taxonomy using the UNITE v9.0 fungal reference database (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). 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) (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). ANOVA with Tukey\u0026rsquo;s HSD (agricolae v1.3-3) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) was used for parametric data, and Wilcoxon rank-sum or Kruskal\u0026ndash;Wallis with Dunn\u0026rsquo;s post hoc tests were applied for non-parametric comparisons.\u003c/p\u003e \u003cp\u003eDifferentially abundant taxa were identified using LEfSe (Kruskal-Wallis \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; LDA\u0026thinsp;\u0026gt;\u0026thinsp;3.0) (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) and edgeR (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Indicator species were determined using indicspecies (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), and core taxa were defined as those present in \u0026ge;\u0026thinsp;80% of samples within each compartment. Microbial co-occurrence networks were generated from filtered ASV tables (relative abundance\u0026thinsp;\u0026gt;\u0026thinsp;0.01%; presence\u0026thinsp;\u0026ge;\u0026thinsp;20% of samples) using Spearman correlations (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.6; FDR-adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Networks were visualized in igraph (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), and node roles were classified according to Zi\u0026ndash;Pi analysis (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) into module hubs, connectors, and peripheral taxa.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAlpha diversity patterns of fungal communities in roots and rhizospheres of corn and wheat\u003c/h2\u003e \u003cp\u003eITS-based amplicon sequencing revealed strong compartmental and host-specific structuring of fungal diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Across both \u003cem\u003eZea mays\u003c/em\u003e (corn) and \u003cem\u003eTriticum aestivum\u003c/em\u003e (wheat), rhizosphere samples consistently exhibited higher richness and diversity than root-associated fungal communities. Shannon and Simpson indices (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B) showed significantly greater fungal diversity and evenness in the rhizosphere (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reflecting a broader and more heterogeneous assemblage of soil fungi. In contrast, the root endosphere displayed lower richness (Chao1, ACE, and Observed Richness; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E) but higher Pielou\u0026rsquo;s evenness (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), suggesting a more selective colonization of endophytic taxa within plant tissues. These patterns mirror those observed for bacterial communities, underscoring compartment identity as the dominant ecological filter with the rhizosphere supporting a diverse, saprotroph-rich fungal pool, while roots maintain a restricted subset of symbiotic or host-adapted lineages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBeta diversity of fungal communities in roots and rhizospheres of corn and wheat\u003c/h3\u003e\n\u003cp\u003ePrincipal coordinates analysis (PCoA) of Bray-Curtis dissimilarities revealed clear segregation between root and rhizosphere fungal communities in both crops (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In corn (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), the first two axes explained 41.85 and 15.91% of total variation, whereas in wheat (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), they accounted for 57.64 and 10.82%, respectively. PERMANOVA analysis confirmed that compartment identity significantly influenced fungal composition (corn: \u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.402, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; wheat: \u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.532, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGenus-level fungal community composition across hosts and treatments\u003c/h2\u003e \u003cp\u003eGenus-level composition varied markedly across hosts, compartments, and bacillin 20 treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In both crops, rhizosphere microbiomes exhibited greater taxonomic heterogeneity and functional turnover than roots, consistent with more dynamic soil environments. In corn, roots were dominated by \u003cem\u003eMyrmecridium\u003c/em\u003e, \u003cem\u003eSetophoma\u003c/em\u003e, \u003cem\u003eTalaromyces\u003c/em\u003e, and \u003cem\u003eCalophoma\u003c/em\u003e, which together represented over 60% of total abundance across treatments. Rhizospheres contained a broader array of genera, including \u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003eChaetomium\u003c/em\u003e, \u003cem\u003eAlternaria\u003c/em\u003e, and \u003cem\u003eExophiala\u003c/em\u003e