Sugar Tech

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Impact of Sugarcane–Legume Intercropping on Diazotrophic Microbiome

  • Manoj Kumar Solanki
  • Fei-Yong Wang
  • Chang-Ning Li
  • Zhen Wang
  • Tao-Ju Lan
  • Rajesh Kumar Singh
  • Pratiksha Singh
  • Li-Tao Yang
  • Yang-Rui LiEmail author
Research Article


The present study discussed the application of the intercropping system to improve land use efficacy and soil microbial activity. We assessed linkages of soil properties and unculturable diazotrophs community under three cultivation systems (monoculture sugarcane, peanut–sugarcane and soybean–sugarcane intercropping). Rhizosphere soil of sugarcane was sampled and DNA was extracted. We amplified the nifH gene and sequenced by high throughput sequencing. The bioinformatics analysis of sequenced data obtained a total of 436,458 nifH gene reads that are classified into 3201 unique operational taxonomic units (OTUs). A higher percentage of exclusive OTUs identified under soybean–sugarcane intercropping (< 375). The microbial structure results showed that Alpha-proteobacteria and Beta-proteobacteria were the dominant groups in all three cultivation systems. While genus such as Bradyrhizobium, Burkholderia, Pelomonas, and Sphingomonas was predominant in the intercropping systems and these diazotrophic bacterial communities were positively correlated to the soil pH and soil enzyme protease. Additionally, a lower quantity of available P in the soil of intercrops indicated a strong link between soil nutrients uptake and microbial activity. The results of the present study concluded some interesting facts of intercropping systems that positively improved the soil microbial activity and this kind of strategy could help to cultivate multiple crops to improve the economic growth of the country by sustainable sugarcane production.


Intercropping Microbial community Next-generation sequencing NifH gene Sugarcane 


Author Contributions

YRL, LTY designed the study. MKS, FYW and CNL conducted the experiments. MKS and ZW did the isolation and characterization of microbes. TJL supported data collection and analysis. MKS, CNL, RKS, PS, LTY and YRL wrote the manuscript.


Present research work supported by Grants from GXAAS (No. GNKB2014021); the Grants from the National High Technology Research and Development Program (“863” Program) of China (2013AA102604), National Natural Science Foundation of China (31171504, 31101122, 31471449), Guangxi Special Funds for Bagui Scholars’s and Distinguished Experts, Guangxi Natural Science Foundation and Guangxi Academy of Agriculture Sciences Fund (2011GXNSFF018002, 2012GXNSFDA053011, 2013NXNSFAA019073 and GuiNongKe2014YD01).

Compliance with Ethical Standards

Conflict of interest

There is no conflict of interest declared by all authors.

Ethical Statement


Informed Consent


Supplementary material

12355_2019_755_MOESM1_ESM.docx (260 kb)
Supplementary material 1 (DOCX 260 kb)


