Response of activity, abundance, and composition of

Biology and Fertility of Soils https://doi.org/10.1007/s00374-018-1320-7

ORIGINAL PAPER

Response of activity, abundance, and composition of anammox bacterial community to different fertilization in a paddy soil San’an Nie 1,2,3

&

Xiumei Lei 1,3 & Lixia Zhao 1,3 & Yi Wang 1,3 & Fei Wang 4 & Hu Li 2 & Wenyan Yang 1,3 & Shihe Xing 1,3

Received: 31 May 2018 / Revised: 24 September 2018 / Accepted: 2 October 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract The anaerobic oxidation of ammonium (anammox) process plays a critical role in the loss of nitrogen (N) in paddy soils, yet the response of anammox to different fertilization is not well documented. In this study, three different fertilized (control, CK; soil treated with inorganic fertilizers, NPK; soil treated with inorganic fertilizer and involving the incorporation of straw, NPKS) paddy soils were selected to investigate the activity, functional gene abundance, diversity, and composition of anammox bacterial community using isotope-tracing technique, quantitative PCR assays, and Illumina sequencing. The anammox rate in the NPKS treatment was 2.4 nmol N g−1 soil h−1, significantly higher than that in CK and NPK treatments (1.7 and 1.8 nmol N g−1 soil h−1, respectively). Potential anammox contributed 6.2–7.8% to total N loss with the remainder being due to denitrification. Significant differences in the number of hydrazine synthase β-subunit (hzsB) gene were observed in three treatments with the highest value being observed in the NPK treatment. The anammox rate of per functional gene in the NPKS treatment (11.4 fmol day−1) was higher than that in CK and NPK treatments (8.3 and 7.0 fmol day−1, respectively). Three genera of anammox bacteria were identified: Candidatus Brocadia, Candidatus Anammoxoglobus, and Candidatus Scalindua, with Candidatus Brocadia being the dominant. Anammox bacteria diversity was significantly lower in the NPK than in CK and NPKS treatments as shown by Shannon, Simpson, Chao 1, and ACE indices (p < 0.05). The results showed that activity, abundance, and composition of anammox bacterial community depended on the type of fertilization. Keywords Anammox bacteria . Different fertilization . Straw . Activity . High-throughput sequencing . q-PCR

Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00374-018-1320-7) contains supplementary material, which is available to authorized users. * San’an Nie [email protected] * Shihe Xing [email protected] 1

Key Lab of Soil Ecosystem Health and Regulation, Fujian Agriculture and Forestry University, Fuzhou 350002, China

2

Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China

3

College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China

4

Soil and Fertilizer Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China

The N cycle in agricultural ecosystems is made more complex by the discovery of anaerobic oxidation of ammonium (anammox) (Mulder et al. 1995; Van de Graaf et al. 1996), which concerns the ammonium oxidation under anaerobic condition coupled with nitrite reduction and the removal of N with production of dinitrogen gas (Thamdrup and Dalsgaard 2002). Since N2 rather than N2O is the direct product, anammox is environmental friendly and an overlooked N removal pathway alternative to heterotrophic denitrification (Zhu et al. 2015a). The wide distribution of anammox in agricultural ecosystems can contribute to N loss, which has generally been overlooked (Nie et al. 2015; Shen et al. 2013, 2016; Yang et al. 2015; Zhu et al. 2011). This significant N loss can explain the gap in N loss from paddy fields that could not be attributed to NH3 volatilization, N2O emission, runoff, or leaching (Zhu 2008), suggesting that anammox may play a significant role in N removal in agricultural ecosystems.

