Crop rotation and native microbiome inoculation restore soil capacity to suppress a root disease

Some soils have a larger capacity to suppress plant disease than others, but the characteristics and management practices that confer this suppressiveness remain unclear. The rhizosphere, enriched with carbon and nutrients, hosts diverse microbial communities that act as a first line of defense against pathogens and influence disease outcomes. In China, peanut is a key crop whose yield is constrained by soil-borne diseases, particularly root rot under intensive monocropping. Crop rotations have been proposed to mitigate disease by breaking host–pathogen cycles, but it is not well understood how management alters rhizosphere microbiomes to affect peanut root rot. Microbial and plant-based tools such as synthetic microbial communities (SynComs) and manipulation of root-derived metabolites may help control soil-borne pathogens, yet their effectiveness under contrasting management regimes is insufficiently understood. This study investigates how monocropping versus rotation shapes the rhizosphere microbiome and soil disease suppression capacity, and whether inoculating depleted native microbes can restore pathogen resistance.

Prior work shows rhizosphere bacteria are central to disease-suppressive soils and can be recruited by plants to suppress fungal pathogens. Synthetic communities and specific microbial metabolites (including VOCs) can inhibit pathogens, while plant root exudates strongly influence microbiome assembly and downstream plant health. Agricultural practices exploiting differences in crop root metabolism can disrupt pathogen persistence. However, the performance and ecological stability of SynComs in real agricultural settings are variable, and there is a need to understand how management regimes (monoculture vs rotation) affect microbiome structure, function, and disease outcomes to guide effective microbiome-based interventions.

- Field experiment (2012–2018, Jiangxi Province, China): Two regimes in randomized blocks with three plots each: (1) peanut monocropping (same cultivar, annually), and (2) rotation (2012 peanut; alternating years with maize, potato, soybean; peanuts again in subsequent peanut years). Disease index (DI) of peanut root rot was assessed at flowering in 2012, 2014, 2016; in 2018 DI assessed at seedling, flowering, and pod-bearing stages. Plant growth metrics were recorded. In 2018, bulk and rhizosphere soils were sampled at stages for microbiome analyses. - Pathogen identification: Endophytic fungal communities in healthy vs diseased roots profiled by ITS1 amplicon sequencing (Illumina MiSeq). Fungi were isolated from diseased roots; 18S rRNA sequencing used for identification. Pathogenicity of Fusarium isolates (F. oxysporum, F. solani) tested by inoculating peanut seedlings and assessing disease incidence. - Pathogen quantification: qPCR quantified F. oxysporum abundance in rhizosphere across growth stages using specific primers. - Rhizosphere bacterial community: 16S rRNA V4 amplicon sequencing of rhizosphere and bulk soils (Illumina MiSeq). Community differences tested by NMDS/ANOSIM; differential OTUs by DESeq2 (FDR adjusted). - Greenhouse pot experiment (2018 soils): Peanuts grown in collected field soils (monocropping vs rotation). Rhizosphere bacterial suspensions prepared to test inhibition of F. oxysporum in vitro via (i) VOC-mediated inverted plate assays and (ii) directed antagonism assays. Percentage inhibition calculated vs controls. - VOC profiling: VOCs from rhizosphere bacterial communities captured by SPME and analyzed by GC-MS. Selected pure VOCs (dimethyl sulfide, 2,5-dimethylcyclohexanone, 6-methyl-3,5-pentadien-2-one, 1,3-xylene) tested for antifungal activity across concentrations. - Cultivable microbiome characterization: Rhizosphere suspensions plated on TSA; all colonies harvested for DNA extraction and 16S sequencing to define cultivable community OTUs. 173 bacterial strains were isolated, purified, and identified by 16S rRNA sequencing; matched to OTUs (>97% identity) representing monocropping-depleted or -enriched taxa. - Metatranscriptomics: After VOC antagonism assays, RNA extracted from cultivable rhizosphere microbiomes, mRNA enriched, cDNA prepared, and sequenced (Illumina HiSeq 4000). KEGG-based functional annotation; differential pathways and representative genes associated with pathogen inhibition quantified (TPM). - Root exudate assays: Root exudates collected from peanuts grown in monocropped vs rotation soils. Growth responses of isolated depleted (e.g., Paenibacillus, Pantoea, Lysinibacillus, Enterobacter, Sporosarcina, Fictibacillus, Pseudomonas) and monocropping-enriched (Burkholderia, Stenotrophomonas) strains to exudates measured by OD600. - PGPR traits and antifungal activity: Isolated strains assessed for phosphate solubilization (PVK agar), siderophore production (CAS agar), IAA production (Salkowski assay), and direct inhibition of F. oxysporum. - Interspecies interactions: Pairwise interactions among depleted strains tested for mutual enhancement or inhibition on TSA. - SynCom design and tests: Synthetic communities composed of 7, 4, or 2 depleted strains constructed (1, 35, and 21 combinations, respectively). In vitro co-culture antagonism against F. oxysporum performed. In vitro supplementation: SynComs added to monocropping rhizosphere community suspensions and tested for enhanced inhibition. In vivo sterile seedling assays: SynComs co-inoculated with monocropping rhizosphere microbiome into vermiculite; seedlings challenged with F. oxysporum; disease incidence and growth measured. Field trial: Monocropped field plots received seed soaking with SynComs (7-, 4-, or 2-strain combinations) vs control; disease incidence and growth assessed at 45 days. - Statistics: Student’s t-tests for pairwise comparisons, ANOVA for multiple comparisons; NMDS/Bray–Curtis; DESeq2 for differential OTUs (FDR); KEGG pathway enrichment analyses. Replication details provided for each assay.

