非共生固氮对青藏高原未施肥高寒草地氮平衡的重要贡献

doi:10.1016/j.jia.2025.09.031

Journal of Integrative Agriculture ›› 2026, Vol. 25 ›› Issue (8): 3483-3494.DOI: 10.1016/j.jia.2025.09.031

非共生固氮对青藏高原未施肥高寒草地氮平衡的重要贡献

收稿日期:2025-05-22 修回日期:2025-09-26 接受日期:2025-08-26 出版日期:2026-08-20 发布日期:2026-07-09

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

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

Received:2025-05-22 Revised:2025-09-26 Accepted:2025-08-26 Online:2026-08-20 Published:2026-07-09
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
Supported by:
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).

摘要/Abstract

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 under future climate change scenario. Beside atmospheric deposition, asymbiotic 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.

Key words: alpine grasslands , asymbiotic biological nitrogen fixation , diazotrophs , DNA stable isotope probing , nitrogen input

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. 非共生固氮对青藏高原未施肥高寒草地氮平衡的重要贡献[J]. Journal of Integrative Agriculture, 2026, 25(8): 3483-3494.

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. Asymbiotic biological nitrogen fixation makes a great contribution to nitrogen balance in unfertilized alpine grasslands across the Qinghai-Tibet Plateau[J]. Journal of Integrative Agriculture, 2026, 25(8): 3483-3494.

参考文献

Aber J, McDowell W, Nadelhoffer K, Magill A, Berntson G, Kamakea M, McNulty S, Currie W, Rustad L, Fernandez I. 1998. Nitrogen saturation in temperate forest ecosystems: Hypotheses revisited. BioScience, 48, 921–934.

Barron A R, Wurzburger N, Bellenger J P, Wright S J, Kraepiel A M L, Hedin L O. 2009. Molybdenum limitation of asymbiotic nitrogen fixation in tropical forest soils. Nature Geoscience, 2, 42–45.

Bei Q, Liu G, Tang H, Cadisch G, Rasche F, Xie Z. 2013. Heterotrophic and phototrophic 15N2 fixation and distribution of fixed 15N in a flooded rice-soil system. Soil Biology and Biochemistry, 59, 25–31.

Berthrong S T, Yeager C M, Gallegos-Graves L, Steven B, Eichorst S A, Jackson R B, Kuske C R. 2014. Nitrogen fertilization has a stronger effect on soil nitrogen-fixing bacterial communities than elevated atmospheric CO2. Applied and Environmental Microbiology, 80, 3103–3112.

Bolyen E, Rideout J R, Dillon M R, Bokulich N A, Abnet C C, Al-Ghalith G A, Alexander H, Alm E J, Arumugam M, Asnicar F, Bai Y, Bisanz J E, Bittinger K, Brejnrod A, Brislawn C J, Brown C T, Callahan B J, Caraballo-Rodríguez A M, Chase J, Cope E K, et al. 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature Biotechnology, 37, 852–857.

Breiman L. 2001. Random forests. Machine Learning, 45, 5–32.

Buckley D H, Huangyutitham V, Hsu S F, Nelson T A. 2007. Stable isotope probing with 15N2 reveals rovel noncultivated diazotrophs in soil. Applied and Environmental Microbiology, 73, 3196–3204.

Che R, Deng Y, Wang F, Wang W, Xu Z, Hao Y, Xue K, Zhang B, Tang L, Zhou H, Cui X. 2018. Autotrophic and symbiotic diazotrophs dominate nitrogen-fixing communities in Tibetan grassland soils. Science of the Total Environment, 639, 997–1006.

Chen H, Ju P, Zhu Q, Xu X, Wu N, Gao Y, Feng X, Tian J, Niu S, Zhang Y, Peng C, Wang Y. 2022. Carbon and nitrogen cycling on the Qinghai-Tibetan Plateau. Nature Reviews Earth & Environment, 3, 701–716.

Chen W, Wang J, Chen X, Meng Z, Xu R, Duoji D, Zhang J, He J, Wang Z, Chen J, Liu K, Hu T, Zhang Y. 2022. Soil microbial network complexity predicts ecosystem function along elevation gradients on the Tibetan Plateau. Soil Biology and Biochemistry, 172, 108766.

Cleveland C C, Townsend A R, Schimel D S, Fisher H, Howarth R W, Hedin L O, Perakis S S, Latty E F, Von Fischer J C, Elseroad A, Wasson M F. 1999. Global patterns of terrestrial biological nitrogen (N2) fixation in natural ecosystems. Global Biogeochemical Cycles, 13, 623–645.