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The hzsA, hzsB, and hzsC are genes used as specific biomarkers for quantifying anammox bacteria (Harhangi et al. 2012; Kartal et al. 2011; Strous et al. 2006). Among these, the hzsB is commonly used in the quantitative assays of anammox bacteria (Wang et al. 2012a, b; Yang et al. 2015). The anammox process is mediated by bacteria affiliated to six genera, Candidatus Brocadia, Candidatus Kuenenia, Candidatus Anammoxoglobus, Candidatus Jettenia, Candidatus Scalindua, and Candidatus Anammoximicrobium of the phylum Planctomycetes (Jetten et al. 2010; Khramenkov et al. 2013; Schmid et al. 2005). The Candidatus Anammoximicrobium was newly discovered and the other five genera have been identified in agricultural soils (Shen et al. 2013, 2015; Wang and Gu 2013; Yang et al. 2015; Zhu et al. 2011). Recently, the community composition of anammox bacteria in soils was found to be affected by the fertilization regime in rice-wheat-cropped soils and upland-cropped soils (Gu et al. 2017; Hui et al. 2017). However, in intensively fertilized paddy soils, the abundance of the functional gene responsible for anammox and thus the distribution of these anammox bacteria are not known. Paddy soils are the largest anthropogenic wetlands and cover 115 million hectares (ha) of the Earth’s surface (Kögel-Knabner et al. 2010). China is the largest rice producer (http://beta.irri.org/statistics) and the most fertilizer consumer (www.fao.org/publications/sofa) in the world. In agricultural production, fertilizer input into agricultural soils may stimulate the growth of anammox bacteria, and this can increase the N loss. In paddy fields, various types and rates of fertilizers are applied to increase rice yields (Yuan et al. 2013). In addition, rice straw is commonly incorporated into paddy fields (Nakamura et al. 2003) and has an effect on N supply and retention (Pan et al. 2017). However, the response of the anammox process to inorganic fertilizer and/or straw incorporation into soil is not known. Hence, in this study, paddy soils with three different fertilization for over 34 years were selected to investigate how the different management with or without straw incorporation can affect the activity, abundance, diversity, and contribution of anammox to N loss. To address this, we carried out a 15Ntracing experiment and q-PCR analysis to evaluate the activity and cell numbers of anammox bacteria, respectively. Community composition of anammox bacteria was analyzed by Illumina sequencing targeting anammox 16S rRNA gene.

a gray yellow paddy soil with heavy loam texture and the main initial chemical properties were as follows: 4.90 of pH (H2O 1:2.5); 21.6 g kg −1 of total organic matter (SOM); 141 mg kg−1 of alkali-hydrolysable N; 12 mg kg−1 of available P; and 41 mg kg−1 of available K. The following three treatments were chosen: control (CK, no fertilizer addition), chemical fertilizer (NPK, addition of 103.5 kg N ha−2 year−1, 11.0 kg P ha−2 year−1, and 109.7 kg K ha−2 year−1), and rice straw incorporation combined with chemical fertilizer (NPKS). The chemical fertilizers were applied as super phosphate + 50% urea + 50% potassium chloride at seeding of rice and the remaining 50% urea + 50% potassium chloride applied at tillering stage of rice growth. The rice straw (about 4500 kg ha −1 , total organic C 373.8 g kg −1 , total N 11.0 g kg−1) used in the NPKS treatment was produced from corresponding plots and was incorporated into soil in April after the harvest of rice. All treated soils were tilled and flooded prior to rice planting. Flooded water was drained at the milky stage of rice growth. There were three replicates for each treatment with a randomized design in the field. Three soil cores (0–20 cm) were sampled from each plot in October 2017. For each plot, the soil samples were mixed thoroughly and separated into three parts. A small fraction was frozen in liquid N2 and brought back to laboratory, freeze-dried, and stored at − 80 °C prior to molecular analysis; another part was incubated for determining anammox and denitrification activity or analyzed for moisture, exchangeable ammonium (NH4+), and nitrate (NO3−) contents; the other part was air-dried, then homogenized and sieved (< 2 mm) prior to the determination of pH, electrical conductivity (EC), total C (TC), and total N. The in situ surface water was also sampled from each plot.