- Disease progression under management: In field trials, peanut root rot DI increased markedly under monocropping from DI2012 = 2.1 to DI2016 = 8.0, while rotation remained relatively low/stable (DI2012 = 1.6; DI2016 = 3.1). Divergence became significant by 2014 (Student’s t-test, t = 8.276, df = 16, P < 0.001). In 2018, DI at seedling stage did not differ (DIrotation = 3.87; DImonocropping = 4.38; P = 0.519), but from flowering onward DI under monocropping rose sharply and was 2.6× higher at pod-bearing (t = −7.901, df = 16, P < 0.001). - Pathogen identification and dynamics: ITS1 sequencing revealed two OTUs enriched in diseased roots: OTU177 (Fusarium oxysporum) and OTU90 (F. solani), with relative abundances 34.1× and 2712.7× higher in diseased vs healthy roots, respectively. Pathogenicity tests showed F. oxysporum caused higher disease incidence (51 ± 11%) than F. solani (35 ± 9%) (t = 5.580, df = 58, P < 0.001). qPCR showed no difference in F. oxysporum abundance at seedling stage (t = −1.525, df = 10, P = 0.158) but higher abundance under monocropping at later stages (t = 2.980, df = 10, P < 0.05), paralleling DI. - Rhizosphere bacterial community shifts: NMDS/ANOSIM indicated significant differences between monocropping and rotation across all stages (P < 0.001). In 2018, monocropping showed 183 enriched and 156 depleted OTUs (Padjusted < 0.05), mainly within Proteobacteria and Actinobacteriota; most discriminant OTUs were low-abundance (<0.1%). - Community-level pathogen suppression: Rhizosphere microbiomes from rotation peanuts inhibited F. oxysporum 45–56% more than monocropping microbiomes in directed and VOC-mediated antagonism assays (Student’s t-test, P < 0.01). - VOC mechanisms: GC-MS detected dimethyl sulfide, 2,5-dimethylcyclohexanone, and 6-methyl-3,5-pentadien-2-one in rotation but not monocropping microbiomes; α-acorenol, dimethyl disulfide, and 1,3-xylene were significantly reduced under monocropping (P < 0.05). Pure standards of selected VOCs significantly inhibited F. oxysporum even at low concentrations (0.5–5.0 µg/mL). - Functional gene expression: Metatranscriptomics of cultivable microbiomes showed enrichment of ABC transporters and Two-component systems. Genes linked to pathogen inhibition (K01823 Isopentenyl-diphosphate Delta-isomerase; K01580 Glutamate decarboxylase; K00294 1-pyrroline-5-carboxylate dehydrogenase) had significantly higher expression in rotation vs monocropping (P < 0.05). - Depleted vs enriched strains: Of 714 cultivable OTUs, 362 were absent in monocropping cultures, 257 were absent in rotation cultures. From 173 isolates, seven strains from rotation matched monocropping-depleted OTUs (>97% identity): Paenibacillus (R60), Pantoea (R05), Lysinibacillus (R06), Enterobacter (R09), Sporosarcina (R07), Fictibacillus (R37), Pseudomonas (R26). Monocropping-enriched OTUs matched Stenotrophomonas (C20) and Burkholderia (C63). - Root exudate responsiveness: Monocropping root exudates promoted growth of enriched Burkholderia and Stenotrophomonas (P < 0.001), while depleted strains responded weakly to monocropping exudates and better to rotation exudates (P < 0.001). Depleted strains exhibited multiple PGPR traits and most inhibited F. oxysporum; enriched Burkholderia did not inhibit the fungus. - SynCom efficacy and synergy: SynComs composed of 2, 4, or 7 depleted strains significantly suppressed F. oxysporum in vitro (ANOVA, F = 38.483, P < 0.001); inhibition increased with SynCom diversity; inactivated SynCom lost activity. Supplementing monocropping rhizosphere microbiome with depleted SynComs enhanced in vitro inhibition (P < 0.001). - Plant assays and field validation: In sterile seedling assays, adding depleted SynComs to the monocropping rhizosphere community significantly reduced disease (ANOVA, F = 71.755, P < 0.001), with the 7-strain SynCom most effective; plant growth was not significantly altered (P > 0.05). In field trials, seed soaking with SynComs significantly decreased root rot incidence (ANOVA, F = 16.169, P < 0.001), with the 7-strain SynCom performing best; no significant growth promotion was observed (P > 0.05).