taxa. Notably, under bacillin 20 treatments T4 and T5, \u003cem\u003eFusarium\u003c/em\u003e and \u003cem\u003eChaetomium\u003c/em\u003e increased in abundance, suggesting treatment-induced enrichment of specific fungal guilds (PERMANOVA, \u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.402, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In wheat, root communities were dominated by \u003cem\u003eSetophoma\u003c/em\u003e, \u003cem\u003eMyrmecridium\u003c/em\u003e, \u003cem\u003ePericonia\u003c/em\u003e and \u003cem\u003eNeosetophoma\u003c/em\u003e, while rhizospheres were enriched in \u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003eCordyceps\u003c/em\u003e and \u003cem\u003eChaetomium\u003c/em\u003e. Rhizosphere assemblages displayed higher variability across treatments, indicative of greater environmental responsiveness and functional plasticity. Statistical analyses confirmed significant differences between compartments (PERMANOVA, \u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.532, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and among treatments (ANOVA, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Together, these results show that spatial niche and host identity jointly structure fungal community assembly, with rhizospheres favoring metabolically versatile taxa involved in nutrient cycling, while roots select for a smaller consortium of endophytic and symbiotic fungi.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCore fungal microbiome in roots and rhizospheres of corn and wheat\u003c/h2\u003e \u003cp\u003eAnalysis of the core fungal microbiome revealed both conserved and host-specific assemblages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Across \u003cem\u003eZea mays\u003c/em\u003e and \u003cem\u003eTriticum aestivum\u003c/em\u003e, core root communities were dominated by \u003cem\u003eSetophoma\u003c/em\u003e, \u003cem\u003eMyrmecridium\u003c/em\u003e, \u003cem\u003ePericonia\u003c/em\u003e, \u003cem\u003eTalaromyces\u003c/em\u003e, \u003cem\u003eFusarium\u003c/em\u003e and \u003cem\u003eNeosetophoma\u003c/em\u003e, whereas rhizospheres were enriched in \u003cem\u003eSolicoccozyma\u003c/em\u003e, \u003cem\u003eExophiala\u003c/em\u003e, \u003cem\u003eAlternaria\u003c/em\u003e and \u003cem\u003eChaetomium\u003c/em\u003e. These dominant members of Ascomycota and Basidiomycota indicated functional partitioning between compartments. In corn, \u003cem\u003eMyrmecridium\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e comprised over half of the core root mycobiome, while \u003cem\u003eFusarium, Talaromyces\u003c/em\u003e and \u003cem\u003eCladosporiunm\u003c/em\u003e were more abundant in rhizospheres. In wheat, \u003cem\u003eSetophoma\u003c/em\u003e, \u003cem\u003ePericonia\u003c/em\u003e, \u003cem\u003eNeostophoma\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e dominated the root core, whereas only three genera \u0026ndash; \u003cem\u003eHoltermannia, Fusidium\u003c/em\u003e and \u003cem\u003eZygotorulaspora\u003c/em\u003e were present in rhizospheres. The overlap between compartments suggests a conserved core of multifunctional fungi, while host-specific enrichments highlight selective filtering and ecological adaptation. Collectively, these patterns indicate that root-associated taxa are specialized for endophytic lifestyles, whereas rhizosphere taxa perform saprotrophic and decomposer functions, contributing to soil carbon and nutrient turnover.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIndicator fungal taxa distinguishing root and rhizosphere compartments\u003c/h2\u003e \u003cp\u003eIndicator species analysis identified distinct compartment-enriched fungal taxa in both crops (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In corn (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), rhizosphere communities were enriched in \u003cem\u003eChaetomium\u003c/em\u003e, \u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003eSolicoccozyma\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e, representing saprotrophic decomposers thriving in nutrient-rich soil habitats. Conversely, roots were dominated by \u003cem\u003eSetophoma\u003c/em\u003e, \u003cem\u003eMyrmecridium\u003c/em\u003e, \u003cem\u003eTalaromyces\u003c/em\u003e and \u003cem\u003eNeosetophoma\u003c/em\u003e, taxa typical of endophytic or weakly pathogenic fungi adapted to plant tissues. In wheat (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), rhizosphere-enriched taxa included \u003cem\u003eExophiala\u003c/em\u003e, \u003cem\u003eCordyceps\u003c/em\u003e and \u003cem\u003eAlternaria\u003c/em\u003e, while roots favored \u003cem\u003eSetophoma\u003c/em\u003e, \u003cem\u003ePericonia\u003c/em\u003e and \u003cem\u003eTalaromyces\u003c/em\u003e. These associations illustrate strong spatial niche filtering, with rhizospheres harboring functionally diverse decomposers and roots supporting specialized symbiotic or host-interactive taxa.