Opportunistic keystone diazotrophs from co-occurrence networks drive biological nitrogen fixation in peanut/cotton intercropping systems

doi:10.1016/j.jia.2025.05.005

Journal of Integrative Agriculture

2026, Vol. 25

Issue (3): 1209-1222 DOI: 10.1016/j.jia.2025.05.005

Agro-ecosystem & Environment

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Opportunistic keystone diazotrophs from co-occurrence networks drive biological nitrogen fixation in peanut/cotton intercropping systems

Shijie Zhang^{1, 3}, Yingchun Han², Guoping Wang², Lu Feng^{1, 2}, Yaping Lei², Shiwu Xiong², Beifang Yang², Xiaoyu Zhi², Minghua Xin², Yahui Jiao², Xiaofei Li^2#, Yabing Li^{1, 2#}, Zhen Jiao^{1, 3#}

¹Zhengzhou Research Base, State Key Laboratory of Cotton Bio-breeding and Integrated Utilization/School of Agriculture and Biomanufacturing, Zhengzhou University, Zhengzhou 450001, China

²State Key Laboratory of Cotton Bio-breeding and Integrated Utilization/Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang 455000, China

³Henan Key Laboratory of Ion-Beam Green Agriculture Bioengineering, Zhengzhou University, Zhengzhou 450001, China

Highlights
● Intercropping and rhizosphere soils significantly enhanced BNF potentials by 7.8–323.0%.
● Heterogeneous selection primarily drove diazotrophic community assembly.
● Opportunistic diazotrophs dominated networks and acted as keystone taxa enhancing BNF.

Abstract
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摘要

豆科作物间作可增强非共生生物固氮（BNF）作用，但其潜在机制，尤其是不同生态位宽度的土壤关键固氮菌类群在其中发挥的作用仍不明确。本研究通过田间试验，评估了花生/棉花间作与各自单作条件下根际与非根际土壤中BNF活性的变化。通过固氮速率、固氮酶活性以及nifH基因丰度，结合微生物系统发育零模型、共现网络及生态位宽度分析，探讨关键固氮类群及其生态功能与BNF的关系。结果显示，与非根际土壤相比，根际土壤的BNF潜力提高了7.8%–125.5%；与单作相比，花生/棉花间作系统中固氮速率、固氮酶活性和nifH基因丰度提升了11.6%–323.0%（P<0.05）。固氮菌群落组成与多样性存在显著差异：间作系统与根际土壤中增加了大部分变形菌门（α-变形菌外）、降低了蓝藻门和厚壁菌门的相对丰度。固氮菌的群落构建主要受确定性过程驱动，尤其是异质性选择过程，在根际（91.9%）和间作土壤（86.3%）中占主导地位。此外，间作系统与根际土壤中的固氮菌共现网络更复杂、连接性更强，其中，优势类群为机会型固氮菌（78.8%–85.9%），其次为特化型（10.2%–18.5%）和泛化型（1.38%–3.80%）。网络关键类群（机会型的Azoarcus、Azohydromonas 和Steroidobacter，泛化型的Pseudomonas 和 Azotobacter），以及土壤微生物量碳和硝态氮，与间作系统和根际土壤BNF活性的增强显著相关。花生/棉花间作通过选择性招募生态功能不同的关键固氮类群，尤其是机会型固氮菌，从而提升农田BNF潜力，促进农业可持续发展。

Abstract

Legume-based intercropping enhances asymbiotic biological nitrogen fixation (BNF); however, the underlying mechanisms remain unclear, including the roles of soil keystone diazotroph taxa with varying niche breadths. A field experiment was conducted to evaluate soil BNF variations between rhizosphere and bulk soils in peanut/cotton intercropping systems and monocultures. BNF activities were measured by nitrogen fixation rates, nitrogenase activity, and nifH gene abundance. Phylogenetic null models, co-occurrence networks, and niche breadth analysis were applied to investigate the roles of diazotrophic keystone taxa and their ecological niches. Rhizosphere soils exhibited 7.8–125.5% higher BNF potentials than bulk soils, whereas intercropping systems showed 11.6–323.0% increases over monocultures for nitrogen fixation rate, nitrogenase activity, and nifH gene abundance (all P<0.05). Diazotrophic community composition and diversity differed significantly, with Proteobacteria (excluding Alphaproteobacteria) enriched in intercropping and rhizosphere soils, while Cyanobacteria and Firmicutes were less abundant. Deterministic processes, particularly heterogeneous selection, dominated community assembly in the rhizosphere (91.9%) and intercropping soils (86.3%). The co-occurrence networks consistently revealed more complex and interconnected communities in intercropping and rhizosphere soils that were dominated by opportunistic diazotrophs (78.8–85.9%), followed by specialists (10.2–18.5%) and generalists (1.38–3.80%). Keystone taxa, including opportunists such as Azoarcus, Azohydromonas, and Steroidobacter, and generalists like Pseudomonas and Azotobacter, correlated positively with microbial biomass carbon and nitrate nitrogen, contributing to enhanced BNF. Peanut/cotton intercropping enhances BNF by selectively enriching the keystone diazotrophic taxa with varying ecological roles, particularly opportunists and generalists. Such targeted intercropping strategies can optimize BNF, improve soil fertility, and promote sustainable agricultural production.