Davies-Barnard T, Friedlingstein P. 2020. The global distribution of biological nitrogen fixation in terrestrial natural ecosystems. Global Biogeochemical Cycles, 34, e2019GB006387.

Durrant M C. 2002. An atomic-level mechanism for molybdenum nitrogenase. Part 1. Reduction of dinitrogen. Biochemistry, 41, 13934–13945.

Feng J, Penton C R, He Z, Van Nostrand J D, Yuan M M, Wu L, Wang C, Qin Y, Shi Z J, Guo X, Schuur E A G, Luo Y, Bracho R, Konstantinidis K T, Cole J R, Tiedje J M, Yang Y, Zhou J. 2019. Long-term warming in alaska enlarges the diazotrophic community in deep soils. mBio, 10, e02521.

Fu G, Zhang G, Zhou H. 2025. Effects of long-term experimental warming on phyllosphere epiphytic bacterial and fungal communities of four alpine plants. Journal of Integrative Agriculture, 24, 799–814.

García-Palacios P, Gross N, Gaitán J, Maestre F T. 2018. Climate mediates the biodiversity-ecosystem stability relationship globally. Proceedings of the National Academy of Sciences of the United States of America, 115, 8400–8405.

Griffith J C, Lee W G, Orlovich D A, Summerfield T C. 2017. Contrasting bacterial communities in two indigenous Chionochloa (Poaceae) grassland soils in New Zealand. PLoS ONE, 12, e0179652.

Gupta V V S R, Kroker S J, Hicks M, Davoren C W, Descheemaeker K, Llewellyn R. 2014. Nitrogen cycling in summer active perennial grass systems in South Australia: Non-symbiotic nitrogen fixation. Crop & Pasture Science, 65, 1044–1056.

Hartmann M, Howes C G, VanInsberghe D, Yu H, Bachar D, Christen R, Henrik Nilsson R, Hallam S J, Mohn W W. 2012. Erratum: Significant and persistent impact of timber harvesting on soil microbial communities in Northern coniferous forests. ISME Journal, 6, 2320–2320.

Heller P, Tripp H J, Turk-Kubo K, Zehr J P. 2014. ARBitrator: A software pipeline for on-demand retrieval of auto-curated nifH sequences from GenBank. Bioinformatics, 30, 2883–2890.

Jean M E, Phalyvong K, Forest-Drolet J, Bellenger J P. 2013. Molybdenum and phosphorus limitation of asymbiotic nitrogen fixation in forests of Eastern Canada: Influence of vegetative cover and seasonal variability. Soil Biology and Biochemistry, 67, 140–146.

Jin D, Ma J, Ma W, Liang C, Shi Y, He J S. 2013. Legumes in Chinese natural grasslands: Species, biomass, and distribution. Rangeland Ecology & Management, 66, 648–656.

Kou D, Yang G, Li F, Feng X, Zhang D, Mao C, Zhang Q, Peng Y, Ji C, Zhu Q, Fang Y, Liu X, Xu R, Li S, Deng J, Zheng X, Fang J, Yang Y. 2020. Progressive nitrogen limitation across the Tibetan alpine permafrost region. Nature Communications, 11, 3331.

Kou Y, Li C, Li J, Tu B, Wang Y, Li X. 2019. Climate and soil parameters are more important than denitrifier abundances in controlling potential denitrification rates in Chinese grassland soils. Science of the Total Environment, 669, 62–69.

Laranjo M, Alexandre A, Oliveira S. 2014. Legume growth-promoting rhizobia: An overview on the Mesorhizobium genus. Microbiology Research, 169, 2–17.

Lauber C L, Hamady M, Knight R, Fierer N. 2009. Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Applied and Environmental Microbiology, 75, 5111–5120.

Li W, Li F, Zeng H, Ma L, Qi L, Wang X, Wang W, Peng Z, Degen A A, Bai Y, Zhang T, Huang M, Han J, Shang Z. 2021. Diversity and variation of asymbiotic nitrogen-fixing microorganisms in alpine grasslands on the Tibetan Plateau. Frontiers in Ecology and Evolution, 9, 702848.

Liang Y, Van Nostrand J D, Deng Y, He Z, Wu L, Zhang X, Li G, Zhou J. 2010. Functional gene diversity of soil microbial communities from five oil-contaminated fields in China. ISME Journal, 5, 403–413.

Lindsay E A, Colloff M J, Gibb N L, Wakelin S A. 2010. The abundance of microbial functional genes in grassy woodlands is influenced more by soil nutrient enrichment than by recent weed invasion or livestock exclusion. Applied and Environmental Microbiology, 76, 5547–5555.

