Fifteen-year cattle manure application reshapes phoD- and gcd-harboring microbiomes, enhancing vegetable yields

Abstract

Long-term cattle manure application significantly influences soil phosphorus (P) cycling and associated microbial communities in agricultural systems. However, the mechanisms by which P-transforming microbial communities and their ecological networks mediate P cycling and crop productivity under sustained organic amendment remain poorly understood. This study investigated the effects of 15-year cattle manure application on soil P forms, P-solubilizing microbial communities, and lettuce (Lactuca sativa) yields across three treatments: no fertilization (control), manure-only (M), and combined manure and chemical fertilizer (M+CF). The M+CF treatment significantly enhanced lettuce yields by 77% compared to control and 41% compared to M treatment, while increasing P content by 3.9% and 2.1%, respectively. Metagenomic analysis revealed that manure application increased the diversity (Shannon index: +32.5%) and abundance (+260%) of phoD-harboring bacteria in the M treatment, while M+CF enhanced both diversity (+45.3%) and abundance (+290%) of gcd-harboring bacteria. Proteobacteria (54.2%−68.8%), Acidobacteria (24.2%−33.2%), and Gemmatimonadetes dominated the P-solubilizing bacterial communities across treatments. Network analysis demonstrated that M+CF treatment increased positive microbial correlations by 74.6% compared to control, with enhanced connectivity among keystone taxa, particularly for gcd-harboring microorganisms. Soil enzyme activities showed strong correlations with gene abundances (R² = 0.92 for gcd-ACP; R² = 0.86 for phoD-ALP), suggesting functional linkages between microbial community composition and P transformation processes. Overall, these findings demonstrate that appropriate long-term fertilization strategies can optimize soil P use efficiency, enhance microbial-mediated P transformations, and improve vegetable yields, providing insights for sustainable nutrient management in intensive cropping systems.

Graphical abstract

Keywords

phosphorus cycling / microbial community structure / network analysis / sustainable agriculture

Highlight

	● 15 years cattle manure addition enhanced soil P availability and lettuce yield.
	● gcd and phoD gene abundances increased under manure treatments, correlating with available P.
	● Combined manure and chemical fertilizers led to increased microbial community diversity.
	● Positive interactions within microbial networks were higher in manure-applied soils.
	● Key microbial taxa were identified as crucial drivers of P transformation.

References

[1] Apel, A.K., Sola-Landa, A., Rodríguez-García, A., Martín, J.F., 2007. Phosphate control of phoA, phoC and phoD gene expression in Streptomyces coelicolor reveals significant differences in binding of PhoP to their promoter regions. Microbiology153, 3527–3537.

[2] Bi, Q.F., Li, K.J., Zheng, B.X., Liu, X.P., Li, H.Z., Jin, B.J., Ding, K., Yang, X.R., Lin, X.Y., Zhu, Y.G., 2020. Partial replacement of inorganic phosphorus (P) by organic manure reshapes phosphate mobilizing bacterial community and promotes P bioavailability in a paddy soil. Science of the Total Environment703, 134977.

[3] Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics30, 2114–2120.

[4] Bremner, J.M., Jenkinson, D.S., 1960. Determination of organic carbon in soil. I. Oxidation by dichromate of organic matter in soil and plant materials. European Journal of Soil Science11, 394–402.

[5] Buchfink, B., Xie, C., Huson, D.H., 2015. Fast and sensitive protein alignment using DIAMOND. Nature Methods12, 59–60.

[6] Bulgarelli, D., Rott, M., Schlaeppi, K., Ver Loren van Themaat, E., Ahmadinejad, N., Assenza, F., Rauf, P., Huettel, B., Reinhardt, R., Schmelzer, E., Peplies, J., Gloeckner, F.O., Amann, R., Eickhorst, T., Schulze-Lefert, P., 2012. Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature488, 91–95.

[7] Casida, L.E.JR., Klein, D.A., Santoro, T., 1964. Soil dehydrogenase activity. Soil Science98, 371–376.