Keywords: biological nitrogen fixation community assembly habitat ecotypes keystone diazotroph taxa

Received: 13 January 2025 Accepted: 21 April 2025 Online: 14 May 2025

Fund:

This work was financially supported by the National Natural Science Foundation of China (32301962 and 31901127), the China Postdoctoral Science Foundation (2024M752947), the Postdoctoral Fellowship Program of China Postdoctoral Science Foundation (GZC20232437), the State Key Laboratory of Cotton Bio-breeding and Integrated Utilization Open Fund, China (CB2023C02), and the Natural Science Foundation of Henan Province, China (252300420222).

About author: Shijie Zhang, E-mail: sjzhang@zzu.edu.cn; #Correspondence Xiaofei Li, E-mail: lixiaofei01@caas.cn; Yabing Li, E-mail: criliyabing1@163.com; Zhen Jiao, E-mail: jiaozhen@zzu.edu.cn

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Shijie Zhang, Yingchun Han, Guoping Wang, Lu Feng, Yaping Lei, Shiwu Xiong, Beifang Yang, Xiaoyu Zhi, Minghua Xin, Yahui Jiao, Xiaofei Li, Yabing Li, Zhen Jiao. 2026. Opportunistic keystone diazotrophs from co-occurrence networks drive biological nitrogen fixation in peanut/cotton intercropping systems . Journal of Integrative Agriculture, 25(3): 1209-1222.

Banerjee S, Schlaeppi K, Van Der Heijden M G A. 2018. Keystone taxa as drivers of microbiome structure and functioning. Nature Reviews Microbiology, 16, 567–576.

Berg G, Smalla K. 2009. Plant species and soil type cooperatively shape the structure and function of microbial communities in the rhizosphere: Plant species, soil type and rhizosphere communities. FEMS Microbiology Ecology, 68, 1–13.

Brooker R W, Bennett A E, Cong W, Daniell T J, George T S, Hallett P D, Hawes C, Iannetta P P M, Jones H G, Karley A J, Li L, McKenzie B M, Pakeman R J, Paterson E, Schöb C, Shen J, Squire G, Watson C A, Zhang C, Zhang F, et al. 2015. Improving intercropping: A synthesis of research in agronomy, plant physiology and ecology. New Phytologist, 206, 107–117.

Chen W, Ren K, Isabwe A, Chen H, Liu M, Yang J. 2019. Stochastic processes shape microeukaryotic community assembly in a subtropical river across wet and dry seasons. Microbiome, 7, 138.

Chen Y J, Leung P M, Wood J L, Bay S K, Hugenholtz P, Kessler A J, Shelley G, Waite D W, Franks A E, Cook P L M, Greening C. 2021. Metabolic flexibility allows bacterial habitat generalists to become dominant in a frequently disturbed ecosystem. The ISME Journal, 15, 2986–3004.

Chi B, Liu J, Dai J, Li Z, Zhang D, Xu S, Nie J, Wan S, Li C, Dong H. 2023. Alternate intercropping of cotton and peanut increases productivity by increasing canopy photosynthesis and nutrient uptake under the influence of rhizobacteria. Field Crops Research, 302, 109059.

Chi B, Zhang Y, Zhang D, Zhang X, Dai J, Dong H. 2019. Wide-strip intercropping of cotton and peanut combined with strip rotation increases crop productivity and economic returns. Field Crops Research, 243, 107617.

Dang P, Lu C, Huang T, Zhang M, Yang N, Han X, Xu C, Wang S, Wan C, Qin X, Siddique K H M. 2024. Enhancing intercropping sustainability: Manipulating soybean rhizosphere microbiome through cropping patterns. Science of the Total Environment, 931, 172714.

