Does added plant residue interfere with the profiling of soil bacterial community by 16S rRNA gene sequencing and qPCR?

doi:10.21203/rs.3.rs-1955158/v1

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Does added plant residue interfere with the profiling of soil bacterial community by 16S rRNA gene sequencing and qPCR?

https://doi.org/10.21203/rs.3.rs-1955158/v1

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Many studies evaluate the effect of plant residue on soil bacteria by comparing the microbial communities in soils with or without added plant residue after a period of incubation. This comparison can be made, however, only when the measured microbial properties are not significantly different between the amended soil and the control on day 0. To confirm this, we mixed plant residue into soil and immediately (within < 10 min; representing day 0) started DNA extraction, and then performed 16S rRNA gene sequencing and quantitative PCR (qPCR) to determine bacterial community composition and abundance. Unamended soil served as a control. Five commonly used DNA extraction kits, 16S rRNA gene primer pairs, and soils, and two types (mature rice straw and alfalfa shoots) and three addition rates (2%, 4%, and 6%; w/w) of plant residue, were tested. In all cases, we found no significant difference in bacterial community composition or abundance between the treatments with or without added plant residue. The results can help us better interpret differences in soil bacterial communities between the treatments with or without added plant residue in soil incubation experiments.

plant residue

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Plant residue is commonly used as a soil additive. Many studies report differences in bacterial communities between soils with or without added plant residue after a period of incubation (Fu et al., 2021). However, if a significant difference in microbial community between soils with or without added plant residue is observed on day 0, that is, if the introduction of plant residue interferes with the characterization of microbial community, we cannot be sure that the observed difference in microbial community after a period of incubation is really caused by the effect of added plant residue on the growth of microorganisms. Plants are known to carry compounds that may be co-extracted and thereafter hinder the PCR (Schrader et al., 2012). However, whether adding plant residue interferes with the profiling of soil bacterial community, which is routinely carried out through 16S rRNA gene sequencing and quantitative PCR (qPCR), has not yet been systematically investigated. The aim of this study is to confirm this, namely, whether there is a significant difference in microbial community between soils with or without added plant residue on day 0.

Here, plant residue was mixed into soil and soil DNA was immediately extracted (the time between the start of mixing plant residue into soil and the start of soil DNA extraction was less than 10 min to represent day 0). Unamended soil served as a control. There were three replicates for each treatment. Then, bacterial community composition and abundance were determined by 16S rRNA gene sequencing and qPCR using the extracted DNA. Whether the presence of plant residue in soil interferes with the profiling of bacterial community was tested with various commonly used DNA extraction kits and 16S rRNA gene primer pairs (Experiment 1), in different soils with a wide range of properties (Experiment 2), as well as with different types (mature rice straw and alfalfa shoots) and addition rates (2%, 4%, and 6%; w/w (oven-dry basis)) of plant residue (Experiment 3). The soils (Table S1) used were air-dried and passed through a 2-mm sieve because plant residue is usually mixed into air-dried soil in laboratory experiments; nevertheless, we also tested two moist soils passed through a 2-mm sieve in Experiment 2. The dry plant residues (Table S2) were finely ground and passed through a 1.5-mm sieve. Each residue was mixed with the soil thoroughly in a plastic bag (Yuan et al., 2019). Soil DNA was extracted with commercial soil DNA extraction kits (Table S3) following the manufacturers’ instructions. The extracted DNA was then sent to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The bacterial 16S rRNA gene in the extracted DNA was amplified with commonly used primer pairs that target different hypervariable regions of the 16S rRNA gene (Table S4) and then sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA). The raw sequences were submitted to the National Center for Biotechnology Information (PRJNA783435). The copy number of bacterial 16S rRNA gene in the extracted DNA was quantified by qPCR using the SYBR Green I detection method with the 338F/806R primer pair (Wang et al., 2020).

The sequencing data were processed (separately for different primer pairs) mainly following the protocol of Yuan et al. (2021) using the USEARCH (Version 10) (Edgar, 2010), VSEARCH (Version 2.13.6) (Rognes et al., 2016), and R (Version 4.0.2) platforms. Briefly, after quality control, operational taxonomic units (OTUs) were generated at the 97% similarity threshold. A rarefied OTU table, wherein all samples had the same number of sequences, was then obtained by random subsampling using the rrarefy function in the ‘vegan’ package. Bacterial communities were ordinated with principal coordinates analysis (PCoA) using the R ‘vegan’ package, based on the Bray–Curtis dissimilarity matrix. The effects of different factors on bacterial community composition were analyzed by permutational multivariate analysis of variance (PERMANOVA) using the adonis function in the ‘vegan’ package. The effects of different factors on bacterial abundance were examined by analysis of variance (ANOVA).