[8] Chen, X.D., Jiang, N., Chen, Z.H., Tian, J.H., Sun, N., Xu, M.G., Chen, L.J., 2017. Response of soil phoD phosphatase gene to long-term combined applications of chemical fertilizers and organic materials. Applied Soil Ecology119, 197–204.

[9] da Silva, L.I., Pereira, M.C., de Carvalho, A.M.X., Buttrós, V.H., Pasqual, M., Dória, J., 2023. Phosphorus-solubilizing microorganisms: a key to sustainable agriculture. Agriculture13, 462.

[10] Dai, Z.M., Liu, G.F., Chen, H.H., Chen, C.R., Wang, J.K., Ai, S.Y., Wei, D., Li, D.M., Ma, B., Tang, C.X., Brookes, P.C., Xu, J.M., 2020. Long-term nutrient inputs shift soil microbial functional profiles of phosphorus cycling in diverse agroecosystems. The ISME Journal14, 757–770.

[11] Delgado-Baquerizo, M., Reich, P.B., Trivedi, C., Eldridge, D.J., Abades, S., Alfaro, F.D., Bastida, F., Berhe, A.A., Cutler, N.A., Gallardo, A., García-Velázquez, L., Hart, S.C., Hayes, P.E., He, J.Z., Hseu, Z.Y., Hu, H.W., Kirchmair, M., Neuhauser, S., Pérez, C.A., Reed, S.C., Santos, F., Sullivan, B.W., Trivedi, P., Wang, J.T., Weber-Grullon, L., Williams, M.A., Singh, B.K., 2020. Multiple elements of soil biodiversity drive ecosystem functions across biomes. Nature Ecology & Evolution4, 210–220.

[12] Deng, Y., Jiang, Y.H., Yang, Y.F., He, Z.L., Luo, F., Zhou, J.Z., 2012. Molecular ecological network analyses. BMC Bioinformatics13, 113.

[13] Fan, K.K., Delgado-Baquerizo, M., Guo, X.S., Wang, D.Z., Zhu, Y.G., Chu, H.Y., 2021. Biodiversity of key-stone phylotypes determines crop production in a 4-decade fertilization experiment. The ISME Journal15, 550–561.

[14] 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.

[15] Fraser, T.D., Lynch, D.H., Bent, E., Entz, M.H., Dunfield, K.E., 2015. Soil bacterial phoD gene abundance and expression in response to applied phosphorus and long-term management. Soil Biology and Biochemistry88, 137–147.

[16] Guo, J.H., Liu, X.J., Zhang, Y., Shen, J.L., Han, W.X., Zhang, W.F., Christie, P., Goulding, K.W. T., Vitousek, P.M., Zhang, F.S., 2010. Significant acidification in major Chinese croplands. Science327, 1008–1010.

[17] Guo, L., Wang, C., Feng, T.Y., Shen, R.F., 2023. Short-term application of organic fertilization impacts phosphatase activity and phosphorus-mineralizing bacterial communities of bulk and rhizosphere soils of maize in acidic soil. Plant and Soil484, 95–113.

[18] Hao, T.X., Zhu, Q.C., Zeng, M.F., Shen, J.B., Shi, X.J., Liu, X.J., Zhang, F.S., de Vries, W., 2019. Quantification of the contribution of nitrogen fertilization and crop harvesting to soil acidification in a wheat-maize double cropping system. Plant and Soil434, 167–184.

[19] Hartmann, M., Six, J., 2023. Soil structure and microbiome functions in agroecosystems. Nature Reviews Earth & Environment4, 4–18.

[20] Haumont, J., Diels, J., Schrevens, E., Cool, S., Lootens, P., Saeys, W., 2023. Assessment of fertilization strategies and policy measures for vegetables by simulation of a long-term cauliflower leek rotation. European Journal of Agronomy149, 126902.