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eNetwork structure of fungal communities in roots and rhizospheres\u003c/h2\u003e \u003cp\u003eCo-occurrence network analysis revealed striking contrasts in network topology between root and rhizosphere fungal communities in both hosts (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Rhizosphere networks exhibited higher connectivity, modularity, and inter-phyla associations, indicating greater ecological complexity and cooperative interactions among fungal taxa. In contrast, root networks were more compact, with fewer but stronger connections, suggesting host-mediated ecological filtering and niche specialization. In corn, root networks (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA) were dominated by \u003cem\u003eAscomycota\u003c/em\u003e taxa such as \u003cem\u003eTalaromyces\u003c/em\u003e, \u003cem\u003eSchizothecium\u003c/em\u003e, and \u003cem\u003eClohesyomyces\u003c/em\u003e, whereas the rhizosphere (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB) displayed increased connectivity among \u003cem\u003eAscomycota\u003c/em\u003e and \u003cem\u003eBasidiomycota\u003c/em\u003e taxa, notably \u003cem\u003eFusidium\u003c/em\u003e, \u003cem\u003eExophiala\u003c/em\u003e, and \u003cem\u003ePullularia\u003c/em\u003e. Similarly, in wheat, root networks (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC) were largely formed by \u003cem\u003eAscomycota\u003c/em\u003e and \u003cem\u003eBasidiomycota\u003c/em\u003e, while rhizosphere networks (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD) included additional nodes from \u003cem\u003eMortierellomycota\u003c/em\u003e and \u003cem\u003eChytridiomycota\u003c/em\u003e, reflecting expanded functional guild diversity. These results suggest that rhizosphere fungal communities form dense, metabolically flexible interaction networks, whereas root assemblages are highly specialized and structurally constrained, consistent with their adaptation to host-controlled environments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFunctional guild composition of fungal communities in corn and wheat\u003c/h2\u003e \u003cp\u003eFUNGuild analysis revealed clear differences in the trophic composition of fungal communities between roots and rhizospheres, as well as between host species (Fig. S2). Across all samples, fungi were assigned to three major trophic guilds, such as, saprotrophs, pathotrophs and symbiotrophs along with mixed guilds (pathotroph-saprotroph, saprotroph-symbiotroph and pathotroph-symbiotroph). In \u003cem\u003eZea mays\u003c/em\u003e (corn) (Fig. S2A), saprotrophic fungi dominated the overall community, accounting for more than 50% of total relative abundance, followed by mixed trophic modes (pathotroph-saprotroph-symbiotroph and pathotroph-symbiotroph) and a smaller fraction of pathotrophs. The rhizosphere exhibited a higher proportion of saprotrophs and mixed guilds, suggesting active decomposition and nutrient cycling processes, while roots harbored a relatively higher abundance of pathotroph-symbiotroph fungi, indicating selective recruitment of functionally flexible taxa within plant tissues. In \u003cem\u003eTriticum aestivum\u003c/em\u003e (wheat) (Fig. S2B), symbiotrophic fungi, including arbuscular mycorrhizal and mutualistic taxa, represented the most abundant functional group, particularly in the rhizosphere, accounting for up to 70% of the total community. Root-associated communities, by contrast, contained greater proportions of pathotrophs and pathotroph\u0026ndash;saprotroph guilds, reflecting niche specialization under host control. Overall, the dominance of saprotrophic guilds in corn and symbiotrophic guilds in wheat underscores host- and compartment-specific functional partitioning of fungal communities, consistent with differences in root architecture, exudation patterns and microhabitat conditions. These results demonstrate that spatial niche differentiation and host genotype jointly influence not only taxonomic composition but also the ecological function of cereal-associated mycobiomes.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides an integrative view of how spatial niche (root vs. rhizosphere), host genotype (corn vs. wheat), and \u003cem\u003eBacillus\u003c/em\u003e-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.