Decadal litter addition mitigates nitrogen enrichment-induced declines of soil microbial biomass in Mongolian pine plantations

Abstract

Anthropogenic activities have concurrently increased atmospheric nitrogen (N) deposition and altered forest litterfall dynamics, both of which can profoundly influence soil microbial communities. The individual influence of these two factors on soil microorganisms is extensively investigated, whereas their interactive effect remains poorly understood. To address this gap, a 10-year full-factorial experiment with N addition (control and 10 g N m^–2 yr^–1) and litter manipulation (removal, ambient, and addition) was conducted in a Pinus sylvestris var. mongolica plantation. We measured 10 variables related to soil acid-base status and N availability, and assessed soil microbial biomass and community composition using phospholipid fatty acid analyses. Results revealed that chronic N addition reduced soil bacterial and fungal biomass, as well as reshaped microbial communities. These changes in soil microorganisms were associated with N addition-induced soil acidification and increases in soil N availability. Litter manipulation alone had minor effects, but its interaction with N addition markedly influenced soil microbial biomass. Specifically, litter addition mitigated N addition-induced declines in soil bacterial and total microbial biomass. This significant interactive effect was attributed to the role of litter addition in mitigating soil acidification caused by N addition. Overall, these results suggest that litter addition could alleviate N enrichment effects on soil acid-base status and consequently on soil microbial biomass likely owing to the rapid N immobilization and followed by slow N mineralization during litter decomposition, and underscore that litterfall conservation is critical for enhancing soil microbial resistance to N deposition.

Graphical abstract

Keywords

litter manipulation / microbial biomass / microbial community composition / nitrogen addition / plant-soil-microbe interaction / soil acidification

Highlight

	● N addition reduced soil microbial biomass and restructured microbial communities.
	● Litter manipulation alone had minor impacts on soil microorganisms.
	● Litter addition mitigated soil acidification caused by N addition.
	● Litter addition alleviated N addition-induced declines in soil microbial biomass.

References

[1] Averill, C., Waring, B., 2018. Nitrogen limitation of decomposition and decay: how can it occur. Global Change Biology24, 1417–1427.

[2] Baldrian, P., López-Mondéjar, R., Kohout, P., 2023. Forest microbiome and global change. Nature Reviews Microbiology21, 487–501.

[3] Bowman, W.D., Cleveland, C.C., Halada, Ĺ., Hreško, J., Baron, J.S., 2008. Negative impact of nitrogen deposition on soil buffering capacity. Nature Geoscience1, 767–770.

[4] Brant, J.B., Myrold, D.D., Sulzman, E.W., 2006. Root controls on soil microbial community structure in forest soils. Oecologia148, 650–659.

[5] Bunn, R.A., Corrêa, A., Joshi, J., Kaiser, C., Lekberg, Y., Prescott, C.E., Sala, A., Karst, J., 2024. What determines transfer of carbon from plants to mycorrhizal fungi. New Phytologist244, 1199–1215.

[6] Chen, D.M., Lan, Z.C., Hu, S.J., Bai, Y.F., 2015. Effects of nitrogen enrichment on belowground communities in grassland: relative role of soil nitrogen availability vs. soil acidification. Soil Biology and Biochemistry89, 99–108.

[7] Cheng, Z.H., Lin, G.G., Zeng, D.H., Cheng, S.F., Xu, W.Q., He, N.P., 2026. Litter removal aggravates the negative effect of long-term nitrogen enrichment on soil bacterial diversity in a Mongolian pine plantation. Plant and Soil518, 639–655.

[8] Cleveland, C.C., Liptzin, D., 2007. C:N:P stoichiometry in soil: is there a “Redfield ratio” for the microbial biomass. Biogeochemistry85, 235–252.

[9] Cui, Y.X., Peng, S.S., Delgado-Baquerizo, M., Rillig, M.C., Terrer, C., Zhu, B., Jing, X., Chen, J., Li, J.Q., Feng, J., He, Y., Fang, L.C., Moorhead, D.L., Sinsabaugh, R.L., Peñuelas, J., 2023. Microbial communities in terrestrial surface soils are not widely limited by carbon. Global Change Biology29, 4412–4429.