Dehling D M, Bender I M A, Blendinger P G, Böhning-Gaese K, Muñoz M C, Neuschulz E L, Quitián M, Saavedra F, Santillán V, Schleuning M, Stouffer D B. 2021. Specialists and generalists fulfil important and complementary functional roles in ecological processes. Functional Ecology, 35, 1810–1821.

Dittmann G, Ding S, Hopmans E C, Schröter S A, Orme A M, Kothe E, Lange M, Gleixner G. 2025. Bioavailable carbon additions to soil promote free-living nitrogen fixation and microbial biomass growth with N-free lipids. Soil Biology & Biochemistry, 203, 109748.

Duchene O, Vian J F, Celette F. 2017. Intercropping with legume for agroecological cropping systems: Complementarity and facilitation processes and the importance of soil microorganisms. A review. Agriculture, Ecosystems & Environment, 240, 148–161.

Edwards J, Johnson C, Santos-Medellín C, Lurie E, Podishetty N K, Bhatnagar S, Eisen J A, Sundaresan V. 2015. Structure, variation, and assembly of the root-associated microbiomes of rice. Proceedings of the National Academy of Sciences of the United States of America, 112, E911–E920.

Fan F, Zhang F, Song Y, Sun J, Bao X, Guo T, Li L. 2006. Nitrogen fixation of faba bean (Vicia faba L.) interacting with a non-legume in two contrasting intercropping systems. Plant and Soil, 283, 275–286.

Fan K, Delgado-Baquerizo M, Guo X, Wang D, Wu Y, Zhu M, Yu W, Yao H, Zhu Y, Chu H. 2019. Suppressed N fixation and diazotrophs after four decades of fertilization. Microbiome, 7, 143.

Feng K, Peng X, Zhang Z, Gu S, He Q, Shen W, Wang Z, Wang D, Hu Q, Li Y, Wang S, Deng Y. 2022. iNAP: An integrated network analysis pipeline for microbiome studies. iMeta, 1, e13.

Fierer N, Bradford M A, Jackson R B. 2007. Toward an ecological classification of soil bacteria. Ecology, 88, 1354–1364.

Fridley J D, Vandermast D B, Kuppinger D M, Manthey M, Peet R K. 2007. Co-occurrence based assessment of habitat generalists and specialists: A new approach for the measurement of niche width. Journal of Ecology, 95, 707–722.

Hu H, Li H, Hao M, Ren Y, Zhang M, Liu R, Zhang Y, Li G, Chen J, Ning T, Kuzyakov Y. 2021. Nitrogen fixation and crop productivity enhancements co-driven by intercrop root exudates and key rhizosphere bacteria. Journal of Applied Ecology, 58, 2243–2255.

Hu W, Wang X M, Wang X, Xu Y, Li R, Zhao L, Ren W, Teng Y. 2023. Enhancement of nitrogen fixation and diazotrophs by long-term polychlorinated biphenyl contamination in paddy soil. Journal of Hazardous Materials, 446, 130697.

Hu W, Wang X M, Xu Y, Wang X, Guo Z, Pan X, Dai S, Luo Y, Teng Y. 2024. Biological nitrogen fixation and the role of soil diazotroph niche breadth in representative terrestrial ecosystems. Soil Biology & Biochemistry, 189, 109261.

Jiang Y, Han S, Luo X. 2024. Role of generalists, specialists and opportunists in Nitrospira community assembly in soil aggregates from nearby cropland and grassland areas in a campus. Applied Soil Ecology, 198, 105377.

Jing B, Shi W, Wang H, Lin F. 2023. 15N labeling technology reveals enhancement of nitrogen uptake and transfer by root interaction in cotton/soybean intercropping. Journal of the Science of Food and Agriculture, 103, 6307–6316.

Li B, Li Y, Wu H M, Zhang F, Li C, Li X, Lambers H, Li L. 2016. Root exudates drive interspecific facilitation by enhancing nodulation and N2 fixation. Proceedings of the National Academy of Sciences of the United States of America, 113, 6496–6501.

Li Y, Gu X, Yong T, Yang W. 2024. A global synthesis reveals additive density design drives intercropping effects on soil N-cycling variables. Soil Biology & Biochemistry, 191, 109318.