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSpatial niche filtering dominates fungal community assembly\u003c/h2\u003e \u003cp\u003eThe strong separation of fungal communities between root and rhizosphere compartments across both hosts underscores the primary role of spatial niche differentiation in microbiome assembly. Rhizospheres supported higher diversity and richness, dominated by saprotrophic and decomposer fungi, while root endospheres exhibited reduced diversity but higher evenness\u0026mdash;an indicator of selective colonization and host control. These findings are consistent with previous reports showing that compartmental filtering exerts a stronger influence on microbial structure than soil type or host genotype (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Enrichment of \u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003eChaetomium\u003c/em\u003e, and \u003cem\u003eAlternaria\u003c/em\u003e in rhizospheres reflects the copiotrophic and decomposer capacities of these taxa, which thrive in exudate-rich, nutrient-dense microenvironments and contribute to carbon and nitrogen cycling (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In contrast, root-enriched taxa such as \u003cem\u003eSetophoma\u003c/em\u003e, \u003cem\u003eTalaromyces\u003c/em\u003e, and \u003cem\u003eMyrmecridium\u003c/em\u003e exemplify endophytic specialization, capable of surviving in low-nutrient conditions and evading plant immune defenses. This compartment-driven ecological partitioning aligns with models describing microhabitat filtering and host selection as fundamental drivers of microbial diversification at the root-soil interface (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHost genotype modulates core and indicator taxa composition\u003c/h2\u003e \u003cp\u003eWhile spatial niche effects dominated, host genotype further refined fungal community structure and functional guild composition. Wheat mycobiomes displayed greater compositional variability and responsiveness to bacillin 20 treatments than those of corn, reflecting higher functional plasticity and environmental sensitivity. Similar host-dependent patterns have been reported in barley (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), rice (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and maize (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), where variations in root exudate profiles and secondary metabolite composition influence fungal recruitment and persistence. The persistence of \u003cem\u003eSetophoma\u003c/em\u003e, \u003cem\u003ePericonia\u003c/em\u003e and \u003cem\u003eTalaromyces\u003c/em\u003e as core taxa across both hosts indicated an evolutionarily conserved endophytic backbone within cereal crops. In contrast, rhizosphere-specific enrichment of \u003cem\u003eExophiala\u003c/em\u003e, \u003cem\u003eChaetomium\u003c/em\u003e and \u003cem\u003eCordyceps\u003c/em\u003e illustrated the functional divergence toward saprotrophic and decomposer roles. Together, these findings supported a model in which cereal microbiomes comprise a stable, multifunctional core complemented by host-specific taxa that respond dynamically to environmental and physiological contexts (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBiocontrol-mediated modulation of fungal guilds and functional traits\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBacillus\u003c/em\u003e-derived peptide bacillin 20 significantly altered fungal community composition and functional guild distribution, primarily within the rhizosphere. Treatments T4 and T5 enriched saprotrophic and mixed trophic guilds (\u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003eChaetomium\u003c/em\u003e), consistent with \u003cem\u003eBacillus\u003c/em\u003e-mediated enhancement of organic matter decomposition and nutrient turnover. These results parallel studies in canola (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), wheat and maize systems (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) where \u003cem\u003eBacillus\u003c/em\u003e inoculants stimulated soil nutrient cycling. The limited structural change observed within root-associated communities highlights the buffering capacity of host filtering, which stabilizes the endophytic microbiome against external interventions. Nonetheless, bacillin 20 induced functional differentiation, favoring biosynthetic and redox-active taxa in roots, consistent with reports that \u003cem\u003eBacillus\u003c/em\u003e-derived bacillin 20 influence fungal metabolism and antioxidant responses without disrupting symbiotic stability (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). These findings underscore the potential of \u003cem\u003eBacillus\u003c/em\u003e-based treatments as ecological modulators rather than community disruptors, capable of enhancing fungal functionality in a compartment-dependent manner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eNetwork organization reflects ecological specialization\u003c/h2\u003e \u003cp\u003eNetwork analyses revealed a clear dichotomy between rhizosphere and root fungal interactions. Rhizosphere networks were highly connected and modular, involving cross-phyla associations among Ascomycota, Basidiomycota, Mortierellomycota, and Chytridiomycota, consistent with the cooperative, resource-sharing nature of soil fungal consortia (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). In contrast, root networks were more compact, dominated by tightly linked \u003cem\u003eAscomycota\u003c/em\u003e taxa, reflecting ecological