2.1. Soil sampling
The soil used in this experiment was sampled from abandoned land for 30 years at the university farm located at the Field Science Centre for Northern Biosphere, Hokkaido University, Japan (43°04¢N, 141°20¢E). The properties of the soil are given in Table 1. The soil type was clay loam with 44.6% sand, 21.5% silt and 33.9% clay.
2.2. Experimental design
A pot experiment was performed in a greenhouse at the Graduate School of Hokkaido University. The sampled soil was air-dried and sieved with 2 mm mesh and subsequently filled into Wagner pots (surface area = 1/5000 a). Each pot contained 1.8 kg air-dried soil. The experimental design was completely randomised, included three fertiliser treatments × four mixed cropping treatments, and conducted in trireplicates. The pots received one of the three types of fertiliser treatments, namely control (‘Ctr’), chemical fertiliser containing P and K (‘CF’) or biochar made from CM (50 g pot−1 carbonised CM; ‘CM’). The application rate for CF was 30 kg P ha−1 and 50 kg K ha−1. The soil and the CM chemical property are described in Table 1.
Each pot then received one of the four types of plant treatment: (1) single maize (Zea mays L.; SM), (2) mixture of cowpea [Vigna unguiculata (L.) Walp.] and maize (VM), (3) mixture of velvet bean [Mucuna pruriens (L.) DC.] and maize (MM) and (4) mixture of common bean (Phaseolus vulgaris L.) and maize (PM). Three replications were included in each treatment. During these treatments, maize and three legume seeds, including cowpea, velvet bean and common bean, were sprouted for 2 weeks in small pots filled with vermiculite before transplantation to Wagner pots. During the experiment, the temperature was maintained at 25°C to 30°C, and the plants were grown for 50 days after transplantation.
2.3. Chemical property analysis
After plant growth soil was sampled from each pot, soils were sampled and measured for pH and extractable NH4+ and NO3− concentrations 50 days after transplantation. For soil pH, 6 g soil was shaken for 30 min with 30 mL Milli-Q water, and pH was subsequently measured by a pH sensor (AS800; ASONE Co., Japan). For extractable NH4+ and NO3−, the samples were extracted with a KCl solution (2 mol L−1), followed by colorimetric analysis using a flow injection analyser system (ACLA-700; Aqualab Co., Ltd., Japan). A two-way analysis of variance (ANOVA) was then performed to investigate the interaction between environmental factors and experimental treatments (Table 2).
2.4. DNA extraction and 16S rRNA sequencing
Using the same sampled soils, DNA was extracted with NucleoSpin® Soil (Takara Bio, Inc., Japan) according to the manufacturer’s instructions. The extracted DNA was subsequently amplified by polymerase chain reaction (PCR) targeting the V4 region of 16S rRNA (amplicon size ~250 bp; forward primer 515F: 5′-GTGCCAGCMGCCGCGGTAA-3′ and reverse primer 806R: 5′-GGACTACHVGGGTWTCTAAT-3′). To perform PCR, 10 µL AmpliTaq Gold® 360 Master Mix (Applied Biosystems, Foster City, CA, USA), 0.4 μL of the forward primer, 0.4 μL of the reverse primer, 7.2 μL nuclease-free water and 1 μL DNA extract were mixed. The first PCR cycle was set to 95°C for 10 min and then 20 cycles at 95°C for 30 s, 57°C for 30 s and 72°C for 1 min, followed by 72°C for 7 min. The PCR products were subsequently purified with Agencourt AMPure XP (Beckman Coulter) according to the given protocol. Then, the purified products were quantified with the QuantiFluor® ONE dsDNA System by a QuantusÔ Fluorometer E6150 (Promega, Madison, WI, USA).