In Experiment 1, a silt loam paddy soil (soil 1 in Table S1) was used. After adding 2% rice straw to the soil, DNA was extracted immediately using five different soil DNA extraction kits (Table S3). The bacterial 16S rRNA gene in the extracted DNA was then amplified by five primer pairs with different targeted regions (Table S4) and sequenced. For all the primer pairs, PERMANOVA showed that DNA extraction kits, rather than the addition of plant residue, significantly affected the bacterial community composition (Table S5); this was also demonstrated by PCoA ordination (Figs. 1a and S1). In addition, two-way ANOVA also showed that DNA extraction kits, rather than the addition of plant residue, significantly influenced bacterial abundance as quantified by qPCR (Fig. 2a and Table S6).

In Experiment 2, five air-dried soils (soils 2–6 in Table S1) and two moist soils (soils 7 and 8 in Table S1, which were taken from soils 2 and 4 after they arrived at the laboratory and were stored at -80 °C without air-drying) were employed to verify the insignificant influence of added plant residue on the profiling of bacterial community in a wider range of soils. After mixing 2% rice straw into each soil, DNA was extracted immediately using the DNeasy PowerSoil Pro Kit (QIAGEN, Germany) (kit II in Table S3). The bacterial 16S rRNA gene in the extracted DNA was then amplified with the 338F/806R (Apprill) primer pair (no. 2 in Table S4) and sequenced. PERMANOVA and PCoA showed that bacterial community composition differed significantly between the soils, rather than between the treatments with or without added plant residue (Figs. 1b and S2; Table S7). The bacterial abundance was not significantly affected by the addition of plant residue either (Figs. 2b and S3; Table S8).

In Experiment 3, the silt loam paddy soil (soil 1 in Table S1) and two types (mature rice straw and alfalfa shoots; Table S2) and three addition rates (2%, 4%, and 6%) of plant residue were employed. Unamended soil served as a control. After mixing plant residue into the soil, DNA was extracted immediately using the DNeasy PowerSoil Pro Kit (QIAGEN, Germany) (Kit II in Table S3). The bacterial 16S rRNA gene in the extracted DNA was then amplified with the 338F/806R (Apprill) primer pair (no. 2 in Table S4) and sequenced. Again, the results showed that for both rice straw and alfalfa, the bacterial community composition and abundance were not significantly affected by the addition of plant residue (Figs. 1c,d and 2c,d; Tables S9 and S10).

The above results show that adding up to 6% plant residue does not interfere with the profiling of soil bacterial community through 16S rRNA gene sequencing and qPCR. This observation stands when using various soil DNA extraction kits and 16S rRNA gene primers and in soils with a wide range of properties. We measured the water-extractable carbon of the soils and plant residues used in this study (Tables S1 and S2) and found that the latter was much higher than the former. Adding 6% alfalfa to the silt loam paddy soil (soil 1 in Table S1), for example, increased the water-extractable carbon of the soil by 46-fold. This implies that the dissolved organic matter released by the added plant residue did not affect soil DNA extraction with current commercial kits, nor the subsequent sequencing and qPCR carried out. With confirming the insignificant influence of added plant residue on the profiling of soil bacterial community, we can now better interpret differences in soil bacterial communities between the treatments with or without added plant residue in soil incubation experiments.

Acknowledgments:

This work was supported by the National Key Research and Development Program of China (2020YFC1807000), the National Natural Science Foundation of China (41977273, U21A20291), and the Major Research plan of the Shandong Science Foundation (ZR2020ZD19). Jing Li also acknowledges funding from the Fundamental Research Funds of Chinese Academy of Forestry (CAFYBB2020QB008) and the Beijing Natural Science Foundation (8214067). We thank Mr. Diansong Yuan and Prof. Zhanhuan Shang for their assistance with soil sampling.

Conflict of Interest: The authors declare that they have no conflict of interest.

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Does added plant residue interfere with the profiling of soil bacterial community by 16S rRNA gene sequencing and qPCR?

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