[10] Eastman, B.A., Adams, M.B., Brzostek, E.R., Burnham, M.B., Carrara, J.E., Kelly, C., McNeil, B.E., Walter, C.A., Peterjohn, W.T., 2021. Altered plant carbon partitioning enhanced forest ecosystem carbon storage after 25 years of nitrogen additions. New Phytologist230, 1435–1448.

[11] Feng, J.G., He, K.Y., Zhang, Q.F., Han, M.G., Zhu, B., 2022. Changes in plant inputs alter soil carbon and microbial communities in forest ecosystems. Global Change Biology28, 3426–3440.

[12] Fierer, N., Schimel, J.P., Holden, P.A., 2003. Variations in microbial community composition through two soil depth profiles. Soil Biology and Biochemistry35, 167–176.

[13] Frostegård, Å., Tunlid, A., Bååth, E., 1993. Phospholipid fatty acid composition, biomass, and activity of microbial communities from two soil types experimentally exposed to different heavy metals. Applied and Environmental Microbiology59, 3605–3617.

[14] Furey, G.N., Tilman, D., 2021. Plant biodiversity and the regeneration of soil fertility. Proceedings of the National Academy of Sciences of the United States of America118, e2111321118.

[15] Galloway, J.N., Townsend, A.R., Erisman, J.W., Bekunda, M., Cai, Z.C., Freney, J.R., Martinelli, L.A., Seitzinger, S.P., Sutton, M.A., 2008. Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science320, 889–892.

[16] Guan, H.L., Zhang, Y.Q., Mao, Q.G., Zhong, B.Q., Chen, W.B., Mo, J.M., Wang, F.M., Lu, X.K., 2023. Consistent effects of nitrogen addition on soil microbial communities across three successional stages in tropical forest ecosystems. Catena227, 107116.

[17] Gurmesa, G.A., Wang, A., Li, S.L., Peng, S.S., de Vries, W., Gundersen, P., Ciais, P., Phillips, O.L., Hobbie, E.A., Zhu, W.X., Nadelhoffer, K., Xi, Y., Bai, E., Sun, T., Chen, D.X., Zhou, W.J., Zhang, Y.P., Guo, Y.R., Zhu, J.J., Duan, L., Li, D.J., Koba, K., Du, E.Z., Zhou, G.Y., Han, X.G., Han, S.J., Fang, Y.T., 2022. Retention of deposited ammonium and nitrate and its impact on the global forest carbon sink. Nature Communications13, 880.

[18] Heděnec, P., Nilsson, L.O., Zheng, H.F., Gundersen, P., Schmidt, I.K., Rousk, J., Vesterdal, L., 2020. Mycorrhizal association of common European tree species shapes biomass and metabolic activity of bacterial and fungal communities in soil. Soil Biology and Biochemistry149, 107933.

[19] Högberg, M.N., Högberg, P., 2002. Extramatrical ectomycorrhizal mycelium contributes one-third of microbial biomass and produces, together with associated roots, half the dissolved organic carbon in a forest soil. New Phytologist154, 791–795.

[20] Holub, S.M., Lajtha, K., Spears, J.D.H., Tóth, J.A., Crow, S.E., Caldwell, B.A., Papp, M., Nagy, P.T., 2005. Organic matter manipulations have little effect on gross and net nitrogen transformations in two temperate forest mineral soils in the USA and central Europe. Forest Ecology and Management214, 320–330.

[21] Huang, W.J., Spohn, M., 2015. Effects of long-term litter manipulation on soil carbon, nitrogen, and phosphorus in a temperate deciduous forest. Soil Biology and Biochemistry83, 12–18.

[22] Jobbágy, E.G., Jackson, R.B., 2004. The uplift of soil nutrients by plants: biogeochemical consequences across scales. Ecology85, 2380–2389.

[23] Joergensen, R.G., 2022. Phospholipid fatty acids in soil—drawbacks and future prospects. Biology and Fertility of Soils58, 1–6.

[24] Jourgholami, M., Sohrabi, H., Venanzi, R., Tavankar, F., Picchio, R., 2022. Hydrologic responses of undecomposed litter mulch on compacted soil: litter water holding capacity, runoff, and sediment. Catena210, 105875.