Liu D, Song X, Hu J, Liu Y, Wang C, Henkin Z. 2024. Precipitation affects soil nitrogen fixation by regulating active diazotrophs and nitrate nitrogen in an alpine grassland of Qinghai-Tibetan Plateau. Science of the Total Environment, 919, 170648.

Liu F, Wu H, Zhao Y, Li D, Yang J-L, Song X, Shi Z, Zhu A X, Zhang G L. 2022. Mapping high resolution national soil information grids of China. Science Bulletin, 67, 328–340.

Morales S E, Cosart T, Holben W E. 2010. Bacterial gene abundances as indicators of greenhouse gas emission in soils. ISME Journal, 4, 799–808.

Neufeld J D, Vohra J, Dumont M G, Lueders T, Manefield M, Friedrich M W, Murrell J C. 2007. DNA stable-isotope probing. Nature Protocols, 2, 860–866.

Pasricha N S, Nayyar V K, Singh R P. 1997. Molybdenum in Agriculture. Cambridge University Press, Ottawa. pp. 245–270.

Pepe-Ranney C, Koechli C, Potrafka R, Andam C, Eggleston E, Garcia-Pichel F, Buckley D H. 2016. Non-cyanobacterial diazotrophs mediate dinitrogen fixation in biological soil crusts during early crust formation. ISME Journal, 10, 287–298.

Pereira e Silva M C, Schloter-Hai B, Schloter M, van Elsas J D, Salles J F. 2013. Temporal dynamics of abundance and composition of nitrogen-fixing communities across agricultural soils. PLoS ONE, 8, e74500.

Petersen D G, Blazewicz S J, Firestone M, Herman D J, Turetsky M, Waldrop M. 2012. Abundance of microbial genes associated with nitrogen cycling as indices of biogeochemical process rates across a vegetation gradient in Alaska. Environmental Microbiology, 14, 993–1008.

Probst P, Wright M N, Boulesteix A L. 2019. Hyperparameters and tuning strategies for random forest. Data Mining and Knowledge Discovery, 9, e1301.

Rädecker N, Pogoreutz C, Gegner H M, Cárdenas A, Perna G, Geißler L, Roth F, Bougoure J, Guagliardo P, Struck U, Wild C, Pernice M, Raina J-B, Meibom A, Voolstra C R. 2022. Heat stress reduces the contribution of diazotrophs to coral holobiont nitrogen cycling. ISME Journal, 16, 1110–1118.

Raven J A. 2012. Protein turnover and plant RNA and phosphorus requirements in relation to nitrogen fixation. Plant Science, 188, 25–35.

Reed S C, Cleveland C C, Townsend A R. 2011. Functional ecology of free-living nitrogen fixation: A contemporary perspective. Annual Review of Ecology Evolution and Systematics, 42, 489–512.

Reed S C, Cleveland C C, Townsend A R. 2013. Relationships among phosphorus, molybdenum and free-living nitrogen fixation in tropical rain forests: Results from observational and experimental analyses. Biogeochemistry, 114, 135–147.

Rehman A N, Kalyanaraman S, Ahmad T, Pervaiz F, Saif U, Subramanian L. 2016. Fine-grained dengue forecasting using telephone triage services. Science Advances, 2, e1501215.

Rousk J, Bååth E, Brookes P C, Lauber C L, Lozupone C, Caporaso J G, Knight R, Fierer N. 2010. Soil bacterial and fungal communities across a pH gradient in an arable soil. ISME Journal, 4, 1340–1351.

Segovia R A, Pennington R T, Baker T R, Coelho de Souza F, Neves D M, Davis C C, Armesto J J, Olivera-Filho A T, Dexter K G. 2020. Freezing and water availability structure the evolutionary diversity of trees across the Americas. Science Advances, 6, eaaz5373.

Shamseldin A, Abdelkhalek A, Sadowsky M J. 2017. Recent changes to the classification of symbiotic, nitrogen-fixing, legume-associating bacteria: A review. Symbiosis, 71, 91–109.

Smercina D N, Evans S E, Friesen M L, Tiemann L K. 2019. To fix or not To fix: Controls on free-living nitrogen fixation in the rhizosphere. Applied and Environmental Microbiology, 85, e02546–18.

Stüeken E E, Buick R, Guy B M, Koehler M C. 2015. Isotopic evidence for biological nitrogen fixation by molybdenum-nitrogenase from 3.2 Gyr. Nature, 520, 666–669.

Tan Q, Jia Y, Wang G. 2021. Decoupling of soil nitrogen and phosphorus dynamics along a temperature gradient on the Qinghai-Tibetan Plateau. Geoderma, 396, 115084.