Another PCR was performed with the utilisation of amplicon-obtained products to make them Ion Torrent sequence sample-specific. To achieve this, the 515F forward primer with the Ion Xpress Barcode Adapters Kit sequence and the 806R reverse primer attached to the Ion Xpress sequence of the Ion P1 adaptor were used (Thermo Fisher Scientific K.K.). The first PCR products were diluted to 2000 ng mL−1, and 1 μL of each product was subsequently mixed with 10 μL AmpliTaq Gold® 360 Master Mix, 0.4 μL of the forward primer, 0.4 μL of the reverse primer and 7.2 μL nuclease-free water. The second PCR cycle was set to 95°C for 10 min and then 5 cycles at 95°C for 30 s, 57°C for 30 s and 72°C for 1 min, followed by 72°C for 7 min. The second PCR products were purified in accordance with the same method outlined above. The final length and concentration of the amplicons were confirmed using a Bioanalyzer DNA 1000 Kit (Agilent Technologies, USA). The library was subsequently diluted to 50 pM and loaded into the Ion 318 chip using Ion Chef Instruments with an Ion PGM Hi-Q Chef Solutions. The samples were sequenced on an Ion PGM Sequencer with Ion PGM Hi-Q View Sequence Solutions (Ion Torrent Life Technologies, USA). Sequence data were deposited in the Sequence Read Archive of the National Center for Biotechnology Information (NCBI) under accession number PRJNA743765.
2.5. Sequence processing
The barcoded 16S rRNA gene sequences were denoised, quality-filtered and assessed using the DADA2 algorithm implemented in Quantitative Insights Into Microbial Ecology (QIIME2) and with its workflow (Bolyen et al., 2019). Rarefaction was performed with minimal reads among all samples, and sequence data were subsampled to 41,095 sequences per sample. The R package Vegan (version 2.5.6) was used to access sample depth and generate the a-rarefaction curve plot (Fig. S1). The rarefaction curve was then evaluated using the interval of a step sample size of 1000.
2.6. Measurement of bacterial abundance
To measure bacterial abundance, quantitative PCR (qPCR) was performed using the extracted DNA, diluted 50 times with nuclease-free water. The 515F/806R primer pairs described above were used to amplify the V4 region of the 16S rRNA. For the standard curve, the PCR products from the DNA extracted from the Ctr pots were used, which were purified with AMPure XP and further diluted to five stages of different concentrations. The samples were prepared with 10.4 μL KAPA SYBR Fast qPCR kit (Kapa Biosystems, USA), 0.08 μL of the forward primer, 0.08 μL of the reverse primer and 2 μL diluted DNA extract. Nuclease-free water was added to achieve the final volume of 20 μL. CFX96 TouchÔ Real (Bio-Rad Laboratories, Inc., USA) was used, and the cycling condition was set to 95°C for 30 s and then 35 cycles at 95°C for 30 s, 58°C for 30 s and 72°C for 1 min, followed by 95°C for 1 min and subsequently 55°C to 95°C by 1°C increment for 10 s. Ct values (threshold cycle) were calculated after quantifying the amplification results using qpcR R package version 1.4.1.
2.7. Statistical analysis
To quantify the diversity of soil microbial communities, the Shannon index (Shannon, 1948) and the Simpson index (Simpson, 1949), an estimation of community a-diversity, were calculated. For each diversity index, two-way ANOVA was performed using fertiliser treatments and plant species as factors with the emmeans R package version 1.4.7 (Lenth et al., 2020). Multiple comparisons were subsequently performed using the Tukey-Kramer method. Also, permutational ANOVA (PERMANOVA; permutation = 9999) was conducted, and significant phyla (p < 0.001) were subsequently identified for the interactions between fertiliser treatments and plant types using two-way ANOVA.
The numbers of operational taxonomy units (OTUs) for each plant treatment within the same fertiliser treatment were illustrated using Venn diagrams. VennDiagram R package version 1.6.20 was applied using the cooccurred OTUs among three replications for each treatment. OTUs that specifically appeared by plants species within each fertilisation were defined as unique OTUs and classified to the phylum. The relative abundance of unique OTUs within the total abundance was plotted for PM and MM treatments that were significantly different in diversity indices within plant species.
Functional profiling of the prokaryotic community was conducted with the Tax4Fun2 R package (Wemheuer et al., 2020). The rarefied OTU table was used for Tax4Fun2 searches, and metagenome functional profiles were predicted to generate a table of Kyoto Encyclopedia of Gene and Genomes orthologs (Kanehisa and Goto, 2000). To evaluate biogeochemical reactions, 36 gene-coding enzymes related to the organic N degradation and N metabolism pathway were selected (Table S1; Harter et al., 2014; He et al., 2021; Sulieman and Tran, 2014; J. Wang et al., 2020) and visualised with ‘ComplexHeatmap’ R package.