2.1 Study site
This study was conducted in a cool-temperate deciduous forest in eastern Hokkaido, Japan (43°24.2′ N, 144°38.5′ E), managed by the Shibecha Branch of the Hokkaido Forest Research Station, Field Science Education and Research Center, Kyoto University. The soils at this site are andosols (IUSS Working Group WRB 2015). The mean annual air temperature and precipitation (1986–2015) at a meteorological station located approximately 9 km south of the study site were 6.3 ℃ and 1,189 mm, respectively. The daily average air temperatures measured at the meteorological station during the experiment are shown in Fig. 1.
Sampling was conducted in the four plots (15 m × 15 m) established in our previous study (Nakayama and Tateno 2018). In the plots, the canopy tree was Q. crispula, and the forest floor was densely covered with dwarf bamboo (S. nipponica). The plots were separated by at least 30 m from each other. The fine root density of Q. crispula at a depth of 0–10 cm in the plots was 132.9 ± 47.8 g m− 2 (Nakayama and Tateno 2018), and that of S. nipponica was 88.0 ± 55.9 g m− 2. More detailed information on the study plots can be found in previous reports (Nakayama and Tateno 2018, 2021).
The soil temperature at 5 cm soil depth in the plots was measured once every 30 min during the experiment using a temperature sensor with a data logger (TR-52i; T & D Corporation, Nagano, Japan). The sensor was removed on November 26, 2019 and April 28 and May 22, 2020, for data collection and a battery change and reburied on the same day. The daily average soil temperatures in the study plots are shown in Fig. 1.
2.2 Soil sampling and treatment
Mineral soils at a 0–10 cm-depth (A horizon) were collected using a shovel from each plot after removing the litter layer by hand. Soil sampling was conducted for 4 days during each sampling season, November 21, 23, 27, and 29, 2019 (beginning of the dormant season; hereafter, early winter); April 29, May 1, 3, and 5 (end of the dormant season; hereafter, early spring); August 23, 25, 28, and 30, 2020 (mid-growing season; hereafter, mid-summer). Soil samples were placed on ice in the field and refrigerated at 4 ℃ until processing.
In the laboratory, soil samples were sieved using a 4 mm mesh sieve. Soil that passed the sieve was considered as non-rhizosphere bulk soil (BS). The fine roots left on the sieve were carefully picked up by hand and forceps and separated into three types, Q. crispula and S. nipponica roots and others, including dead roots and roots of other species, based on their morphological traits (e.g., mycorrhizal type, color, and branching pattern). The ‘other’ roots constituted a small proportion and were discarded. Next, the fine roots of Q. crispula and S. nipponica were gently shaken, after which the soil adhering to their roots was collected as the rhizosphere (Phillips and Fahey 2006) and considered as the canopy tree rhizosphere (TR) and understory rhizosphere (UR), respectively.
After sieving and separation, the samples were divided into wet, oven-dry, and frozen subsamples. Oven-dried subsamples were dried at 60 ℃ for more than 72 h. Wet and frozen subsamples were stored at 4 and − 20 ℃, respectively, until further processing.
2.3 Soil chemical analysis
To measure the gravimetric water content of the samples, the oven-dried subsamples were weighed before and after oven-drying. Total N and C contents of oven-dried samples were then measured using an elemental analyzer (Sumigraph NC-900; Sumika Chemical Analysis Service, Ltd., Osaka, Japan). Next, 2 g of dried soil was extracted in 5 mL of deionized water, and then the pH of the extracts was measured using a pH meter (HORIBA D-51; Horiba, Ltd., Kyoto, Japan).
A portion of the frozen subsample was extracted in 2 M KCl (extracted from 1 g of the wet weight of the frozen subsample into 5 mL of 2 M KCl) to measure the concentration of total extractable N (TEN), NO3−-N, NH4+-N, and extractable organic N (EON). The concentration of NO3−-N and NH4+-N were colorimetrically measured using the Griess assay and indophenol blue method (Miranda et al. 2001), using a microplate reader (Synergy HXT; BioTek, Winooski, VT, USA), at wavelengths of 540 and 636 nm, respectively. To measure the concentration of TEN, 1 mL of 2 M KCl extracts was mixed with 1 mL of deionized water and 200 µL of alkaline potassium persulfate solution and then autoclaved (121 ℃, 20 min). Thereafter, TEN concentration was measured as NO3−-N concentration using the aforementioned method. The concentration of EON was calculated by subtracting the sum of inorganic N (NO3−-N and NH4+-N) from TEN.
2.4 Extracellular enzymatic activity measurements
The enzyme assays were conducted within 24 h of sampling using wet subsamples. The activities of six extracellular enzymes (Online Resource 1) involved in C, N, and P cycles were measured following the method of Saiya-Cork et al. (2002) with a few modifications. For the hydrolytic enzyme assays, i.e., β-xylosidase (XY), β-glucosidase (GL), acid phosphatase (AP), and β-1,4-N-acetylglucosaminidase (NAG), 1.0 g of wet soil sample was suspended in 100 mL of 50 mM sodium acetate (pH 5.0). The suspensions were mixed well using a magnetic stirrer for 1 min. Afterward, 800 µL of aliquots was dispensed into sample wells (four replicate wells per sample per enzyme) and quenching standard wells of 96-well deep-well plates. For the quenching standard wells, 200 µL of 4-methylumbelliferone (MUB) was added (concentrations: 0, 1, 2, 4, 10, 20, 40, and 100 µM). In the negative control well, 800 µL of acetate buffer was added. Subsequently, 200 µL of a 200 µM-substrate was added to the sample wells, after which the deep-well plates were incubated at 20 ℃ in the dark for 1 h. After incubation, the deep-well plates were centrifuged for 3 min at max speed (600 × g) (Bell et al. 2013). Thereafter, 250 µL supernatant was transferred into a 96-well black microplate; then the fluorescence was measured using a microplate reader (Synergy HXT, BioTek). The wavelengths of excitation and emission were 360 and 460 nm, respectively.
For the oxidative enzyme assays, phenol oxidase (PO) and peroxidase (PE), 800 µL of soil suspensions and 200 µL of 5 mM L-3,4-dihydroxyphenylalanine (DOPA) as the substrate were added to sample wells. Negative control wells contained 800 µL of acetate buffer and 200 µL of DOPA, and blank control wells contained 800 µL of soil suspensions and 200 µL of acetate buffer. For the PE assay, 40 µL of 0.3% H2O2 was further added to each well. Next, the deep-well plates were incubated in the dark at 20 ℃ for 10 h. The deep-well plates were then centrifuged for 3 min at max speed, and 250 µL of supernatants were transferred into a flat-bottom clear microplate. Enzyme activity was quantified by measuring absorbance at 450 nm using the same microplate reader.
2.5 DNA extraction and quantification of microbial gene abundance
Soil DNA was extracted from 0.25 g wet weight of frozen soil subsamples using a DNA extraction kit (DNeasy PowerSoil Pro Kit; QIAGEN, Hilden, Germany), following the manufacturer’s protocol. The extracted DNA solution was stored at -20 ℃ until further analysis.
Microbial gene abundance quantification by real-time quantitative polymerase chain reaction (qPCR) was performed using a LightCycler 96 System (Roche Diagnostics K.K., Mannheim, Germany), with the intercalating dye SYBR Green I (FastStart Essential DNA Green Master; Roche Diagnostics K.K.). The bacterial and archaeal 16S rRNA genes and fungal ITS regions of the rRNA genes were quantified to estimate total abundances of bacteria, archaea, and fungi, respectively. In addition, we estimated the abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) by quantifying the bacterial and archaeal ammonia monooxygenase gene (amoA), respectively. The subsequent steps and primer set used for qPCR have been described in Nakayama et al. (2021).
2.6 Statistical analyses
To test the difference in soil chemical properties, enzymatic activity, and microbial gene abundance among sampling seasons (early winter, early spring, and mid-summer) and sample positions (BS, TR, and UR), a two-way analysis of variance (ANOVA) with random effects (random effects: sampling date and plots) was used. We conducted multiple comparisons using the emmeans function in R with Tukey’s adjusted P-value to test the difference in soil chemical properties, enzymatic activity, and microbial gene abundance. The magnitude of the RPE was calculated as the percentage difference between the paired rhizosphere and bulk soil sample for each variable. To determine whether the magnitude of the RPE significantly differed from zero, a t-test was used. The differences in the RPE magnitude among sampling seasons and between sampling positions (TR vs. UR) were tested using a two-way ANOVA followed by multiple comparisons as described above. All statistical analyses were conducted in R ver. 4.0.5 (R Core Team 2021).