Tian P, Liu S, Zhao X, Sun Z, Yao X, Niu S, Crowther T W, Wang Q. 2021. Past climate conditions predict the influence of nitrogen enrichment on the temperature sensitivity of soil respiration. Communications Earth & Environment, 2, 251.

Tian Z, Liu F, Liang Y, Zhu X. 2022. Mapping soil erodibility in southeast China at 250 m resolution: Using environmental variables and random forest regression with limited samples. International Soil and Water Conservation Research, 10, 62–74.

Tu Q, Deng Y, Yan Q, Shen L, Lin L, He Z, Wu L, Van Nostrand J D, Buzzard V, Michaletz S T, Enquist B J, Weiser M D, Kaspari M, Waide R B, Brown J H, Zhou J. 2016. Biogeographic patterns of soil diazotrophic communities across six forests in the North America. Molecular Ecology, 25, 2937–2948.

Uroz S, Buée M, Murat C, Frey-Klett P, Martin F. 2010. Pyrosequencing reveals a contrasted bacterial diversity between oak rhizosphere and surrounding soil. Environmental Microbiology Reports, 2, 281–288.

Wang Y, Li C, Kou Y, Wang J, Tu B, Li H, Li X, Wang C, Yao M. 2017. Soil pH is a major driver of soil diazotrophic community assembly in Qinghai-Tibet alpine meadows. Soil Biology and Biochemistry, 115, 547–555.

Wang Z H, Jiang Y, Zhang M H, Chu C J, Chen Y F, Fang S, Jin G, Jiang M, Lian J Y, Li Y, Liu Y, Ma K, Mi X, Qiao X, Wang X, Wang X, Xu H, Ye W, Zhu L, Zhu Y, et al. 2023. Diversity and biogeography of plant phyllosphere bacteria are governed by latitude-dependent mechanisms. New Phytologist, 240, 1534–1547.

Wurzburger N, Bellenger J P, Kraepiel A M, Hedin L O. 2012. Molybdenum and phosphorus interact to constrain asymbiotic nitrogen fixation in tropical forests. PLoS ONE, 7, e33710.

Xiao J, Dong S, Shen H, Zhang R, Shi H, He F, Li W, Li X, Li Y, Ding C. 2025. Short-term P addition may improve the stimulating effects of N deposition on N2O emissions in alpine grasslands on the Qinghai-Tibet Plateau. Journal of Integrative Agriculture, 24, 900–912.

Zhang B, Yang L, Wang W, Li Y, Li H. 2010. Quantification and comparison of soil elements in the Tibetan Plateau Kaschin-Beck disease area. Biological Trace Element Research, 138, 69–78.

Zhang Y, Zhang F, Abalos D, Luo Y, Hui D, Hungate B A, García-Palacios P, Kuzyakov Y, Olesen J E, Jørgensen U, Chen J. 2022. Stimulation of ammonia oxidizer and denitrifier abundances by nitrogen loading: Poor predictability for increased soil N2O emission. Global Change Biology, 28, 2158–2168.

Zheng M, Chen H, Li D, Luo Y, Mo J. 2020. Substrate stoichiometry determines nitrogen fixation throughout succession in southern Chinese forests. Ecology Letters, 23, 336–347.

Zhou J, Deng Y, Shen L, Wen C, Yan Q, Ning D, Qin Y, Xue K, Wu L, He Z, Voordeckers J W, Nostrand J D V, Buzzard V, Michaletz S T, Enquist B J, Weiser M D, Kaspari M, Waide R, Yang Y, Brown J H. 2016. Temperature mediates continental-scale diversity of microbes in forest soils. Nature Communications, 7, 12083.

Zhou S, Xue K, Zhang B, Tang L, Pang Z, Wang F, Che R, Ran Q, Xia A, Wang K, Li L, Dong J, Du J, Hu R, Hao Y, Cui X, Wang Y. 2021. Spatial patterns of microbial nitrogen-cycling gene abundances along a precipitation gradient in various temperate grasslands at a regional scale. Geoderma, 404, 115236.

Zhu J, Wang Q, He N, Smith M D, Elser J J, Du J, Yuan G, Yu G, Yu Q. 2016. Imbalanced atmospheric nitrogen and phosphorus depositions in China: Implications for nutrient limitation. Journal of Geophysical Research (Biogeosciences), 121, 1605–1616.

Zong N, Song M, Zhao G, Shi P. 2020. Nitrogen economy of alpine plants on the north Tibetan Plateau: Nitrogen conservation by resorption rather than open sources through biological symbiotic fixation. Ecology and Evolution, 10, 2051–2061.

非共生固氮对青藏高原未施肥高寒草地氮平衡的重要贡献

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics