Experiment design and sample collection. A fertilization and irrigation experiment was established in 2008 and double cropping rice (Oryza sativa L.) was planted annually at Baisha Experimental Station (119°04′10″E, 26°13′31″N), Minhou County, Fuzhou City, Fujian Province, China. The early and late cultivars of rice (O. sativa L.) are conventional rice varieties 78-30 and 428, respectively, from south China. This region has a subtropical monsoonal climate with an average annual air temperature of 19.5 ℃ and mean annual precipitation of 1 350 mm. The soil is a typic Hapli-Stagnic Anthrosol (USDA soil system). At the beginning of the experiment, the soil had a pH 6.19, 14.16 g kg-1 soil organic matter (SOM), 0.66 g kg-1 total N (TN), 0.30 g kg-1 total P (TP), 3.8 mg kg-1 NO3--N, 12 mg kg-1 NH4+-N, 3.358 and 0.83 mg kg-1 of available P (AP) and K (AK), respectively, in the 0 − 20 cm soil layer. A randomized complete block design with three treatments was conducted in 9 plots (4.0 m long × 5.0 m wide). Each treatment had three duplicates. The treatments included control (no fertilization with traditional irrigation, T0), traditional fertilization with traditional irrigation (T1, based on local practices), and optimum fertilization with water-saving irrigation (T2, based on both fertilizer recommendation from local agriculture committee and water saving by shallow intermittent irrigation). The water and fertilizer practices used in this experiment are described in Table 1. The chemical compound fertilizer (15% N, 15% P2O5, 15% K2O) was produced by China Petroleum and Chemical Co., Ltd. N, P, and K fertilizers were applied in the form of urea (46.4 % N), superphosphate (12 % P2O5), and potassium chloride (60 % K2O) and used according to the amount of these fertilizers as shown in Table 1. The 100% of the total amount of P, 60% of N, and 40% of K fertilizers were used as basal fertilizers before transplanting of rice seedlings, and the 40% N and 60% K fertilizers as topdressing fertilizers after tillering, respectively (Table 1). Traditional irrigation was needed to be maintained at a depth of 1.0 − 6.0 cm during the rice-growing season, and water-saving irrigation at a depth of -3.0 − 3.0 cm in the field. To avoid water and fertilizer exchange between adjacent experimental units, a cement concrete border, with dimensions (length × width × height) 40 cm × 30 cm × 20 cm, was constructed. The early rice was transplanted with a 20.0 cm × 23.0 cm row spacing on 21 April and harvested on 25 July 2018. The late rice was transplanted with the same row spacing on 30 July and harvested on 1 December 2018.
The rice straw and grain were sampled. In addition, five samples of 0−20 cm soil layer were collected and mixed from each plot after late rice harvest. The fresh soil samples were transported immediately on ice to the laboratory. Plant residues and stones were manually removed from soil samples. The soil samples were then mixed and sieved to < 2.0 mm. One subsample was stored at - 80 ℃ for soil microbial analysis, while the other subsample was air-dried for chemical analysis.
Plant, soil physiochemical properties, and Potential ammonia oxidation (PAO). The rice straw and grain yields were measured at harvest from each plot, separately (rice grain weights were adjusted to 13.5% moisture content). In addition, the rice grain and straw materials were dried at 60 ℃ for 72 h, and weighed. Meanwhile, soil moisture was calculated as the difference between oven - dry (24 h at 105 ℃) and fresh weight. Soil pH was analyzed by a pH meter (EL20 K, Mettler - Toledo, Greifensee, Switzerland) in a 1:2.5 (m:v) soil - water suspension. The SOC content was measured by means of the oxidation-reduction titration. The TN content was analyzed using a Kjeldahl digestion. Both NH4+-N and NO3−-N in fresh soils were extracted with 2 M KCl (1:10 (m:v) soil/extract), and the extract was analyzed by using UV spectrophotometry 26. The PAO activity was measured according to Kurola et al. (2005) 27 with minor modifications. Briefly, 5 g of fresh soil was incubated in 20 mL phosphate-buffered saline (PBS) and 1 mM of (NH4)2SO4 at room temperature in the dark for 24 h. Then 10 mg L−1 of KClO3 addition inhibited nitrite oxidation. At the end of incubation, soil NO2−-N was extracted with 5 mL of 2 M KCl. The optical density (at 540 nm) of the supernatant was determined after each centrifugation in order to calculate the NO2--N content using sulfonamide and naphthylethylene diamide as reagents. The PAO activity was estimated by the slope of the NO2--N accumulation.
Soil ammonia-oxidizing archaeal analysis. DNA was extracted from 0.25 g of fresh soils per sample by the E.Z.N.A Soil DNA Kit (Omega Bio-tek, Norcross, Georgia, USA) on the basis of the manufacturer’s instructions 28. The DNA purity and concentration were detected using a Nanodrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA).