  1. Asis Jr., C.A., M. Kubota, V.K. Chebotar, H. Ohta, Y. Arima, K. Nishiyama, K.I. Tsuchiya, and S. Akao. 2000. Endophytic bacterial population in Philippine sugarcane cultivars and isolation of nitrogen-fixing strains. Microbes and Environments, 15: 209–216. CrossRefGoogle Scholar
  2. Bao, S.D. 2002. Soil agricultural chemical analysis, 3rd ed. Beijing: China Agricultural Press.Google Scholar
  3. Blais, M., J.É. Tremblay, A.D. Jungblut, J. Gagnon, J. Martin, M. Thaler, and C. Lovejoy. 2012. Nitrogen fixation and identification of potential diazotrophs in the Canadian Arctic. Global Biogeochemical Cycles 26: GB3022. Scholar
  4. Bremmer, J.M., and C.S. Mulvaney. 1982. “Total nitrogen”, in methods of soil analysis—Part 2. In Chemical and microbiological properties, ed. C.A. Bluck, 595–624. Madison: American Society of Agronomy.Google Scholar
  5. Carter, Martin R. 1993. Soil sampling and methods of analysis. Boca Raton: Lewis Publishers.Google Scholar
  6. Coelho, Marcia R.R., Ivanildo E. Marriel, Sasha N. Jenkins, Clare V. Lanyon, Lucy Seldin, and Anthony G. O’Donnell. 2009. Molecular detection and quantification of nifH gene sequences in the rhizosphere of sorghum (Sorghum bicolor) sown with two levels of nitrogen fertilizer. Applied Soil Ecology 42: 48–53. Scholar
  7. Cole, James R., Qiong Wang, Jordan A. Fish, Benli Chai, Donna M. McGarrell, C. Yanni Sun, Titus Brown, Andrea Porras-Alfaro, Cheryl R. Kuske, and James M. Tiedje. 2014. Ribosomal database project: Data and tools for high throughput rRNA analysis. Nucleic Acids Research 42: D633–D642. Scholar
  8. Edgar, Robert C. 2013. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nature Methods 10: 996–998. Scholar
  9. Edgar, Robert C., Brian J. Haas, Jose C. Clemente, Christopher Quince, and Rob Knight. 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27: 2194–2200. Scholar
  10. Fadrosh, D.W., B. Ma, P. Gajer, N. Sengamalay, S. Ott, R.M. Brotman, and J. Ravel. 2014. An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome 2: 6. Scholar
  11. Gaby, John Christian, and Daniel H. Buckley. 2014. A comprehensive aligned nifH gene database: A multipurpose tool for studies of nitrogen-fixing bacteria. Database 2014: bau001. Scholar
  12. Guan, S.Y., D. Zhang, and Z. Zhang. 1986. Soil enzyme and its research methods. Beijing: Agricultural.Google Scholar
  13. Hammer, Ř., D.A.T. Harper, and P.D. Ryan. 2001. Past: Paleontological statistics software package for education and data analysis. Palaeontologia Electronica 4: 178.Google Scholar
  14. Hardy, R.W., R.D. Holsten, E.K. Jackson, and R.C. Burns. 1968. The acetylene-ethylene assay for n(2) fixation: Laboratory and field evaluation. Plant Physiology 43: 1185–1207. Scholar
  15. Heller, P., H.J. Tripp, K. Turk-Kubo, and J.P. Zehr. 2014. ARBitrator: A software pipeline for on-demand retrieval of auto-curated nifH sequences from GenBank. Bioinformatics 30: 2883–2890. Scholar
  16. Kaur, J., M. Verma, and R. Lal. 2011. Rhizobium rosettiformans sp. nov., isolated from a hexachlorocyclohexane dump site, and reclassification of Blastobacter aggregatus Hirsch and Muller 1986 as Rhizobium aggregatum comb. nov. International Journal of Systematic and Evolutionary Microbiology 61: 1218–1225. Scholar
  17. Kent, A.D., and E.W. Triplett. 2002. Microbial communities and their interactions in soil and rhizosphere ecosystems. Annual Review of Microbiology 56: 211–236. Scholar
  18. Li, C., Y. Dong, H. Li, J. Shen, and F. Zhang. 2016. Shift from complementarity to facilitation on P uptake by intercropped wheat neighboring with faba bean when available soil P is depleted. Scientific Reports 6: 18663. Scholar