Liu C, Li X, Mansoldo F R P, An J, Kou Y, Zhang X, Wang J, Zeng J, Vermelho A B, Yao M. 2022. Microbial habitat specificity largely affects microbial co-occurrence patterns and functional profiles in wetland soils. Geoderma, 418, 115866.

Lu J, Liu Y, Zou X, Zhang X, Yu X, Wang Y, Si T. 2024. Rotational strip peanut/cotton intercropping improves agricultural production through modulating plant growth, root exudates, and soil microbial communities. Agriculture, Ecosystems & Environment, 359, 108767.

Luo J, Banerjee S, Ma Q, Liao G, Hu B, Zhao H, Li T. 2023. Organic fertilization drives shifts in microbiome complexity and keystone taxa increase the resistance of microbial mediated functions to biodiversity loss. Biology and Fertility of Soils, 59, 441–458.

Ma J, Bei Q, Wang X, Lan P, Liu G, Lin X, Liu Q, Lin Z, Liu B, Zhang Y, Jin H, Hu T, Zhu J, Xie Z. 2019. Impacts of Mo application on biological nitrogen fixation and diazotrophic communities in a flooded rice-soil system. Science of the Total Environment, 649, 686–694.

Magoč T, Salzberg S L. 2011. FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics, 27, 2957–2963.

De Matos G F, Rouws L F M , Simões-Araújo J L, Baldani J I. 2021. Evolution and function of nitrogen fixation gene clusters in sugarcane associated Bradyrhizobium strains. Environmental Microbiology, 23, 6148–6162.

Nuccio E E, Starr E, Karaoz U, Brodie E L, Zhou J, Tringe S G, Malmstrom R R, Woyke T, Banfield J F, Firestone M K, Pett-Ridge J. 2020. Niche differentiation is spatially and temporally regulated in the rhizosphere. The ISME Journal, 14, 999–1014.

Pandit S N, Kolasa J, Cottenie K. 2009. Contrasts between habitat generalists and specialists: An empirical extension to the basic metacommunity framework. Ecology, 90, 2253–2262.

Philippot L, Chenu C, Kappler A, Rillig M C, Fierer N. 2024. The interplay between microbial communities and soil properties. Nature Reviews Microbiology, 22, 226–239.

Philippot L, Raaijmakers J M, Lemanceau P, van der Putten W H. 2013. Going back to the roots: The microbial ecology of the rhizosphere. Nature Reviews Microbiology, 11, 789.

Poly F, Monrozier L J, Bally R. 2001. Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil. Research in Microbiology, 152, 95–103.

Qiao M, Sun R, Wang Z, Dumack K, Xie X, Dai C, Wang E, Zhou J, Sun B, Peng X, Bonkowski M, Chen Y. 2024. Legume rhizodeposition promotes nitrogen fixation by soil microbiota under crop diversification. Nature Communications, 15, 2924.

Qiu Y, Li X, Tang Y, Xiong S, Han Y, Wang Z, Feng L, Wang G, Yang B, Lei Y, Du W. 2023. Directly linking plant N, P and K nutrition to biomass production in cotton-based intercropping systems. European Journal of Agronomy, 151, 126960.

Salazar G. 2022. EcolUtils: utilities for community ecology analysis. R package version 0.1.

Shi S, Nuccio E E, Shi Z J, He Z, Zhou J, Firestone M K. 2016. The interconnected rhizosphere: High network complexity dominates rhizosphere assemblages. Ecology Letters, 19, 926–936.

Shu D, Banerjee S, Mao X, Zhang J, Cui W, Zhang W, Zhang B, Chen S, Jiao S, Wei G. 2024. Conversion of monocropping to intercropping promotes rhizosphere microbiome functionality and soil nitrogen cycling. Science of the Total Environment, 949, 174953.

Solanki M K, Wang Z, Wang F, Li C, Gupta C L, Singh R K, Malviya M K, Singh P, Yang L, Li Y. 2020. Assessment of diazotrophic Proteobacteria in sugarcane rhizosphere when intercropped with legumes (peanut and soybean) in the Field. Frontiers in Microbiology, 11, 1814.

Stegen J C, Lin X, Fredrickson J K, Chen X, Kennedy D W, Murray C J, Rockhold M L, Konopka A. 2013. Quantifying community assembly processes and identifying features that impose them. The ISME Journal, 7, 2069–2079.

Vitousek P M, Menge D N L, Reed S C, Cleveland C C. 2013. Biological nitrogen fixation: Rates, patterns and ecological controls in terrestrial ecosystems. Philosophical Transactions of the Royal Society (B: Biological Sciences), 368, 20130119.

