Asymbiotic biological nitrogen fixation makes a great contribution to nitrogen balance in unfertilized alpine grasslands across the Qinghai-Tibet Plateau

doi:10.1016/j.jia.2025.09.031

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Ke Zhang¹, Feng Zhang¹, Yaoming Li^1#, Anna Du¹, Qingpu Wang¹, Zilong Liu¹, Fengcai He¹, Shengnan Wu¹, Shengmei Li¹, Chunhui Ma¹, Xianqi Zhou¹, Juejie Yang¹, Huaiying Yao², Richard D Bardgett³, Shikui Dong^1#

¹School of Grassland Science, Beijing Forestry University, Beijing 100083, China

²School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China

³Department of Earth and Environmental Sciences, The University of Manchester, Manchester M13 9PT, UK

Highlights

l Mesorhizobium spp., known for their ability to fix nitrogen symbiotically, can also actively fix nitrogen in asymbiotic conditions.

l Asymbiotic nitrogen fixation results in ~0.50 Tg nitrogen input each year, which represents nearly 25% of the total nitrogen input in the QTP grasslands.

l Soil properties, especially molybdenum concentration, are highlighted as the most significant factor affecting potential nitrogen fixation rates.

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摘要

青藏高原草地的氮限制已经得到了充分的证实，这对未来气候变化情景下青藏高原草地植物生长和固碳潜力的预测具有重要影响。除了大气氮沉降，由于缺乏人工施肥和豆科植物，非共生固氮可能成为青藏高原高寒草地氮输入的关键途径。然而，非共生固氮对青藏高原氮输入的贡献鲜为人知。为填补这一知识空白，我们通过样带采样、DNA稳定性同位素探针、扩增子测序、随机森林算法建模和数字制图等多种方法，系统研究了青藏高原高寒草地固氮菌的群落组成、多样性及固氮活性。研究发现Skermanella和Mesorhizobium是最主要的固氮菌属，土壤pH值和全磷浓度是驱动其群落组成与多样性的主导因素。稳定性同位素探针技术揭示Mesorhizobium是活性最高的固氮菌。这些固氮菌潜在固氮速率的范围为0-18.1 kg N ha^-1 yr^-1，据此估算青藏高原高寒草地的年固氮量约为0.50 Tg，约占年氮输入总量的25%。影响非共生固氮速率的最关键因素是土壤微量元素钼，可解释其24.64%的变异。这些发现表明非共生固氮菌在维持青藏高原草地氮平衡中发挥着重要作用，并拓展了我们对Mesorhizobium属微生物超越传统共生关系的生态功能认知。

Abstract

Nitrogen limitation has been well documented in grasslands on the Qinghai- Tibet Plateau (QTP), significantly affecting predictions of plant growth and carbon sequestration potential here under future climate change scenario. Beside atmospheric deposition, asymbiotic biological nitrogen fixation (ANF) may be crucial for nitrogen input in QTP grasslands, due to the lack of artificial fertilization and legume plants. However, little is known about the ANF’s contribution to nitrogen input on the QTP. To fill this knowledge gap, we studied the composition, diversity and activity of ANF diazotrophs across the QTP grasslands by using multiple methods of transect sampling, ¹⁵N-labeling and DNA stable isotope probing (SIP), amplicon sequencing, Random Forest algorithm modelling and digital mapping. We found that Skermanella and Mesorhizobium were the most abundant diazotrophic genera. Soil pH and total phosphorus concentration were the dominant driving factors for their composition and diversity. DNA stable isotope probing with ¹⁵N₂ revealed that Mesorhizobium were the most active nitrogen-fixing microorganisms. The potential N-fixation rates of these diazotrophs ranged from 0 to 18.1 kg N ha^-1 yr^-1, resulting in an estimated annual input of approximately 0.50 Tg N across the entire QTP’s alpine grasslands (i.e. ~25% of annual nitrogen input). The most important factor affecting the ANF rate was soil micronutrient molybdenum, a cofactor in the nitrogen-fixing nitrogenase, accounting for 24.64% of the variance. These findings suggested that ANF diazotrophs play important roles in maintaining nitrogen balance in the QTP grasslands and expand our understanding of Mesorhizobium’s ecological roles beyond traditional symbiotic interactions.

Keywords: alpine grasslands asymbiotic biological nitrogen fixation diazotrophs DNA stable isotope probing nitrogen input

Online: 26 September 2025

Fund:

This work was supported by the National Key Research and Development Program of China (2021YFE0112400 and 2023YFF1304303) and the National Natural Science Foundation of China (32361143870).

About author: Ke Zhang, Mobile: +86-19800322120, E-mail: zkcoral@bjfu.edu.cn; #Correspondence Yaoming Li, Mobile: +86-13401064151, E-mail: yaomingli@bjfu.edu.cn; Shikui Dong, Mobile: +86-13811445835, E-mail: dongshikui@bjfu.edu.cn

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Ke Zhang, Feng Zhang, Yaoming Li, Anna Du, Qingpu Wang, Zilong Liu, Fengcai He, Shengnan Wu, Shengmei Li, Chunhui Ma, Xianqi Zhou, Juejie Yang, Huaiying Yao, Richard D Bardgett, Shikui Dong. 2025. Asymbiotic biological nitrogen fixation makes a great contribution to nitrogen balance in unfertilized alpine grasslands across the Qinghai-Tibet Plateau. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.09.031

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