[25] Kieft, T.L., Ringelberg, D.B., White, D.C., 1994. Changes in ester-linked phospholipid fatty acid profiles of subsurface bacteria during starvation and desiccation in a porous medium. Applied and Environmental Microbiology60, 3292–3299.

[26] Kramer, C., Gleixner, G., 2008. Soil organic matter in soil depth profiles: distinct carbon preferences of microbial groups during carbon transformation. Soil Biology and Biochemistry40, 425–433.

[27] LeBauer, D.S., Treseder, K.K., 2008. Nitrogen limitation of net primary productivity in terrestrial ecosystems is globally distributed. Ecology89, 371–379.

[28] Li, S.C., Tang, S.M., Ju, X.T., Zhu, Z.H., Zhang, Y.J., Chen, H.Y., Jin, K., 2024. Soil acidification drives the negative effects of nitrogen enrichment on soil microbial biomass at the global scale. Plant and Soil503, 517–528.

[29] Lin, G.G., Gao, M.X., Zeng, D.H., Fang, Y.T., 2020. Aboveground conservation acts in synergy with belowground uptake to alleviate phosphorus deficiency caused by nitrogen addition in a larch plantation. Forest Ecology and Management473, 118309.

[30] Lin, G.G., Mao, R., Zhao, L., Zeng, D.H., 2013. Litter decomposition of a pine plantation is affected by species evenness and soil nitrogen availability. Plant and Soil373, 649–657.

[31] Lin, G.G., Yuan, Z.Q., Zhang, Y.S., Zeng, D.H., Wang, X.G., 2022. Dominant tree mycorrhizal associations affect soil nitrogen transformation rates by mediating microbial abundances in a temperate forest. Biogeochemistry158, 405–421.

[32] Liu, J., Wang, J., Morreale, S.J., Schneider, R.L., Li, Z.G., Wu, G.L., 2023. Contributions of plant litter to soil microbial activity improvement and soil nutrient enhancement along with herb and shrub colonization expansions in an arid sandy land. Catena227, 107098.

[33] Liu, R., Zhang, Y., Hu, X.F., Wan, S.Z., Wang, H.M., Liang, C., Chen, F.S., 2021. Litter manipulation effects on microbial communities and enzymatic activities vary with soil depth in a subtropical Chinese fir plantation. Forest Ecology and Management480, 118641.

[34] Lu, X.K., Vitousek, P.M., Mao, Q.G., Gilliam, F.S., Luo, Y.Q., Zhou, G.Y., Zou, X.M., Bai, E., Scanlon, T.M., Hou, E.Q., Mo, J.M., 2018. Plant acclimation to long-term high nitrogen deposition in an N-rich tropical forest. Proceedings of the National Academy of Sciences of the United States of America115, 5187–5192.

[35] Manzoni, S., Trofymow, J.A., Jackson, R.B., Porporato, A., 2010. Stoichiometric controls on carbon, nitrogen, and phosphorus dynamics in decomposing litter. Ecological Monographs80, 89–106.

[36] Meng, C., Tian, D.S., Zeng, H., Li, Z.L., Yi, C.X., Niu, S.L., 2019. Global soil acidification impacts on belowground processes. Environmental Research Letters14, 074003.

[37] Miura, T., Owada, K., Nishina, K., Utomo, M., Niswati, A., Kaneko, N., Fujie, K., 2016. The effects of nitrogen fertilizer on soil microbial communities under conventional and conservation agricultural managements in a tropical clay-rich ultisol. Soil Science181, 68–74.

[38] Parton, W., Silver, W.L., Burke, I.C., Grassens, L., Harmon, M.E., Currie, W.S., King, J.Y., Adair, E.C., Brandt, L.A., Hart, S.C., Fasth, B., 2007. Global-scale similarities in nitrogen release patterns during long-term decomposition. Science315, 361–364.

