Unveiling of active diazotrophs in a flooded rice soil by combination of NanoSIMS and 15N2-DNA-stable isotope probing

Abstract

The biological nitrogen fixation in planted and nonplanted paddy soils was quantified using a chamber-based 15N2-labeling technique, and the active diazotrophs of soil were assessed by 15N2-DNA-stable isotope probing (SIP). In addition, the nanometer scale secondary ion mass spectrometry (NanoSIMS) was applied to analysis the 15N-enrichment of soil DNA in SIP fractions. 15N2-labeling experiment showed that BNF was 11.33 ± 1.90 kg N ha−1 in the rice-planted soil and 3.55 ± 1.18 kg N ha−1 in the nonplanted soil after 28-day labeling. The biologically fixed 15N was mainly (> 95%) recovered in the surface layer (0–0.5 cm) in the rice-planted soil. High throughput sequencing of nifH genes extracted from surface soil showed that the presence of rice affected the community composition of diazotrophs. The relative abundance of Nostocales and Stigonematales was significantly higher in rice-planted soil than in nonplanted soil (P < 0.05). After CsCl gradient ultracentrifugation, NanoSIMS images clearly showed that 15N was incorporated into soil DNA in the 15N2-labeling SIP gradient fractions. Analyses of nifH genes in 15N-enriched SIP gradient fractions suggested that Nostocales and Stigonematales were the major contributors to BNF in the rice-soil system. Taken together, these results have highlighted the contributions of cyanobacteria to the BNF in paddy fields and improved our understanding of the close relationship between rice plants and cyanobacteria.

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Acknowledgments

The authors thank Yufang Sun for help on 15N-enrichment analysis of gas samples, and our colleagues from the research group (other than the authors) for help on the DNA-SIP experiment. We are grateful for computation resources from the High Performance Computing System at National Engineering Laboratory of Soil Pollution Control and Remediation Technologies, CAS Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences. We would like to express our sincere gratitude to Prof Paolo Nannipieri and the two anonymous reviewers for their valuable comments and suggestions.

Funding

This work was supported by the Natural Science Foundation of China (31870500, 41501273), the Special Project on the Basis of National Science and Technology of China (2015FY110700), the Knowledge Innovation Program of Chinese Academy of Sciences (KZCX2-EW-409), and Technology Supporting Project of Jiangsu Province (BE2013451).

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Fig. S1

An overview of the experimental setup and used procedures (PDF 340 kb)

Fig. S2

15N-enrichment of N2 in the 15 N2-labeled chamber (DOCX 93 kb)

Fig. S3

NanoSIMS images (12C14N and 12C15N/12C14N) of soil DNA in the 15 N2-labeling gradient fractions (#1~#12) from 15 N2-DNA-SIP. The colored bars indicate the total ion counts of 12C14N− and the isotopic ratio of 12C15N−/12C14N− in a 30 × 30 μm2 area. Scale bars represent 2 μm. The white outlines indicate regions of hot spots, which were used to estimate the highest 15 N−/14 N− ratios of the DNA. The 15 N enrichment of DNA in SIP gradient fractions was depicted in atom% 15 N (DOCX 1973 kb)

Fig. S4

The nifH gene copy numbers in each SIP gradient fraction derived from 15 N2-labeling and nonlabeling treatments (DOCX 46 kb)

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Wang, X., Bei, Q., Yang, W. et al. Unveiling of active diazotrophs in a flooded rice soil by combination of NanoSIMS and 15N2-DNA-stable isotope probing. Biol Fertil Soils 56, 1189–1199 (2020). https://doi.org/10.1007/s00374-020-01497-2

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Keywords

  • 15N2-labeling
  • DNA-SIP
  • NanoSIMS
  • Cyanobacteria
  • Rice soil