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

Sustainable food production is a critical global challenge, especially in densely populated regions with limited arable land like China. Intercropping, the practice of growing two or more crops simultaneously in the same field, offers a promising solution. This method frequently leads to increased productivity, improved resource use efficiency, better pest and disease control, and enhanced ecological sustainability. Peanut (*Arachis hypogaea* L.)/maize (*Zea mays*) intercropping is prevalent in China, proving more effective and ecologically sound than peanut monoculture, particularly for smallholder farmers. Peanuts are a crucial oilseed legume, with China accounting for a significant portion of global production. However, peanut yield and quality in Northern China Plain are significantly hampered by widespread iron (Fe) deficiency in the alkaline and calcareous soils prevalent in the region. The high pH of these soils renders iron insoluble and biologically unavailable to plants. Intercropping peanuts with maize effectively mitigates this iron deficiency, improving peanut iron nutrition, photosynthetic efficiency, yield, and overall land-use efficiency. While the benefits of this intercropping system are well-documented, the underlying mechanisms, especially the contribution of the rhizosphere microbiome, remain largely unexplored. This study proposes to investigate this crucial aspect of intercropping, focusing on how the plant root-associated microbiota contributes to the increased iron nutrition observed in peanut when intercropped with maize. It is known that plants employ different strategies for iron acquisition, with peanuts utilizing strategy I (direct Fe(III) reduction to Fe(II)) and maize employing strategy II (phytosiderophore secretion to chelate Fe(III)). Maize generally outperforms peanuts in iron acquisition under alkaline conditions. A prevailing hypothesis suggests that intercropping benefits peanuts by enhancing the secretion of deoxymugineic acid (DMA) from maize roots, which solubilizes Fe(III) for subsequent absorption by nearby peanut plants. However, this model lacks integration of root-microbiome interactions, which are well-established as crucial for plant fitness and iron acquisition. Beneficial rhizobacteria often secrete siderophores to dissolve insoluble Fe(III), suggesting a potential mechanism underlying the observed improvement in iron nutrition in intercropping systems. This study, therefore, hypothesizes that peanut/maize intercropping modifies the rhizosphere microbiome, promoting the exchange of root exudates and microbiome members, thereby enhancing iron acquisition opportunities for peanuts. The study employs a multifaceted approach combining microbiome profiling, functional strain characterization, metabolite identification, and greenhouse and field experiments to determine the precise links between intercropping, belowground root-microbiome interactions, and improved iron acquisition in peanuts.

Existing literature supports the positive impact of intercropping on crop productivity and resource utilization. Studies have shown that intercropping systems often lead to increased yields compared to monoculture, primarily due to improved resource partitioning and complementary resource use by different plant species. The role of root exudates in mediating plant-plant interactions belowground has also been investigated. Studies indicate that root exudates can alter the soil environment and influence the rhizosphere microbiome composition. Previous research on iron acquisition in plants has highlighted the importance of two primary strategies. Dicots and non-graminaceous monocots employ strategy I, reducing Fe(III) to Fe(II) before absorption. Graminaceous monocots, such as maize, utilize strategy II, secreting phytosiderophores to chelate insoluble Fe(III) for subsequent absorption. The role of rhizosphere microorganisms in plant nutrient acquisition is well-established. Many studies have demonstrated the importance of plant growth-promoting rhizobacteria (PGPR) in improving nutrient uptake, including iron. Siderophore-producing bacteria, which secrete iron-chelating molecules, are particularly crucial in enhancing iron availability in soils. Previous research has shown that intercropping can lead to microbiome shifts and an increase in the abundance of certain beneficial bacteria, although the specific mechanisms driving these changes and their impact on nutrient acquisition remain unclear. This work aimed to build upon this existing knowledge base by focusing on the peanut-maize intercropping system and explicitly considering the role of microbiome convergence in enhancing iron nutrition.

