5:15 PM - 7:15 PM
[MIS22-P07] Elucidating the soil microbial community that drives the priming effect of Andosols
-Differences in the microbial flora depending on soil depth as seen through DNA analysis-
Keywords:Andisol, priming effect, microbial composition, 16S amplicon sequencing
In Japan, Andosols, which are rich in organic matter, are widely distributed. These soils accumulate large amounts of carbon not only in the surface horizons but also in the buried humic horizons in the subsoil. Generally, microbial biomass and decomposition activity decrease with soil depth. However, when deep plowing or inversion tillage brings the buried humic soil to the surface, the addition of labile organic C from applied organic materials or fine roots may enhance the decomposition of recalcitrant organic C through the priming effect (PE; Kuzyakov, 2000). This could accelerate the decomposition of buried humic substances. If this PE can be applied to facilitate the decomposition and release of nitrogen and phosphorus from recalcitrant compost according to crop demands, it may enable agricultural land management with reduced chemical fertilizer use. However, the PE also poses a risk of reducing the carbon storage capacity of Andosols. Therefore, further research is needed to elucidate the mechanism of the PE for its potential application. Since the PE is driven by soil microorganisms, it is necessary to identify the microbial communities responsible for this process. In this study, we aimed to identify the microbial communities triggering the PE using amplicon sequencing analysis of 16S rRNA from the incubated soils.
The tested soils were collected from forest soils in Shibecha, Hokkaido Forest Research Station, Field Science Education and Research Center, Kyoto University. The sampled layers included surface soil (0–9 cm), humified soil 1 (40–70 cm), and humified soil 2 (90–103 cm). These soils were incubated for 187 days with three treatments: no addition, low addition, and high addition of ¹³C-labeled cellulose. Sampling was performed periodically (0, 7, 14, 21, 53, 128, and 187 days) in triplicate. DNA extraction followed the method of Hori et al. (2014). The extracted DNA was used as a template for polymerase chain reaction (PCR) amplification. The V4 region of the 16S rRNA gene was amplified using the primers 515F/806R (Parada et al., 2016; Apprill et al., 2015). The PCR temperature profile was optimized based on Hori et al. (2014), with an initial denaturation at 98°C for 90 sec, followed by 32 cycles of denaturation at 98°C for 10 sec, annealing at 54°C for 30 sec, extension at 72°C for 30 sec, and a final extension at 72°C for 120 sec. The PCR products were subjected to ligation, gel extraction, and bead purification before quantifying DNA concentration. The DNA was then adjusted to 2 nM with sterile water and analyzed using next-generation sequencing (MiSeq). The obtained sequences were identified using the Silva-138 16S rRNA database with Qiime 2 for taxonomic classification, principal coordinate analysis (PCoA), alpha diversity analysis.
Hayakawa et al. (2020) observed that microbial biomass significantly increased in response to ¹³C-labeled cellulose addition during specific incubation periods: surface soil (days 7–14), humified soil 1 (days 7–14), and humified soil 2 (days 7–21). These periods were considered as the PE activation periods in this study. We examined the relationship between changes in microbial community structure and these PE activation periods.Using Weighted UniFrac data from next-generation sequencing, we conducted PCoA. In surface soil, clear differences were observed at day 14 during the PE activation period, suggesting changes in microbial diversity. In contrast, humified soil 1 (day 14) and humified soil 2 (day 7) did not show significant changes, possibly due to the lower microbial biomass and decomposition activity in these layers.In alpha diversity analysis, significant reductions (p < 0.05) were observed in the number of the shannon index (Observed microbial count and evenness index in the sample) during the PE activation period for surface soil (low and high cellulose addition), humified soil 1 (high addition), and humified soil 2 (low addition). This suggests that the PE activation period is associated with the proliferation of specific microbial groups, leading to changes in microbial composition. Taxonomic classification analysis revealed that during the PE activation period, the dominant microbial taxa were Bacillus in surface soil (day 14),Bacillus in low addition of humified soil 1 (day 14), Staphylococcus in high addition of humified soil 1 (day 14), and the AD3 class of the Chloroflexi phylum (day 7) and Staphylococcus (day 14) in humified soil 2. These findings suggest that these microorganisms play a role in PE in Andosols.
The tested soils were collected from forest soils in Shibecha, Hokkaido Forest Research Station, Field Science Education and Research Center, Kyoto University. The sampled layers included surface soil (0–9 cm), humified soil 1 (40–70 cm), and humified soil 2 (90–103 cm). These soils were incubated for 187 days with three treatments: no addition, low addition, and high addition of ¹³C-labeled cellulose. Sampling was performed periodically (0, 7, 14, 21, 53, 128, and 187 days) in triplicate. DNA extraction followed the method of Hori et al. (2014). The extracted DNA was used as a template for polymerase chain reaction (PCR) amplification. The V4 region of the 16S rRNA gene was amplified using the primers 515F/806R (Parada et al., 2016; Apprill et al., 2015). The PCR temperature profile was optimized based on Hori et al. (2014), with an initial denaturation at 98°C for 90 sec, followed by 32 cycles of denaturation at 98°C for 10 sec, annealing at 54°C for 30 sec, extension at 72°C for 30 sec, and a final extension at 72°C for 120 sec. The PCR products were subjected to ligation, gel extraction, and bead purification before quantifying DNA concentration. The DNA was then adjusted to 2 nM with sterile water and analyzed using next-generation sequencing (MiSeq). The obtained sequences were identified using the Silva-138 16S rRNA database with Qiime 2 for taxonomic classification, principal coordinate analysis (PCoA), alpha diversity analysis.
Hayakawa et al. (2020) observed that microbial biomass significantly increased in response to ¹³C-labeled cellulose addition during specific incubation periods: surface soil (days 7–14), humified soil 1 (days 7–14), and humified soil 2 (days 7–21). These periods were considered as the PE activation periods in this study. We examined the relationship between changes in microbial community structure and these PE activation periods.Using Weighted UniFrac data from next-generation sequencing, we conducted PCoA. In surface soil, clear differences were observed at day 14 during the PE activation period, suggesting changes in microbial diversity. In contrast, humified soil 1 (day 14) and humified soil 2 (day 7) did not show significant changes, possibly due to the lower microbial biomass and decomposition activity in these layers.In alpha diversity analysis, significant reductions (p < 0.05) were observed in the number of the shannon index (Observed microbial count and evenness index in the sample) during the PE activation period for surface soil (low and high cellulose addition), humified soil 1 (high addition), and humified soil 2 (low addition). This suggests that the PE activation period is associated with the proliferation of specific microbial groups, leading to changes in microbial composition. Taxonomic classification analysis revealed that during the PE activation period, the dominant microbial taxa were Bacillus in surface soil (day 14),Bacillus in low addition of humified soil 1 (day 14), Staphylococcus in high addition of humified soil 1 (day 14), and the AD3 class of the Chloroflexi phylum (day 7) and Staphylococcus (day 14) in humified soil 2. These findings suggest that these microorganisms play a role in PE in Andosols.