  19. Li, Z.G., Y.M. Luo, and Y. Teng. 2008. Research methods for soil and environmental microbiology. Beijing: Science Press.Google Scholar
  20. Li, X., P. Penttinen, Y. Gu, and X. Zhang. 2012. Diversity of nifH gene in rhizosphere and non-rhizosphere soil of tobacco in Panzhihua, China. Annals of Microbiology 62: 995–1001. Scholar
  21. Li, Y., H. Wen, L. Chen, and T. Yin. 2014. Succession of bacterial community structure and diversity in soil along a chronosequence of reclamation and re-vegetation on coal mine spoils in China. Edited by Daniele Daffonchio. PLoS ONE 9: e115024. Scholar
  22. Li, Y.R., and L.T. Yang. 2015. Sugarcane agriculture and sugar industry in China. Sugar Tech 17: 1–8. Scholar
  23. Li, Y.R., X.Z. Zhou, and L.T. Yang. 2015. Biological nitrogen fixation in sugarcane and nitrogen transfer from sugarcane to cassava in an intercropping system. International Journal of Science and Nature 6: 214–218.Google Scholar
  24. Magoc, T., and S.L. Salzberg. 2011. FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27: 2957–2963. Scholar
  25. Orr, C.H., A. James, C. Leifert, J.M. Cooper, and S.P. Cummings. 2011. Diversity and activity of free-living nitrogen-fixing bacteria and total bacteria in organic and conventionally managed soils. Applied and Environmental Microbiology 77: 911–919. Scholar
  26. Perin, L., L. Martínez-Aguilar, G. Paredes-Valdez, J.I. Baldani, P. Estrada-de Los Santos, V.M. Reis, and J. Caballero-Mellado. 2006. Burkholderia silvatlantica sp. nov., a diazotrophic bacterium associated with sugarcane and maize. International Journal of Systematic and Evolutionary Microbiology 56: 1931–1937. Scholar
  27. Robertson, G.P., and P.M. Vitousek. 2009. Nitrogen in agriculture: balancing the cost of an essential resource. Annual Review of Environment and Resources 34: 97–125. Scholar
  28. Ruan, A., R. He, S. Xu, and T. Lin. 2009. Effect of dissolved oxygen on nitrogen purification of microbial ecosystem in sediments. Journal of Environmental Science and Health, Part A 44: 397–405. Scholar
  29. Sachs, J.L., S.W. Kembel, A.H. Lau, and E.L. Simms. 2009. In situ phylogenetic structure and diversity of wild Bradyrhizobium communities. Applied and Environmental Microbiology 75: 4727–4735. Scholar
  30. Solanki, M.K., F.Y. Wang, Z. Wang, C.N. Li, T.J. Lan, R.K. Singh, P. Singh, L.T. Yang, and Y.R. Li. 2018. Rhizospheric and endospheric diazotrophs mediated soil fertility intensification in sugarcane–legume intercropping systems. Journal of Soil and Sediments. Scholar
  31. Solanki, M.K., Z. Wang, F.Y. Wang, C.N. Li, T.J. Lan, R.K. Singh, P. Singh, L.T. Yang, and Y.R. Li. 2017. Intercropping in sugarcane cultivation influenced the soil properties and enhanced the diversity of vital diazotrophic bacteria. Sugar Tech 19: 136–147. Scholar
  32. Song, B., and B.B. Ward. 2003. Nitrite reductase genes in halobenzoate degrading denitrifying bacteria. FEMS Microbiology Ecology 43: 349–357. Scholar
  33. Tai, X.S., W.L. Mao, G.X. Liu, T. Chen, W. Zhang, X.K. Wu, H.Z. Long, B.G. Zhang, and T.P. Gao. 2014. Distribution of ammonia oxidizers in relation to vegetation characteristics in the Qilian Mountains, northwestern China. Biogeosciences Discussions 11: 5123–5146. Scholar
  34. Tai, X.S., W.L. Mao, G.X. Liu, T. Chen, W. Zhang, X.K. Wu, H.Z. Long, B.G. Zhang, and Y. Zhang. 2013. High diversity of nitrogen-fixing bacteria in the upper reaches of the Heihe River, northwestern China. Biogeosciences 10: 5589–5600. Scholar
  35. Walkley, A., and I.A. Black. 1934. An examination of the Degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method. Soil Science 37: 29–38.CrossRefGoogle Scholar