[39] Peng, Y., Song, S.Y., Li, Z.Y., Li, S., Chen, G.T., Hu, H.L., Xie, J.L., Chen, G., Xiao, Y.L., Liu, L., Tang, Y., Tu, L.H., 2020. Influences of nitrogen addition and aboveground litter-input manipulations on soil respiration and biochemical properties in a subtropical forest. Soil Biology and Biochemistry142, 107694.

[40] Peng, Y.F., Guo, D.L., Yang, Y.H., 2017. Global patterns of root dynamics under nitrogen enrichment. Global Ecology and Biogeography26, 102–114.

[41] Romaní, A.M., Fischer, H., Mille-Lindblom, C., Tranvik, L.J., 2006. Interactions of bacteria and fungi on decomposing litter: differential extracellular enzyme activities. Ecology87, 2559–2569.

[42] 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. The ISME Journal4, 1340–1351.

[43] Sayer, E.J., 2006. Using experimental manipulation to assess the roles of leaf litter in the functioning of forest ecosystems. Biological Reviews81, 1–31.

[44] Schimel, J., Balser, T.C., Wallenstein, M., 2007. Microbial stress-response physiology and its implications for ecosystem function. Ecology88, 1386–1394.

[45] Shabtai, I.A., Wilhelm, R.C., Schweizer, S.A., Höschen, C., Buckley, D.H., Lehmann, J., 2023. Calcium promotes persistent soil organic matter by altering microbial transformation of plant litter. Nature Communications14, 6609.

[46] Shi, K.L., Sun, Y.Z., Zeng, D.H., Sheng, Z.M., Zhang, Y.S., Lin, G.G., 2025a. Oxidative enzymes underlie tree species effects on soil organic carbon stocks: a common garden test with eight tree species. Soil Biology and Biochemistry202, 109715.

[47] Shi, Z.Q., Fan, C.K., Zhao, S.M., Xiao, R., Miao, R.H., Yang, Z.L., Wan, S.Q., 2025b. Effect of litter changes on soil microbial community and respiration in a coniferous-broadleaf mixed forest. Ecosystems28, 26.

[48] Smith, A.P., Marín-Spiotta, E., Balser, T., 2015. Successional and seasonal variations in soil and litter microbial community structure and function during tropical postagricultural forest regeneration: a multiyear study. Global Change Biology21, 3532–3547.

[49] Soong, J.L., Fuchslueger, L., Marañon-Jimenez, S., Torn, M.S., Janssens, I.A., Penuelas, J., Richter, A., 2020. Microbial carbon limitation: the need for integrating microorganisms into our understanding of ecosystem carbon cycling. Global Change Biology26, 1953–1961.

[50] Sun, X.L., Zhao, J., You, Y.M., Sun, O.J., 2016. Soil microbial responses to forest floor litter manipulation and nitrogen addition in a mixed-wood forest of northern China. Scientific Reports6, 19536.

[51] Tanner, E.V.J., Sheldrake, M.W.A., Turner, B.L., 2016. Changes in soil carbon and nutrients following 6 years of litter removal and addition in a tropical semi-evergreen rain forest. Biogeosciences13, 6183–6190.

[52] Treseder, K.K., 2008. Nitrogen additions and microbial biomass: a meta-analysis of ecosystem studies. Ecology Letters11, 1111–1120.

[53] Treseder, K.K., 2023. Ecological strategies of microbes: thinking outside the triangle. Journal of Ecology111, 1832–1843.

[54] van Diepen, L.T.A., Lilleskov, E.A., Pregitzer, K.S., Miller, R.M., 2010. Simulated nitrogen deposition causes a decline of intra- and extraradical abundance of arbuscular mycorrhizal fungi and changes in microbial community structure in northern hardwood forests. Ecosystems13, 683–695.

[55] Wang, C., Liu, D.W., Bai, E., 2018a. Decreasing soil microbial diversity is associated with decreasing microbial biomass under nitrogen addition. Soil Biology and Biochemistry120, 126–133.

[56] Wang, C., Lu, X.K., Mori, T., Mao, Q.G., Zhou, K.J., Zhou, G.Y., Nie, Y.X., Mo, J.M., 2018b. Responses of soil microbial community to continuous experimental nitrogen additions for 13 years in a nitrogen-rich tropical forest. Soil Biology and Biochemistry121, 103–112.