[21] Hedley, M.J., Stewart, J.W.B., Chauhan, B.S., 1982. Changes in inorganic and organic soil phosphorus fractions induced by cultivation practices and by laboratory incubations. Soil Science Society of America Journal46, 970–976.

[22] Ho, A., Di Lonardo, D.P., Bodelier, P.L.E., 2017. Revisiting life strategy concepts in environmental microbial ecology. FEMS Microbiology Ecology93, fix006.

[23] Hu, Y.J., Xia, Y.H., Sun, Q., Liu, K.P., Chen, X.B., Ge, T.D., Zhu, B.L., Zhu, Z.K., Zhang, Z.H., Su, Y.R., 2018. Effects of long-term fertilization on phoD-harboring bacterial community in Karst soils. Science of the Total Environment628–629, 53–63.

[24] Huo, W.G., Peng, Y., Maimaitiaili, B., Batchelor, W.D., Feng, G., 2023. Phosphorus fertilizer recommendation based on minimum soil surplus for cotton growing in salt-affected soils. Field Crops Research291, 108799.

[25] Hyatt, D., Chen, G.L., LoCascio, P.F., Land, M.L., Larimer, F.W., Hauser, L.J., 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics11, 119.

[26] Kang, L.Y., Fan, B.Q., Chen, S., Chen, Q., 2018. Fertigation combined with catch crop maximize vegetable yield and minimize N and P surplus. Nutrient Cycling in Agroecosystems112, 87–99.

[27] Lagos, L.M., Acuña, J.J., Maruyama, F., Ogram, A., de la Luz Mora, M., Jorquera, M.A., 2016. Effect of phosphorus addition on total and alkaline phosphomonoesterase-harboring bacterial populations in ryegrass rhizosphere microsites. Biology and Fertility of Soils52, 1007–1019.

[28] Lang, M., Li, H.M., Lakshmanan, P., Chen, Y.X., Chen, X.P., 2022. phoD-harboring bacterial community composition dominates organic P mineralization under long-term P fertilization in acid purple soil. Frontiers in Microbiology13, 1045919.

[29] Langmead, B., Salzberg, S.L., 2012. Fast gapped-read alignment with Bowtie 2. Nature Methods9, 357–359.

[30] Li, D.H., Liu, C.M., Luo, R.B., Sadakane, K., Lam, T.W., 2015. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics31, 1674–1676.

[31] Louhisuo, A., Yli-Halla, M., Termonen, M., Kykkänen, S., Järvenranta, K., Virkajärvi, P., 2024. Long-term changes in soil phosphorus in response to fertilizer application and negative phosphorus balance under grass rotation in mineral soils in Nordic conditions. Soil Use and Management40, e13013.

[32] Luan, L., Liang, C., Chen, L.J., Wang, H.T., Xu, Q.S., Jiang, Y.J., Sun, B., 2020. Coupling bacterial community assembly to microbial metabolism across soil profiles. mSystems5, e00298–20.

[33] Mander, C., Wakelin, S., Young, S., Condron, L., O’Callaghan, M., 2012. Incidence and diversity of phosphate-solubilising bacteria are linked to phosphorus status in grassland soils. Soil Biology and Biochemistry44, 93–101.

[34] Mao, X.L., Xu, X.L., Lu, K.P., Gielen, G., Luo, J.F., He, L.Z., Donnison, A., Xu, Z.X., Xu, J., Yang, W.Y., Song, Z.L., Wang, H.L., 2015. Effect of 17 years of organic and inorganic fertilizer applications on soil phosphorus dynamics in a rice–wheat rotation cropping system in eastern China. Journal of Soils and Sediments15, 1889–1899.

[35] Mao, Y.T., Hu, W., Li, Y.M., Li, Y., Lei, B.K., Zheng, Y., 2023. Long-term cattle manure addition enhances soil-available phosphorus fractions in subtropical open-field rotated vegetable systems. Frontiers in Plant Science14, 1138207.

[36] Miao, Y.X., Stewart, B.A., Zhang, F.S., 2011. Long-term experiments for sustainable nutrient management in China. A review. Agronomy for Sustainable Development31, 397–414.

[37] Mohanty, S., Paikaray, N.K., Rajan, A.R., 2006. Availability and uptake of phosphorus from organic manures in groundnut (Arachis hypogea L. )–corn (Zea mays L.) sequence using radio tracer technique. Geoderma133, 225–230.

[38] Molla, M.A.Z., Chowdhury, A.A., Islam, A., Hoque, S., 1984. Microbial mineralization of organic phosphate in soil. Plant and Soil78, 393–399.

[39] Murphy, J., Riley, J.P., 1962. A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta27, 31–36.

[40] Olesen, J.M., Bascompte, J., Dupont, Y.L., Jordano, P., 2007. The modularity of pollination networks. Proceedings of the National Academy of Sciences of the United States of America104, 19891–19896.

[41] Olsen, S.R., Cole, C.V., Watanabe, F.S., 1954. Estimation of Available Phosphorus in Soils by Extraction with Sodium Bicarbonate. Washington: US Government Printing Office.

[42] Patro, R., Duggal, G., Love, M.I., Irizarry, R.A., Kingsford, C., 2017. Salmon provides fast and bias-aware quantification of transcript expression. Nature Methods14, 417–419.

[43] Penn, C.J., Camberato, J.J., 2019. A critical review on soil chemical processes that control how soil pH affects phosphorus availability to plants. Agriculture9, 120.

[44] Pold, G., Melillo, J.M., DeAngelis, K.M., 2015. Two decades of warming increases diversity of a potentially lignolytic bacterial community. Frontiers in Microbiology6, 480.

[45] Qin, X.C., Guo, S.F., Zhai, L.M., Pan, J.T., Khoshnevisan, B., Wu, S.X., Wang, H.Y., Yang, B., Ji, J.H., Liu, H.B., 2020. How long-term excessive manure application affects soil phosphorous species and risk of phosphorous loss in fluvo-aquic soil. Environmental Pollution266, 115304.

[46] Qin, Y.Q., Zhang, W., Feng, Z.W., Feng, G.D., Zhu, H.H., Yao, Q., 2022. Arbuscular mycorrhizal fungus differentially regulates P mobilizing bacterial community and abundance in rhizosphere and hyphosphere. Applied Soil Ecology170, 104294.

[47] Randall, K., Brennan, F., Clipson, N., Creamer, R., Griffiths, B., Storey, S., Doyle, E., 2019. Soil bacterial community structure and functional responses across a long-term mineral phosphorus (Pi) fertilisation gradient differ in grazed and cut grasslands. Applied Soil Ecology138, 134–143.

[48] Rawat, P., Das, S., Shankhdhar, D., Shankhdhar, S.C., 2021. Phosphate-solubilizing microorganisms: mechanism and their role in phosphate solubilization and uptake. Journal of Soil Science and Plant Nutrition21, 49–68.

[49] Richardson, A.E., Barea, J.M., McNeill, A.M., Prigent-Combaret, C., 2009. Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms. Plant and Soil321, 305–339.

[50] Rogers, M.B., Firek, B., Shi, M., Yeh, A., Brower-Sinning, R., Aveson, V., Kohl, B.L., Fabio, A., Carcillo, J.A., Morowitz, M.J., 2016. Disruption of the microbiota across multiple body sites in critically ill children. Microbiome4, 66.

[51] Siles, J.A., Starke, R., Martinovic, T., Parente Fernandes, M.L., Orgiazzi, A., Bastida, F., 2022. Distribution of phosphorus cycling genes across land uses and microbial taxonomic groups based on metagenome and genome mining. Soil Biology and Biochemistry174, 108826.

[52] Steinegger, M., Söding, J., 2017. MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nature Biotechnology35, 1026–1028.

[53] Tabatabai, M.A., 1994. Soil enzymes. In: Bottomley, P.S., Angle, J.S. Weaver, R.W., eds. Methods of Soil Analysis: Part 2—Microbiological and Biochemical Properties. Madison: Soil Science Society of America, 775–833.

[54] Tian, D.S., Niu, S.L., 2015. A global analysis of soil acidification caused by nitrogen addition. Environmental Research Letters10, 024019.

[55] Tiessen, H., Moir, J.O., 1993. Characterization of available P by sequential extraction. Soil Sampling and Methods of Analysis824, 75–87.

[56] Wang, F., Wei, X., Zhang, L.Q., Feng, G., 2023. Long-term fertilisation management changes bacterial phoD and gcd gene communities and abundances in the rhizosphere of cotton (Gossypium hirsutum L.) grown in a grey desert soil. Rhizosphere28, 100797.

[57] Wei, X.M., Hu, Y.J., Razavi, B.S., Zhou, J., Shen, J.L., Nannipieri, P., Wu, J.S., Ge, T.D., 2019. Rare taxa of alkaline phosphomonoesterase-harboring microorganisms mediate soil phosphorus mineralization. Soil Biology and Biochemistry131, 62–70.

[58] Whalen, J.K., Chang, C., Clayton, G.W., Carefoot, J.P., 2000. Cattle manure amendments can increase the pH of acid soils. Soil Science Society of America Journal64, 962–966.

[59] Wu, X.J., Cui, Z.L., Peng, J.J., Zhang, F.S., Liesack, W., 2022a. Genome-resolved metagenomics identifies the particular genetic traits of phosphate-solubilizing bacteria in agricultural soil. ISME Communications2, 17.

[60] Wu, X.J., Rensing, C., Han, D.F., Xiao, K.Q., Dai, Y.X., Tang, Z.X., Liesack, W., Peng, J.J., Cui, Z.L., Zhang, F.S., 2022b. Genome-resolved metagenomics reveals distinct phosphorus acquisition strategies between soil microbiomes. mSystems7, e01107–21.

[61] Xiang, Y.Z., Li, Y., Luo, X.Q., Liu, Y., Yue, X.J., Yao, B., Xue, J.M., Zhang, L.Y., Fan, J., Xu, X.Y., Li, Y.H., 2022. Manure properties, soil conditions and managerial factors regulate greenhouse vegetable yield with organic fertilizer application across China. Frontiers in Plant Science13, 1009631.

[62] Xie, Y.Y., Wang, F.H., Wang, K., Yue, H.Z., Lan, X.F., 2020. Responses of bacterial phoD gene abundance and diversity to crop rotation and feedbacks to phosphorus uptake in wheat. Applied Soil Ecology154, 103604.

[63] Yahya, M., Rasul, M., Sarwar, Y., Suleman, M., Tariq, M., Hussain, S.Z., Sajid, Z.I., Imran, A., Amin, I., Reitz, T., Tarkka, M.T., Yasmin, S., 2022. Designing synergistic biostimulants formulation containing autochthonous phosphate-solubilizing bacteria for sustainable wheat production. Frontiers in Microbiology13, 889073.

[64] Zhang, W.S., Li, H.P., Li, Y.L., 2019. Spatio-temporal dynamics of nitrogen and phosphorus input budgets in a global hotspot of anthropogenic inputs. Science of the Total Environment656, 1108–1120.

[65] Zhao, Y.N., Mao, X.X., Zhang, M.S., Yang, W., Di, H.J., Ma, L., Liu, W.J., Li, B.W., 2021. The application of Bacillus Megaterium alters soil microbial community composition, bioavailability of soil phosphorus and potassium, and cucumber growth in the plastic shed system of North China. Agriculture, Ecosystems & Environment307, 107236.

[66] Zhou, G.P., Gao, S.J., Lu, Y.H., Liao, Y.L., Nie, J., Cao, W.D., 2020. Co-incorporation of green manure and rice straw improves rice production, soil chemical, biochemical and microbiological properties in a typical paddy field in southern China. Soil and Tillage Research197, 104499.

RIGHTS & PERMISSIONS

Higher Education Press