constraint and host-mediated filtering. This compartmental divergence illustrates a trade-off between metabolic versatility and symbiotic stability. The rhizosphere network\u0026rsquo;s complexity confers adaptability to environmental fluctuations, whereas the root\u0026rsquo;s constrained structure ensures stability and efficient host-microbe cooperation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Similar patterns in rice and maize demonstrate that high network modularity enhances functional robustness in open environments, while tightly linked endophyte networks underpin nutrient exchange and host defense (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEcological and practical implications\u003c/h2\u003e \u003cp\u003eTogether, our results establish a mechanistic framework in which spatial compartmentalization defines the foundation of cereal mycobiomes, host genotype refines community composition, and biocontrol treatments act as functional modulators within this hierarchy. The rhizosphere emerges as a metabolically dynamic and interaction-rich hub supporting nutrient turnover, while the root endosphere functions as a selective, host-regulated niche harboring symbiotic and defensive lineages. From an applied perspective, this compartmental understanding underscores the potential for targeted microbiome engineering. Rhizosphere-directed \u003cem\u003eBacillus\u003c/em\u003e-based formulations may enhance soil nutrient cycling and suppress pathogens, whereas root-focused inoculants or breeding strategies could reinforce beneficial symbioses and stress tolerance. Integration of multi-omics approaches-metagenomics, transcriptomics, and metabolomics will be essential to link community composition with function and to mechanistically resolve how Bacillus-derived metabolites orchestrate microbial interactions within plant-associated ecosystems.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLimitations and future directions\u003c/h2\u003e \u003cp\u003eWhile this study provides a detailed view of fungal community structuring in cereal roots and rhizospheres, several limitations warrant mention. Amplicon sequencing, while effective for taxonomic profiling, does not capture functional gene expression or metabolite dynamics. Combining shotgun metagenomics and metatranscriptomics would provide deeper insights into the functional mechanisms of bacillin 20 mediated modulation. Additionally, the co-occurrence networks presented here are correlative, and future validation through synthetic community experiments and perturbation assays is necessary to confirm interaction types (mutualistic, competitive, or neutral). Multi-site, multi-year field trials encompassing diverse soil types and genotypes would help determine the broader ecological generality of these findings. Future research should also link microbial shifts with plant physiological and immune responses to better understand how microbiome restructuring translates into crop performance and resilience.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study establishes that spatial compartmentalization is the dominant determinant of fungal community assembly in cereals, with host genotype and \u003cem\u003eBacillus\u003c/em\u003e-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.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eASV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmplicon Sequence Variant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLEfSe\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLinear discriminant analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh-performance liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRPM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003erevolutions per minute\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used in this study are available online with the accession number \u0026nbsp;PRJNA1366560 in the NCBI SRA database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest and any commercial or financial relationships.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research does not include or report the results of a clinical trial; therefore, trial registration and ethical approval was not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no competing interests among authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research project was funded by CXC Montreal in Quebec, Canada.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBA: conceptualization, experimentation, processing sequencing data, statistical analysis and draft the manuscript. MN: experimentation and sampling. RE and JL: analysis of sequencing data. AK: draft and revision of the manuscript. MH: conceptualization, and manuscript revision. DS: conceptualization, fund acquisition, supervision, and manuscript drafting. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are grateful to the molecular biology laboratory facilities at the Institut de Recherche en biologie vegetale (IRBV), Montreal.