Microbiome convergence enables siderophore-secreting-rhizobacteria to improve iron nutrition and yield of peanut intercropped with maize

Fe deficiency commonly limits peanut yield and quality in alkaline calcareous soils in northern China. Intercropping peanut with maize is widely practiced and improves peanut iron nutrition, photosynthetic efficiency, and land-use efficiency, thereby increasing yield. Classical models attribute this benefit to belowground plant–plant facilitation: maize (a Strategy II plant) secretes phytosiderophores such as deoxymugineic acid (DMA) that solubilize Fe(III), which nearby peanut (a Strategy I plant) can take up. However, these models overlook root–microbiome interactions, although rhizosphere microbes are known to aid plant iron acquisition, often via secretion of siderophores that chelate Fe(III). The authors hypothesize that peanut/maize intercropping modulates and converges the rhizosphere microbiomes between species, enabling exchange of beneficial taxa and their metabolites (notably siderophores), thereby boosting iron availability and peanut iron nutrition. The study aims to test whether intercropping benefits depend on a functional microbiome, to identify key microbial taxa and metabolites involved, and to validate their causal role under greenhouse and field conditions.

Background work shows Strategy I plants (dicots, non-graminaceous monocots) reduce Fe(III) to Fe(II) for uptake, while Strategy II graminaceous plants exude mugineic acid family phytosiderophores (e.g., DMA) to chelate and take up Fe(III). Previous studies proposed that maize-derived DMA improves peanut iron uptake in intercropping on calcareous soils. The rhizosphere microbiome is recognized as critical for plant fitness and iron acquisition, with plants recruiting beneficial rhizobacteria under iron limitation and bacteria aiding iron solubilization via siderophores. Siderophores, including pyoverdines, can both suppress pathogens through iron competition and potentially serve as iron sources for plants. Despite this, prior intercropping models rarely incorporated plant–microbe interactions, leaving a knowledge gap about microbiome-mediated mechanisms in intercropping-driven iron nutrition improvements.

The study combined greenhouse pot experiments, soil sterilization assays, rhizosphere microbiome profiling (16S rRNA amplicon sequencing), culture-based isolation and functional screening of siderophore-producing rhizobacteria, chemical characterization and purification of bacterial siderophores, and validation assays in both greenhouse and field conditions.