[57] Wang, C.Q., Kuzyakov, Y., 2024. Mechanisms and implications of bacterial-fungal competition for soil resources. The ISME Journal18, wrae073.

[58] Wang, Q.K., He, T.X., Wang, S.L., Liu, L., 2013. Carbon input manipulation affects soil respiration and microbial community composition in a subtropical coniferous forest. Agricultural and Forest Meteorology178–179, 152–160.

[59] Xu, S., Liu, L.L., Sayer, E.J., 2013. Variability of above-ground litter inputs alters soil physicochemical and biological processes: a meta-analysis of litterfall-manipulation experiments. Biogeosciences10, 7423–7433.

[60] Yang, A., Song, B., Zhang, W.X., Zhang, T.N., Li, X.W., Wang, H.T., Zhu, D., Zhao, J., Fu, S.L., 2024. Chronic enhanced nitrogen deposition and elevated precipitation jointly benefit soil microbial community in a temperate forest. Soil Biology and Biochemistry193, 109397.

[61] Yang, L., Wang, N., Chen, Y., Yang, W., Tian, D.S., Zhang, C.Y., Zhao, X.H., Wang, J.S., Niu, S.L., 2020. Carbon management practices regulate soil bacterial communities in response to nitrogen addition in a pine forest. Plant and Soil452, 137–151.

[62] Yang, Y., Chen, X.L., Liu, L.X., Li, T., Dou, Y.X., Qiao, J.B., Wang, Y.Q., An, S.S., Chang, S.X., 2022. Nitrogen fertilization weakens the linkage between soil carbon and microbial diversity: a global meta-analysis. Global Change Biology28, 6446–6461.

[63] Yokobe, T., Tokuchi, N., Hyodo, F., Tateno, R., Hiura, T., 2022. Response of microorganisms to a 5-year large-scale nitrogen loading in immature volcanic ash soil in an oak-dominated forest. Applied Soil Ecology177, 104537.

[64] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils29, 111–129.

[65] Zeng, J., Liu, X.J., Song, L., Lin, X.G., Zhang, H.Y., Shen, C.C., Chu, H.Y., 2016. Nitrogen fertilization directly affects soil bacterial diversity and indirectly affects bacterial community composition. Soil Biology and Biochemistry92, 41–49.

[66] Zhang, J.L., Lin, G.G., Zeng, D.H., 2024. Long-term nitrogen addition modifies fine root growth and vertical distribution by affecting soil nutrient availability in a Mongolian pine plantation. Science of the Total Environment921, 171168.

[67] Zhang, T.A., Chen, H.Y.H., Ruan, H.H., 2018. Global negative effects of nitrogen deposition on soil microbes. The ISME Journal12, 1817–1825.

[68] Zhang, Y.S., Zeng, D.H., Lei, Z.Y., Li, X., Lin, G.G., 2023. Microbial properties determine dynamics of topsoil organic carbon stocks and fractions along an age-sequence of Mongolian pine plantations. Plant and Soil483, 441–457.

[69] Zhao, L., Hu, Y.L., Lin, G.G., Gao, Y.C., Fang, Y.T., Zeng, D.H., 2013. Mixing effects of understory plant litter on decomposition and nutrient release of tree litter in two plantations in Northeast China. PLoS One8, e76334.

[70] Zhao, Q., Classen, A.T., Wang, W.W., Zhao, X.R., Mao, B., Zeng, D.H., 2017. Asymmetric effects of litter removal and litter addition on the structure and function of soil microbial communities in a managed pine forest. Plant and Soil414, 81–93.

[71] Zhou, Q., Fransen, A., de Winde, H., 2025. Lignin-degrading enzymes and the potential of Pseudomonas putida as a cell factory for lignin degradation and valorization. Microorganisms13, 935.

[72] Zhou, Z.H., Wang, C.K., Zheng, M.H., Jiang, L.F., Luo, Y.Q., 2017. Patterns and mechanisms of responses by soil microbial communities to nitrogen addition. Soil Biology and Biochemistry115, 433–441.

Rights & permissions

Higher Education Press