The study employed a multi-faceted approach to investigate the role of the rhizosphere microbiome in iron nutrition improvement in peanut/maize intercropping systems. The methodology comprised several key stages: 1. **Greenhouse Experiments:** Experiments were conducted in pots containing naturally iron-limited calcareous soils. Monocropping and intercropping treatments were established, with peanuts and maize grown separately and together. Chlorophyll levels (SPAD values) and active iron concentrations in young leaves were measured at various time points (days post-sowing, dps). Available iron concentrations in the rhizosphere were also determined. To assess the involvement of the rhizosphere microbiome, experiments were repeated in both normal and sterilized soils. 2. **Microbiome Profiling:** 16S rRNA amplicon sequencing was used to profile the rhizosphere microbiome of peanuts and maize under monocropping and intercropping conditions at multiple time points. Data analysis involved principal coordinate analysis (PCoA) to visualize microbiome community structures, linear discriminant analysis effect size (LEfSe) to identify key bacterial taxa linked to improved iron nutrition, and correlation analysis to assess relationships between microbiome composition and iron nutrition metrics. 3. **Strain Isolation and Characterization:** Siderophore-secreting rhizobacteria were isolated from the intercropping peanut rhizosphere using a chrome azurol S (CAS) plate assay. High siderophore-producing strains were identified and characterized using 16S rRNA gene sequencing and whole-genome sequencing. 4. **Siderophore Identification and Characterization:** The primary siderophore produced by a high-producing *Pseudomonas* strain (*Pseudomonas* sp. 1502IPR-01) was isolated and characterized using various techniques including HPLC, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. The siderophore's capacity to chelate Fe(III) from insoluble Fe(OH)₃ was determined. 5. **Functional Validation:** Greenhouse and field experiments were conducted to validate the role of *Pseudomonas* sp. 1502IPR-01 and its siderophore (pyoverdine) in improving peanut iron nutrition. Treatments involved inoculating plants with the bacterial strain, applying purified pyoverdine, and comparing these to controls. SPAD values, active iron concentrations, available iron in the rhizosphere, biomass, and yield were measured. A pyoverdine null mutant (*P. aeruginosa* PAO1 Δ*pvdDpchEF*) was used to confirm the functional role of pyoverdine. Ferric-chelate reductase (FCR) activity in peanut and deoxymugineic acid (DMA) production in maize were measured to assess the impact of pyoverdine on plant iron metabolism. 6. **Statistical Analysis:** Appropriate statistical tests (parametric and non-parametric) were used to analyze the data, taking into account data distribution and variance. Multiple comparisons were corrected using the Benjamini-Hochberg (BH) algorithm.

The study yielded several significant findings: 1. **Intercropping Improves Peanut Iron Nutrition:** Greenhouse experiments confirmed that intercropping peanuts with maize significantly improved peanut iron nutrition, as evidenced by increased SPAD values and active iron concentrations in young leaves, starting from 53 dps. This improvement was also reflected in higher available iron levels in the rhizosphere. Maize, however, did not show similar improvements. 2. **Rhizosphere Microbiome Plays a Crucial Role:** Experiments conducted in sterilized soil revealed that the observed improvement in peanut iron nutrition in intercropping systems was dependent on the presence of a functional rhizosphere microbiome. Sterilization negated the positive effects of intercropping on peanut iron nutrition, indicating the critical role of the microbiome in this process. 3. **Microbiome Convergence:** 16S rRNA amplicon sequencing showed that intercropping induced both changes in microbiome composition and a convergence between the peanut and maize microbiomes. The microbiomes of intercropped peanuts and maize were more similar than those of monocropped plants. 4. ***Pseudomonas* as a Keystone Taxon:** Linear discriminant analysis (LDA) effect size (LEfSe) identified *Pseudomonas* as a top biomarker enriched in intercropped peanuts compared to monocropped peanuts, and present in higher abundance in monocropped maize. The abundance of *Pseudomonas*, and other genera, positively correlated with both active iron in leaves and available iron in the rhizosphere. 5. **High Siderophore-Secreting *Pseudomonas* Strains:** Numerous siderophore-secreting bacteria were isolated from intercropping peanuts, with a large proportion being *Pseudomonas* spp. One particular *Pseudomonas* strain, *Pseudomonas* sp. 1502IPR-01, exhibited exceptionally high siderophore production. 6. **Pyoverdine as the Primary Siderophore:** The *Pseudomonas* sp. 1502IPR-01 strain was found to secrete pyoverdine as its primary siderophore. Pyoverdine was identified and characterized through various techniques including MS, NMR, and chemical analysis. It showed a high capacity to solubilize Fe(III) from insoluble Fe(OH)₃. 7. **Pyoverdine Improves Iron Nutrition in Greenhouse and Field Experiments:** Both *Pseudomonas* sp. 1502IPR-01 and its pyoverdine significantly improved iron nutrition in monocropped and intercropped peanuts in both greenhouse and field experiments. The effects were evident in increased SPAD values, active iron, and available iron, as well as higher biomass and yields. In field experiments, the positive effect of *Pseudomonas* sp. 1502IPR-01 and pyoverdine treatment on peanut nutrition and yield was comparable to, and in some cases exceeded, that of traditional EDTA-Fe treatment. 8. **Pyoverdine's Direct Effect on Plant Iron Metabolism:** Experiments using a pyoverdine null mutant demonstrated that pyoverdine is essential for improving peanut iron nutrition. The presence of pyoverdine resulted in a downregulation of FCR activity in peanuts and DMA production in maize, suggesting pyoverdine as the primary iron acquisition route.