  36. Wang, Q., G.M. Garrity, J.M. Tiedje, and J.R. Cole. 2007. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology 73: 5261–5267. Scholar
  37. Wang, Z.G., X. Jin, X.G. Bao, X.F. Li, J.H. Zhao, J.H. Sun, P. Christie, and L. Li. 2014. Intercropping enhances productivity and maintains the most soil fertility properties relative to sole cropping. Edited by Ben Bond-Lamberty. PLoS ONE 9: e113984. Scholar
  38. Xie, C.H., and A. Yokota. 2005. Reclassification of Alcaligenes latus strains IAM 12599T and IAM 12664 and Pseudomonas saccharophila as Azohydromonas lata gen. nov., comb. nov., Azohydromonas australica sp. nov. and Pelomonas saccharophila gen. nov., comb. nov., respectively. International Journal of Systematic and Evolutionary Microbiology 55: 2419–2425. Scholar
  39. Xie, C.H., and A. Yokota. 2006. Sphingomonas azotifigens sp. nov., a nitrogen-fixing bacterium isolated from the roots of Oryza sativa. International Journal of Systematic and Evolutionary Microbiology 56: 889–893. Scholar
  40. Yang, W., Z. Li, J. Wang, P. Wu, and Y. Zhang. 2013. Crop yield, nitrogen acquisition and sugarcane quality as affected by interspecific competition and nitrogen application. Field Crops Research 146: 44–50. Scholar
  41. Yousuf, B., R. Kumar, A. Mishra, and B. Jha. 2014. Differential distribution and abundance of diazotrophic bacterial communities across different soil niches using a gene-targeted clone library approach. FEMS Microbiology Letters 360: 117–125. Scholar
  42. Zehr, J.P., M.T. Mellon, and S. Zani. 1998. New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nifH) genes. Applied and Environmental Microbiology 64: 3444–3450.Google Scholar
  43. Zehr, J.P., B.D. Jenkins, S.M. Short, and G.F. Steward. 2003. Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environmental Microbiology 5(7): 539–554.CrossRefGoogle Scholar
  44. Zeng, Q., X. Wu, J. Wang, and X. Ding. 2017. Phosphate solubilization and gene expression of phosphate-solubilizing bacterium Burkholderia multivorans WS-FJ9 under different levels of soluble phosphate. Journal of Microbiology and Biotechnology 27: 844–855. Scholar
  45. Zhang, L.H., and S.F. Chen. 2012. Pseudacidovorax intermedius NH-1, a novel marine nitrogen-fixing bacterium isolated from the South China Sea. World Journal of Microbiology & Biotechnology 28: 2839–2847. Scholar
  46. Zou, Y., J. Zhang, D. Yang, X. Chen, J. Zhao, W. Xiu, X. Lai, and G. Li. 2011. Effects of different land use patterns on nifH genetic diversity of soil nitrogen-fixing microbial communities in Leymus Chinensis steppe. Acta Ecologica Sinica 31: 150–156. Scholar

Copyright information

© Society for Sugar Research & Promotion 2019

Authors and Affiliations

  1. 1.Guangxi Crop Genetic Improvement and Biotechnology LabGuangxi Academy of Agricultural SciencesNanningChina
  2. 2.Horticulture InstituteGuangxi Academy of Agricultural SciencesNanningChina
  3. 3.Guangxi Key Laboratory of Sugarcane Genetic Improvement/Key Laboratory of Sugarcane Biotechnology and Genetic Improvement (Guangxi)Ministry of Agriculture/Sugarcane Research Institute of Guangxi Academy of Agricultural Sciences/Sugarcane Research Center of Chinese Academy of Agricultural SciencesNanningChina
  4. 4.Agricultural College, State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresourcesGuangxi UniversityNanningChina
  5. 5.Agricultural Research Organization, Department of Food Quality & Safety, Institute for Post-harvest and Food SciencesThe Volcani CenterRishon LezionIsrael

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