• Greenhouse intercropping/monocropping: Peanut (Arachis hypogaea L. cv. Luhua14) and maize (Zea mays L. cv. Zhengdan958) were grown in pots containing 8 kg of iron-deficient calcareous sandy soil collected from Beijing. Treatments: monocropped peanut (6 plants/pot), monocropped maize (3 plants/pot), and intercropped peanut+maize (3+3 plants/pot). Environmental conditions were controlled (28–33 °C, natural light ~400–450 μmol m−2 s−1, 70–75% RH). Peanut samples were harvested at 46, 53, 63, and 73 dps for plant and rhizosphere assessments.
• Soil sterilization: To test microbiome dependence, parallel experiments used γ-irradiated soil (20 kGy) versus unsterilized soil, with peanut/maize intercropping and peanut monocropping assessed at 73 dps.
• Plant and soil iron-related measurements: Chlorophyll content measured as SPAD in young leaves; HCl-extractable “active iron” in young leaves quantified by ICP-OES; DTPA-extractable available iron in rhizosphere soil measured by ICP-OES. Peanut root ferric-chelate reductase (FCR) activity and maize DMA secretion were assayed with established colorimetric methods.
• Microbiome profiling: DNA extracted from rhizosphere soil; V3–V4 16S rRNA region amplified (338F/806R) and sequenced (Illumina MiSeq PE300). Reads processed in QIIME2 with DADA2 to define ASVs; taxonomy assigned using a Naive Bayes classifier trained on SILVA 138. Features annotated as plastids/mitochondria removed; low-abundance/low-prevalence ASVs filtered; counts normalized by SRS to 17,063 reads/sample. Beta-diversity analyzed by UniFrac/Bray-Curtis with PCoA and PERMANOVA (Adonis). Differential taxa identified by LEfSe (LDA>3, p<0.05). Spearman correlations linked biomarker abundances with plant iron metrics.
• Isolation and screening of siderophore producers: From intercropped peanut rhizosphere, 1 g soil was suspended and plated on CAS indicator medium to isolate siderophore producers; 324 isolates were obtained, and 46 high-producers (halo >5 mm) were selected. Siderophore production quantified using a modified CAS assay with calibration to desferrioxamine B (DFOB) equivalents.
• Identification and genomics: Isolates identified via full-length 16S rRNA sequencing; a representative high producer, Pseudomonas sp. 1502IPR-01 (97.7% identical to ASV487), underwent whole-genome sequencing (PacBio + Illumina). Pyoverdine biosynthesis/secretion genes were identified by TBLASTN comparisons to P. aeruginosa PAO1.
• Pyoverdine purification and structural elucidation: Pyoverdine was produced in iron-limited standard succinic medium, extracted via XAD-4 resin, separated by semi-preparative HPLC, and characterized by UV/Vis, HR-MS (Q-TOF, Orbitrap), MS/MS fragmentation, and 1D/2D NMR (COSY, NOESY, HSQC, HMBC). The molecular formula was determined (C57H85N16O25) and peptide sequence inferred from MS/MS series (including FoOHOrn, Ser, Lys) consistent with known pyoverdines.
• Iron solubilization assay: Purified pyoverdine was tested for its ability to chelate Fe(III) from Fe(OH)3 suspensions; released iron quantified by ICP-OES, and color change monitored.
• Functional validation in greenhouse and field: In greenhouse, peanut (monocropped and intercropped) grown in sterilized and normal soils were treated via rhizosphere irrigation with Pseudomonas sp. 1502IPR-01 (10^10 CFU plant−1 at 50, 64, 75 dps) or purified iron-free pyoverdine (20 μM; 2 μmol plant−1), or water (control). Maize responses were also monitored. In field trials (Beijing and Puyang, Henan; calcareous soils), monocropped peanut received repeated treatments: water control, 1502IPR-01 (10^8 CFU mL−1; 150 mL per plant), pyoverdine (48 μM; 7.2 μmol per plant), or EDTA-Fe foliar spray; SPAD, active iron, rhizosphere available iron, and yield were measured. Intercropped peanut in field also tested but showed no additional benefit beyond intercropping.
• Genetic validation: A pyoverdine/pyochelin-deficient mutant of P. aeruginosa PAO1 (ΔpvdD ΔpchEF) was compared with wild-type PAO1 and Pseudomonas sp. 1502IPR-01 in greenhouse pots to confirm the necessity of siderophore production.
• Statistics: Normality (Shapiro–Wilk) and homoscedasticity (Levene) were tested; BoxCox transformations applied as needed. Parametric tests (t-test, ANOVA + LSD) used when assumptions held; otherwise non-parametric tests (Wilcoxon, Kruskal–Wallis with Dunnett T3, Scheirer–Ray–Hare). BH correction applied for multiple comparisons; two-sided tests were used.

• Intercropping improved peanut iron nutrition but not maize under greenhouse conditions: SPAD increased by 17.6–51.5% and active iron in young leaves by 23.7–60.3% from 53 dps onward; rhizosphere available iron increased by 14.3–21.4% in intercropped peanut versus monocropped peanut. • Microbiome dependence: In sterilized soil, intercropping no longer improved peanut SPAD, active iron, or rhizosphere available iron, with significant crop type × sterilization interactions, demonstrating a requirement for a functional rhizosphere microbiome. • Microbiome convergence: 16S profiling showed distinct communities for monocropped vs intercropped rhizospheres, with intercropped peanut and maize communities becoming more similar than their monocropped counterparts (PERMANOVA significant across time points), indicating cross-enrichment between plant species. • Biomarker taxa: LEfSe identified 10 genera enriched in intercropped peanut and more abundant in monocropped maize; five (including Pseudomonas, Pseudoxanthomonas, Luteolibacter, Allorhizobium–Neorhizobium–Pararhizobium–Rhizobium, Sphingobium) positively correlated with both iron metrics. Pseudomonas showed the strongest association with rhizosphere iron availability (Spearman rho ~0.81). • Culture-based validation: Of 46 top siderophore producers isolated from intercropped peanut rhizosphere, 58.7% were Pseudomonas spp.; many matched an amplicon variant ASV487 that was undetectable in monocropped peanut, enriched in intercropped peanut, and abundant in monocropped maize. • Pseudomonas sp. 1502IPR-01 characterization: Genomics revealed a pyoverdine biosynthetic/secretion gene cluster (51.4–84.1% protein identity to P. aeruginosa PAO1 homologs). Chemical analyses confirmed the primary siderophore as a pyoverdine (C57H85N16O25), and 90.5% ± 0.9% of the supernatant’s iron-chelating activity was attributable to siderophores. • Fe(OH)3 solubilization: Purified pyoverdine solubilized Fe(III) from insoluble Fe(OH)3 in a dose-dependent manner, matching reference pyoverdine–Fe(III) color changes, indicating high capacity to mobilize iron in calcareous-like conditions. • Greenhouse functional tests: In sterilized and normal soils, applying either Pseudomonas sp. 1502IPR-01 or its iron-free pyoverdine significantly increased peanut SPAD, active leaf iron, rhizosphere available iron, and biomass in both monocropping and intercropping setups. Maize iron metrics did not improve, though intercropped maize biomass increased. • Field validation: In two calcareous field sites (Beijing and Henan), monocropped peanut treated with Pseudomonas sp. 1502IPR-01 or pyoverdine showed marked improvements relative to controls: SPAD +45.9–67.6%, active leaf iron +78.2–107.5%, rhizosphere available iron +53.6–73.2%, and yield +44.8–89.8%. Effects were similar to or exceeded foliar EDTA-Fe in some cases. In intercropped peanut under field conditions, additional treatment had no effect, consistent with intercropping already alleviating Fe deficiency. • Genetic validation: Wild-type P. aeruginosa PAO1 and Pseudomonas sp. 1502IPR-01 improved peanut iron nutrition and biomass, whereas the siderophore-deficient mutant PAO1 ΔpvdD ΔpchEF did not, confirming the necessity of pyoverdine for the beneficial effect. • Plant iron metabolism feedback: Peanut FCR activity was downregulated in the presence of pyoverdine-producing strains or added pyoverdine, and upregulated with the siderophore-deficient mutant; maize DMA secretion was similarly downregulated by Pseudomonas sp. 1502IPR-01 or pyoverdine, indicating that bacterial pyoverdine directly modulates plant iron acquisition pathways.