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKumar R, Farda B, Mignini A, Djebaili R, Koolman L, Paul A, et al. Microbial Solutions in Agriculture: Enhancing Soil Health and Resilience Through Bio-Inoculants and Bioremediation. Bacteria. 2025;4(3):28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichl K, Berg G, Cernava T. 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Harnessing root-soil-microbiota interactions for drought-resilient cereals. Microbiol Res. 2024;283:127698.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumawat KC, Razdan N, Saharan K. Rhizospheric microbiome: Bio-based emerging strategies for sustainable agriculture development and future perspectives. Microbiol Res. 2022;254:126901.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bacillin 20, Rhizosphere, Cereal crops, Community assembly, Network ecology, FUNGuild","lastPublishedDoi":"10.21203/rs.3.rs-8164785/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8164785/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe phytomicrobiome plays a crucial role in nutrient cycling, plant growth promotion, and stress resilience in cereals. However, how spatial niche differentiation (root vs. rhizosphere), host genotype and microbial biocontrol agents jointly shape fungal community structure and function remains poorly understood.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUsing ITS-based amplicon sequencing, we examined the effects of compartmental identity, host species (\u003cem\u003eZea mays\u003c/em\u003e and \u003cem\u003eTriticum aestivum\u003c/em\u003e) and a \u003cem\u003eBacillus thuringiensis\u003c/em\u003e-derived bacteriocin (bacillin 20) on fungal community diversity, composition and co-occurrence networks. Alpha and beta diversity analyses revealed strong compartmental segregation, with rhizosphere communities exhibiting greater richness and taxonomic heterogeneity than roots, which harbored more even and host-filtered assemblages. \u003cem\u003eSetophoma\u003c/em\u003e, \u003cem\u003eMyrmecridium\u003c/em\u003e, \u003cem\u003ePericonia\u003c/em\u003e, and \u003cem\u003eTalaromyces\u003c/em\u003e as dominant root taxa, while \u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003eChaetomium\u003c/em\u003e, \u003cem\u003eAlternaria\u003c/em\u003e, and \u003cem\u003eExophiala\u003c/em\u003e were enriched in rhizospheres. Bacillin 20 treatments, particularly at higher concentrations, enhanced the abundance of saprotrophic guilds and increased network complexity in rhizosphere communities. In contrast, root-associated networks were more compact and modular, reflecting stronger host-mediated ecological filtering. Functional annotation using FUNGuild revealed that saprotrophic fungi predominated in corn, whereas symbiotrophic guilds dominated wheat, highlighting host-specific functional partitioning.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur findings demonstrated that spatial compartmentalization is the primary driver of fungal community assembly in cereals, with host genotype and \u003cem\u003eBacillus\u003c/em\u003e-mediated modulation exerting secondary but significant effects on composition and trophic function. Bacillin 20 acted as an ecological modulator, promoting saprotrophic activity and metabolic flexibility in rhizospheres while maintaining stable, symbiotic networks within roots. This study establishes a hierarchical model of cereal mycobiome organization, providing a framework for phytomicrobiome-informed biocontrol and sustainable crop management strategies.\u003c/p\u003e","manuscriptTitle":"Spatial niche differentiation and bacillin 20 mediated modulation shape fungal community diversity and network structure in roots and rhizospheres of corn and wheat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-18 13:52:09","doi":"10.21203/rs.3.rs-8164785/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-01-13T08:58:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-27T05:42:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204169795528630222378959768843028753688","date":"2025-12-23T03:04:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143933261386485123109420807506706221124","date":"2025-12-17T00:49:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-16T10:52:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-27T09:06:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-26T09:01:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-11-26T08:49:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"278e6804-8c9d-404a-af8b-ab85d6e6e8c6","owner":[],"postedDate":"December 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-18T13:52:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-18 13:52:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8164785","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8164785","identity":"rs-8164785","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}