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Elucidating functional partitioning and inhibitory regulation of ammonia oxidizers in agricultural ecosystems under long-term fertilization regimes

Beibei Zhang1*, Manman Chen1*, Meng Wei2, Yanhong Lou1, Haojie Feng1, Hui Wang1, Quangang Yang1, Hongjie Di1, Yuping Zhuge1#, Hong Pan1#

1 National Engineering Research Center for Efficient Utilization of Soil and Fertilizer Resources, College of Resources and Environment, Shandong Agricultural University, Tai’an 271018, China

2 Xuzhou Institute of Agricultural Sciences of the Xuhuai District of Jiangsu Province/Xuzhou Sweetpotato Research Center of Jiangsu Province, Xuzhou 221131, China

 Highlights 

l AOB mediated the majority of autotrophic nitrification in fertilized soils.

Nitrososphaera (AOA) and Nitrosospira cluster 3a (AOB) were identified as the principal nitrifiers.

l 1-octyne (C8H14) exhibited broad-spectrum inhibition, significantly suppressing AOB but also AOA activity.

l Simvastatin could exhibite clade-level selectivity, preferentially inhibiting comammox Nitrospira clade B.

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摘要  

氨氧化微生物(AOMs)在农田生态系统氮循环中发挥着关键作用,但其代谢功能与生态位分化机制尚不明确。本研究采用DNA稳定同位素探针(DNA-SIP)技术,对长期不同施肥制度土壤中驱动氨氧化的核心微生物类群进行了解析。结合DNA-SIP技术与靶向抑制乙炔、辛伐他汀、1-辛炔),系统解析了历经数十年不同施肥处理的土壤中,氨氧化古菌(AOA)、氨氧化细菌(AOB)及全程氨氧化菌(comammox Nitrospira)的功能分化特征。结果表明,施肥土壤中AOB(主要为Nitrosospira cluster 3a)占据绝对功能优势,驱动了超过85%的自养硝化活性。相比之下,AOA的显著活性仅存在于施肥(CK)和单施矿质氮肥(N)的土壤中。值得注意的是,全程氨氧化菌未检出明显的标记DNA,表明其对土壤硝化活性的贡献可忽略。抑制剂试验发现,1-辛炔不仅显著抑制AOB活性(抑制率61.9–88.9%),对AOA产生显著抑制(最高达42.7%),这一发现挑战了其作为AOB特异性抑制剂的既有认知;辛伐他汀则优先抑制全程氨氧化菌clade B。高通量测序进一步明确NitrososphaeraAOA)和Nitrosospira cluster 3aAOB)为活性硝化功能类群。综上,本研究质疑了全程氨氧化菌在农业生态系统中具普遍代谢优势的假设,揭示长期施肥通过高铵有效性驱动微生物类群筛选,重塑土壤硝化微生物网络,最终促使AOB成为硝化过程的竞争优势类群。研究结果为精准氮素管理、减缓环境风险提供了分子层面的理论依据。



Abstract  

Ammonia-oxidizing microorganisms (AOMs) mediate a pivotal yet poorly understood step in agricultural nitrogen cycling under long-term fertilization. To identify the key microbial drivers of ammonia oxidation, two DNA stable isotope probing (DNA-SIP) experiments were conducted on soils under long-term fertilization regimes. Through integrated DNA-SIP and targeted inhibition approaches (C2H2, simvastatin, C8H14), we intended to resolve functional partitioning among ammonia-oxidizing archaea (AOA), bacteria (AOB), and comammox Nitrospira in soils subjected to multi-decadal fertilization regimes. SIP revealed the exclusive functional dominance of AOB (primarily Nitrosospira cluster 3a), which drove over 85% of autotrophic nitrification in fertilized soils. In contrast, AOA activity was significant only in non-fertilized (CK) and mineral N-only (N) soils. Notably, comammox Nitrospira showed negligible functional engagement, with no labeled DNA detected. The inhibitor 1-octyne (C8H14) broadly suppressed both AOB (by 61.9–88.9%) and, unexpectedly, AOA (by up to 42.7%), challenging its specificity. Simvastatin preferentially inhibited comammox Nitrospira clade B. High-throughput sequencing confirmed Nitrososphaera (AOA) and Nitrosospira 3a as keystone nitrifiers, with no active comammox phylotypes detected. These findings challenge assumptions of comammox metabolic prominence in agroecosystems, demonstrating that long-term fertilization restructures nitrification networks through species sorting driven by high ammonium availability, leading to the competitive dominance of AOB. Our work establishes AOB as primary nitrogen cycle engineers in intensively managed soils, providing a molecular blueprint for precision nitrogen management to mitigate environmental impacts.

Keywords:  nitrification       ammonia oxidizing archaea/bacteria (AOA/AOB)       complete nitrifying bacteria (comammox)       DNA stable isotope probing  
Online: 18 March 2026  
Fund: 

This work was funded by Shandong Provincial Natural Science Foundation (ZR2023MD006, ZR2024MD014 and ZR2019BD032), the Natural Science Foundation of China (41907026), China Postdoctoral Science Foundation (2020T130387 and 2019M652448).

About author:  Beibei Zhang, E-mail: zhbbbei@163.com; Manman Chen, E-mail: chenmanman313@163.com; #Correspondence Yuping Zhuge, E-mail: zhugeyp@sdau.edu.cn; Hong Pan, E-mail: panhong6239@163.com

Cite this article: 

Beibei Zhang, Manman Chen, Meng Wei, Yanhong Lou, Haojie Feng, Hui Wang, Quangang Yang, Hongjie Di, Yuping Zhuge, Hong Pan. 2026. Elucidating functional partitioning and inhibitory regulation of ammonia oxidizers in agricultural ecosystems under long-term fertilization regimes. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2026.03.038

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