The study demonstrates that peanut/maize intercropping enhances peanut iron nutrition through a microbiome-mediated mechanism involving rhizosphere microbiome convergence. Intercropping facilitates cross-enrichment of Pseudomonas spp. from maize to the peanut rhizosphere, where they secrete pyoverdine capable of solubilizing Fe(III) from insoluble Fe(OH)3, increasing bioavailable iron and alleviating peanut iron-deficiency chlorosis. The necessity of a functional microbiome is underscored by the loss of intercropping benefits in sterilized soil. Functional and genetic validations show that pyoverdine production is required for improved iron nutrition, and that pyoverdine alters plant iron-deficiency responses (downregulating peanut FCR and maize DMA secretion), suggesting a shift toward microbially mediated iron acquisition. Field trials confirm that both the Pseudomonas inoculant and purified pyoverdine can substantially improve iron status and yield in calcareous soils, rivaling or surpassing EDTA-Fe. Beyond iron, intercropping may also promote nitrogen-related benefits through cross-enrichment of nitrogen-fixing taxa, suggesting reciprocal advantages to both crops. The work advances understanding of plant–microbe interactions in intercropping systems and positions pyoverdine as a key microbial metabolite driving improved nutrient acquisition and yield.

Intercropping peanut with maize promotes rhizosphere microbiome convergence that cross-enriches siderophore-secreting Pseudomonas spp. in the peanut rhizosphere. These bacteria produce pyoverdine, which mobilizes Fe(III) in calcareous soils and substantially improves peanut iron nutrition, growth, and yield. The effect requires a functional microbiome and is recapitulated by applying either a characterized Pseudomonas strain (1502IPR-01) or purified pyoverdine in greenhouse and field conditions. The findings provide a mechanistic basis for intercropping benefits and suggest practical, eco-friendly biofertilization strategies leveraging beneficial microbes or their metabolites as alternatives to synthetic chelates. Future research should clarify plant uptake mechanisms for pyoverdine-bound iron, evaluate contributions from other candidate taxa (e.g., Sphingobium, Luteolibacter), explore multi-kingdom interactions (including fungi), and test generalization across soils, climates, and other intercropping combinations.

• Soil specificity: Results are derived from alkaline calcareous soils; performance may vary across soil types with different chemistry and buffering capacities. • Microbiome variability: Beneficial taxa abundance and community function can differ across locations and soil properties (e.g., NH4+, NO3−, organic carbon), potentially affecting outcomes. • Mechanistic uncertainty: The biochemical pathway by which plants access iron from pyoverdine (direct uptake of Fe–pyoverdine vs. extracellular reduction/degradation) remains unresolved. • Scope of taxa: While Pseudomonas was validated, other enriched genera associated with improved iron metrics were not functionally tested; roles of non-bacterial microbiota (e.g., mycorrhizal fungi) were not addressed. • Experimental constraints: Sterilization eliminates complex microbial networks beyond bacteria, and while efforts minimized cross-contamination during rhizosphere sampling, root proximity in intercropping inherently complicates complete separation. • Field translation: Intercropping already mitigated Fe deficiency in field conditions, limiting additive benefit testing; broader multi-year, multi-site validations would strengthen generalizability.