Wahdan S F M, Ji L, Schädler M, Wu Y T, Sansupa C, Tanunchai B, Buscot F, Purahong W. 2023. Future climate conditions accelerate wheat straw decomposition alongside altered microbial community composition, assembly patterns, and interaction networks. The ISME Journal, 17, 238–251.

Wang J, Zou Y, Di Gioia D, Singh B K, Li Q. 2020. Conversion to agroforestry and monoculture plantation is detrimental to the soil carbon and nitrogen cycles and microbial communities of a rainforest. Soil Biology & Biochemistry, 147, 107849.

Wang Y, Li C, Tu B, Kou Y, Li X. 2021. Species pool and local ecological assembly processes shape the β-diversity of diazotrophs in grassland soils. Soil Biology & Biochemistry, 160, 108338.

Wen T, Xie P, Yang S, Niu G, Liu X, Ding Z, Xue C, Liu Y, Shen Q, Yuan J. 2022. ggClusterNet: An R package for microbiome network analysis and modularity-based multiple network layouts. iMeta, 1, e32.

Wolińska A, Kuźniar A, Zielenkiewicz U, Banach A, Błaszczyk M. 2018. Indicators of arable soils fatigue - Bacterial families and genera: A metagenomic approach. Ecological Indicators, 93, 490–500.

Wu Z, Han X, Chen X, Lu X, Yan J, Wang W, Zou W, Yan L. 2024. Application of organic manure as a potential strategy to alleviate the limitation of microbial resources in soybean rhizospheric and bulk soils. Journal of Integrative Agriculture, 23, 2065–2082.

Xiao Y, Li L, Zhang F. 2004. Effect of root contact on interspecific competition and N transfer between wheat and fababean using direct and indirect 15N techniques. Plant and Soil, 262, 45–54.

Xu Q, Vandenkoornhuyse P, Li L, Guo J, Zhu C, Guo S, Ling N, Shen Q. 2022. Microbial generalists and specialists differently contribute to the community diversity in farmland soils. Journal of Advanced Research, 40, 17–27.

Yang Y, Chai Y, Xie H, Zhang L, Zhang Z, Yang X, Hao S, Gai J, Chen Y. 2023. Responses of soil microbial diversity, network complexity and multifunctionality to three land-use changes. Science of the Total Environment, 859, 160255.

Zhang L, Yuan L, Wen Y, Zhang M, Huang S, Wang S, Zhao Y, Hao X, Li L, Gao Q, Wang Y. 2024. Maize functional requirements drive the selection of rhizobacteria under long-term fertilization practices. New Phytologist, 242, 1275–1288.

Zhang L, Zhang M, Huang S, Li L, Gao Q, Wang Y, Zhang S, Huang S, Yuan L, Wen Y, Liu K, Yu X, Li D, Zhang L, Xu X, Wei H, He P, Zhou W, Philippot L, Ai C. 2022. A highly conserved core bacterial microbiota with nitrogen-fixation capacity inhabits the xylem sap in maize plants. Nature Communications, 13, 3361.

Zhang S, Han Y, Wang G, Feng L, Lei Y, Xiong S, Yang B, Zhi X, Xin M, Jiao Y, Li X, Li Y, Jiao Z. 2025. Peanut–cotton intercropping to enhance soil ecosystem multifunctionality: Roles of microbial keystone taxa, assembly processes, and C-cycling profiles. Agriculture, Ecosystems & Environment, 377, 109254.

Zhang T, Liu Y, Ge S, Peng P, Tang H, Wang J. 2024. Sugarcane/soybean intercropping with reduced nitrogen addition enhances residue-derived labile soil organic carbon and microbial network complexity in the soil during straw decomposition. Journal of Integrative Agriculture, 23, 4216–4236.

Zhou G, Fan K, Li G, Gao S, Chang D, Liang T, Li S, Liang H, Zhang J, Che Z, Cao W. 2023. Synergistic effects of diazotrophs and arbuscular mycorrhizal fungi on soil biological nitrogen fixation after three decades of fertilization. iMeta, 2, e81.

Zhou J, Jiang X, Zhou B, Zhao B, Ma M, Guan D, Li J, Chen S, Cao F, Shen D, Qin J. 2016. Thirty four years of nitrogen fertilization decreases fungal diversity and alters fungal community composition in black soil in northeast China. Soil Biology & Biochemistry, 95, 135–143.

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