This research provides compelling evidence for the significant role of rhizosphere microbiome convergence in enhancing iron nutrition in peanut/maize intercropping systems. The study successfully identified a specific mechanism—the contribution of siderophore-producing *Pseudomonas*—that explains the observed improvements. The findings address the research question by demonstrating that the increased iron nutrition in peanuts during intercropping is not solely due to direct plant-plant interactions, such as DMA secretion from maize roots, but also significantly involves the rhizosphere microbiome. The microbiome convergence observed leads to the enrichment of beneficial *Pseudomonas* strains, which in turn secrete pyoverdine to increase iron bioavailability. The significance of these findings lies in their relevance to sustainable agriculture and food security. The identification of pyoverdine as a key factor, and its direct impact on plant iron metabolism, provides a foundation for the development of novel biofertilizer strategies that could replace or supplement traditional iron fertilizers. The potential for improving iron nutrition in peanuts and reducing reliance on synthetic fertilizers would have significant environmental and economic benefits. Future research could focus on exploring other intercropping systems to determine the extent to which microbiome convergence and siderophore production are generalizable to other plant combinations and soil types. Further investigation into the specific mechanisms by which plants utilize pyoverdine-chelated iron, the interactions between plant and microbial iron acquisition systems, and the long-term effects of pyoverdine application on soil health and microbial communities is warranted.

This study provides strong evidence for the importance of microbiome convergence in mediating the benefits of intercropping on plant nutrition. Specifically, the translocation of siderophore-producing *Pseudomonas* strains from maize to peanut rhizosphere, resulting in increased pyoverdine secretion and enhanced iron bioavailability for peanuts, was demonstrated. The findings have significant implications for sustainable agriculture, suggesting a potential for developing environmentally friendly biofertilizers based on *Pseudomonas* sp. 1502IPR-01 and pyoverdine. Future studies should explore the potential of this approach in other intercropping systems and soil types, and investigate the complex interactions between plant and microbial iron acquisition pathways.

While this study provides strong evidence for the role of *Pseudomonas* and pyoverdine in improving peanut iron nutrition, several limitations should be considered. The study focused primarily on bacterial members of the rhizosphere microbiome, while other microbes, such as arbuscular mycorrhizal fungi, may also play a role in intercropping benefits. The research was conducted in specific soil types (calcareous soils) and geographical locations, and the findings may not be fully generalizable to other soil types or environments. The long-term effects of pyoverdine application on soil health and microbial community dynamics were not extensively investigated. Finally, the exact mechanisms by which plants acquire iron from pyoverdine-chelated complexes remain unclear and need further investigation.