Decoding the impact of relic DNA on soil microbiomes: A new soil relic DNA removal method

Highlights

● Benzonase removes relic DNA twice as efficiently as PMA and is adaptable across more types of soil than DNase I.

● Relic DNA removal leads to an approximately 10% reduction in soil microbial diversity and richness on average.

● The abundance of soil relic DNA is higher than previously expected.

Abstract

Microbes play a crucial ecological role in soils, but the presence of relic DNA left by previous microorganisms can lead to inaccurate estimations of viable microbial function and diversity. To address this, we proposed a new method for removing relic DNA in soil using Benzonase endonuclease and compared it with propidium monoazide (PMA) and DNase I, which have been widely applied in viable microbiome studies. Unlike PMA, Benzonase does not require light activation and is suitable for use in opaque media such as soil. Therefore, its efficiency (40%−60%) in removing soil relic DNA was twice that of PMA (0−30%). Moreover, our results showed that Benzonase outperformed DNase I in most soils, probably due to its broader range of operating conditions compared to DNase I. In addition to higher relic DNA removal efficiency, Benzonase exhibited a weak impact on soil viable microbial communities. Subsequently, Benzonase was used to remove relic DNA in natural soils, and the results showed that relic DNA removal led to an approximately 10% reduction in microbial diversity and richness on average. Notably, it caused significant changes in the relative abundance of specific taxa, such as Bacillus and Sphingomonas. These findings reveal disparities between total and viable microbiomes in soils. Our study not only provides a reliable method for soil relic DNA removal but also highlights the necessity of relic DNA removal for viable soil microbiome assessments, laying the methodological foundation for advancing soil microbial ecology research.

Graphical abstract

Keywords

relic DNA / viable soil microbiome / PMA / DNase I / Benzonase / high-throughput sequencing

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 32100094 to Y. Wang and 42020104003 to Q. Huang). We thank Nan Xiao (Huazhong Agricultural University) and Fei Qin (Huazhong Agricultural University) for technical support on the analysis of soil physico-chemical properties.

Declaration of competing interest

The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All raw sequences were deposited in NCBI Sequence Read Archive under the accession number of PRJNA961957.

Electronic supplementary material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s42832-024-0263-1 and is accessible for authorized users.

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