The study demonstrates that intensive monocropping of peanut alters rhizosphere microbiome assembly, depleting key low-abundance bacterial taxa and reducing the soil’s inherent capacity to suppress root rot disease. Crop rotation maintains or enriches beneficial rhizosphere communities that inhibit pathogen invasion, partly via production of antifungal VOCs and upregulation of functional genes associated with pathogen suppression. Root exudates under monocropping favored opportunistic taxa (e.g., Burkholderia, Stenotrophomonas), which likely occupied niches and resources, leading to depletion of beneficial taxa (e.g., Paenibacillus, Pantoea, Lysinibacillus, Enterobacter, Sporosarcina, Fictibacillus, Pseudomonas) with weaker responses to these exudates. Reintroducing the depleted native strains restored pathogen suppression in vitro, in vivo, and in field conditions, with stronger effects from higher-diversity SynComs, indicating synergistic interactions and the importance of phylogenetic/functional diversity. These findings link management-driven microbiome assembly to disease outcomes and support microbiome-based interventions to regenerate soil suppressiveness.

This work shows that crop rotation preserves rhizosphere microbiome functions that suppress peanut root rot, while monocropping depletes key taxa and weakens disease suppression. Targeted inoculation with native, monocropping-depleted bacterial strains can restore rhizosphere resistance to Fusarium oxysporum across laboratory, greenhouse, and field settings, primarily through antagonistic activity rather than direct plant growth promotion. The study underscores management practices and microbiome engineering as complementary strategies to combat soil-borne diseases and promote plant health. Future work can refine SynCom composition and delivery, and further elucidate mechanisms (e.g., VOCs, functional gene pathways, root exudate–microbiome interactions) to enhance robustness and scalability in diverse agroecosystems.