The archaeal amoA gene was amplified by the primers Arch-amoAF (5′ STAATGGTCTGGCTTAGACG 3′) and Arch-amoAR (5′GCGGCCATCCATCTGTATGT 3′) 29. Base on the preliminary experiment, the reaction systems and cycling conditions such as DNA amounts, annealling temperatures, and circular times were further optimized. The preliminary PCR amplification was performed for 27 cycles. All PCR products after 2.0% (w/v) agarose gel electrophoresis on a 2.0% (w/v) agarose gel is used to verify ammonia-oxidizing bacterial and archaeal size and quality. The band numbers and relative intensities of PCR products were analyzed using Quantity One analysis software (Bio-Rad). However, only the ammonia-oxidizing archaeal community was found and further analyzed under the following conditions. Each 20-μL qPCR reaction mixture contained 10 μL 2X Taq Plus Master Mix (VazymeBiotech, Nanjing, China), 0.8 μL forward and reverse primers (5 μM), 1 μL DNA template and 7.4 μL ddH2O. The qPCR of ammonia-oxidizing archaeal amoA gene was conducted on an ABI 7300 thermocycler (Applied Biosystems, California, USA) at 95 °C for 5 min, 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. The purified PCR products were ligated into the plasmid pMD19-T Vector (Takara, Dalian, China) carrying AOA amoA insert. Individual clones were grown in Luria-Bertani (LB) medium at 37 °C for 18 h. Plasmid DNA from a 5-mL culture was extracted with a TIANprep Mini Plasmid Kit (Tiangen, Beijing, China) and quantified by a Nanodrop 2000 UV-Vis spectrophotometer 17. Each sample and each standard were quantified in triplicate. The qPCR was performed with three times, and the amplification efficiency of the qPCR was 89.09% (R2 = 0.9998) for AOA. The cell numbers of AOA were calculated from the quantified numbers of the amoA gene on the basis of the fact that each cell in AOA contains one copy of the amoA genes 30.
The primers Arch-amoAF and Arch-amoAR 29 were used to amplify amoA gene fragments by a GeneAmp PCR system 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). The PCR reactions for AOA were performed with the following conditions: denaturation at 95 °C for 3 min , 27 cycles of 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 45 s, and a final extension at 72 °C for 10 min. The PCR reaction mixture (20 μL) was comprised of 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of 5 μM each primer, 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. The PCR products were run on 2% agarose gels and purified with an AxyPrep DNA Gel Extraction Kit (Axygen, USA), and quantified using QuantiFluor™-ST (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions. The purified amplicons were pooled in equimolar concentrations and paired-end sequenced (2 × 300) using an Illumina MiSeq platform (Illumina, San Diego, USA) on the basis of the standard protocols by the Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China) 3.
Raw fastq files were demultiplexed, filtered using Trimmomatic software (Version 3.29) and merged by FLASH (Version 1.2.7) with the criteria: (i) The sequences were trimmed at any site receiving an average quality score < 20 over a 50 bp sliding window. (ii) Sequences with mismatches to either the primer (> 2) or with ambiguous bases (> 1) were discarded. (iii) Sequences with their overlap > 10 bp or length < 200 bp were deleted. The remaining sequences were selected for chimeras by UCHIME 31. The high-quality sequences were assigned to operational taxonomic units (OTUs) according to 97% sequence identity by the UPARSE pipeline. The taxonomy of each amoA gene sequence was identified by the Ribosomal Database Project (RDP) Classifier tool (http://rdp.cme.msu.edu/) against the fgr/amoA database (GeneBank Release 7.3 http://fungene.cme.msu.edu) with a confidence threshold of 70%. Furthermore, the rarefaction curve and other OTUs-based parameters, including coverage estimators (ACE), Chao1, Shannon-Wiener index (H′), and Simpson's index (D) were analyzed by the mothur software package 32. Chao1 and ACE were used to evaluate the ammonia-oxidizing archaeal community richness on the basis of the degree of sequence dissimilarity. H′ and D were used to evaluate to the diversity within each individual sample 33. In addition, a Venn diagram showing the number of shared and unique archaeal OTUs was used to describe the similarities and differences among the archaeal communities associated with three treatments. A heatmap analysis was performed to compare the relative abundance of the top 10 archaeal genera. Moreover, a heatmap of relationship between the relative genus abundances of ammonia-oxidizing archaea and soil properties (e.g., pH, SOC, and TN) was conducted by Canoco for Windows 4.5 package. In addition, environmental factors were selected by the functions of envfit (permu = 999) and vif.cca, and the environmental factors such as SOC, TN, NH4+–N, NO3-–N, and PAO with P < 0.05 or vif < 10 were retained. The distance ‐ based redundancy analysis (db‐RDA) and partial least squares discriminant analysis (PLS - DA ) were processed by R software (version 3.2.1). The phylogenetic analysis on the basis of the sequences acquired from this study and reference sequences from the NCBI GenBank was made using the software MEGA version 5.0 34 to construct a phylogenetic tree by the neighbor-joining method. All bioinformatics analyses for soil ammonia-oxidizing archaeal communities were performed on online “I-Sanger” (http://www.i-sanger.com/) developed by Majorbio Bio-Pharm Technology Co. Ltd. All original nucleotide sequence reads were deposited at the NCBI Sequence Read Archive (SRA) with the accession number of SRP293735.
One-way analysis of variance (ANOVA) and Duncan's multiple range tests were used to estimate the statistical significance of the differences of edaphic characteristics, rice yields, archaeal amoA gene abundance and alpha-diversity under different water and fertilizer regimes by SAS Version 8.02 (SAS Institute Inc, Carey, North Carolina, USA). All data were expressed as mean ± SD (n=3).