Materials and methods

15

Site description and soil sampling

Different 15N isotopes were added for determining anammox and denitrification rates according to Risgaard-Petersen et al. (2004) and Thamdrup and Dalsgaard (2002), with some modifications. Approximately 3.5 g of homogenized soil was transferred to glass vials (Exetainer, Buckinghamshire, UK) together

The long-term (34 years) fertilizer experiment was located in the field research station (119° 04′ 10″ E, 26° 13′ 31″ N) at the Fujian Academy of Agricultural Sciences (Fig. S1). The soil is

Physicochemical analyses Moisture was analyzed by a MA100 moisture meter (Sartorius Company, Göttingen, Germany). Soil pH was determined by a pH meter (INESA, Shanghai, China) in a 1:2.5 soil/H2O suspension. Soil total C and total N were determined by a total C/ N analyzer (LECO Corporation, MI, USA). Exchangeable NH4+ and NO3− were analyzed by a microflow automated continuous flow analyzer (SYSTEA S.p.A., FR, Italy) after extraction of a 1:5 soil/2 M KCl mixture. Salinity (EC) was analyzed in a 1:5 soil/H2O suspension by a conductivity meter (INESA, Shanghai, China).

N isotope-tracing technique

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with N2-purged water from in situ surface water and preincubated to deplete residual NOx− and O2. Subsequently, 100 μL of N2-purged stock solution was added to the following isotope treatments, i.e., (1) negative control ((15NH4)2SO4 at 99.14%); (2) positive control ((15NH4)2SO4 + K14NO3); and (3) K15NO3 at 98.15%. All solutions were flushed with helium prior to addition. At 0, 3, 6, 12, and 24 h, the reactions were stopped by adding 200 μL 7 M ZnCl2 solution. To prevent atmospheric contamination, 2 mL of headspace solutions was transferred into new vials in an ultra-pure helium-filled anaerobic glovebox (Shel Lab Bactron VI, Cornelius, USA). Meanwhile, N2 standard curve was also prepared with 0, 30, 60, and 100 μL air injected into vials together with 2 mL N2purged water. The vials were shaken vigorously and stored upright to allow N2 to equilibrate prior to the measurement of N2. A linear regression was obtained between total signal and N2 production (Fig. S2). The rates and potential contributions to N2 production by anammox and denitrification were calculated by determining the produced 29N2 and 30N2, which were analyzed by isotope-ratio mass spectrometers (MAT253 with Gasbench II and autosampler (GC-PAL), Bermen, Thermo Electron Corporation, Finnigan, Germany). The multiple correlation coefficient (R2) for linear regression of 28/29/30N2 concentration was R > 0.90. The used equations are shown in Table S1.

unique 7-bp barcode; then, the barcode and primer sequence were truncated. Raw sequences from original DNA fragments were merged with FLASH (Version 1.2.7) (Magoc and Salzberg 2011) and then filtered with QIIME (Version 1.7.0) (Caporaso et al. 2010). Then, the high-quality clean tags were obtained and compared with the database using UCHIME algorithm. To accurately detect and correct frameshifts, only the sequences containing no ambiguous bases were selected into the downstream analysis. An averaged and rounded rarefied OUT table was generated to minimize the difference in sequence depth. Then, all effective tags were clustered by Uparse software (Version 7.0). Preprocessed sequences were clustered into OTUs based on their sequence similarity. The representative sequences were selected using the Uparse software. The OTU representative sequences were then classified taxonomically using the QIIME-based wrapper. The OTU phylogenetic relationship data of the top ten genera were selected and combined with the relative abundance of OTUs and confidence information of the species annotation (Bastian et al. 2015). The α-diversity indices including Shannon, Simpson, Chao 1, and ACE were extracted with QIIME (Version 1.7.0). The BChao 1^ and BACE^ assessed the richness of phylotypes; BShannon index^ estimated both richness and evenness, and BSimpson^ evaluated dominant OTUs.

DNA extraction, PCR, and Illumina sequencing and analysis

Quantitative real-time PCR

Soil DNA was extracted from 0.25 g freeze-dried soil using the TIANamp Soil DNA Kit (Tiangen Biotech, Beijing, China) according to the operation protocol. The DNA concentration was determined by a ND-2000 spectrophotometer (Thermo Scientific, Schwerte, Germany). Finally, the DNA was diluted with sterile water to a concentration of 1 ng μL−1. A nested PCR assay was developed to amplify the anammox bacterial 16S rRNA gene. The first amplication used the PLA46f-630r primer (Juretschko et al. 1998; Neef et al. 1998) to amplify the Planctomycetales 16S rRNA gene according to the following thermal profile: 96 °C for 10 min, followed by 35 cycles of 60 s at 96 °C, 1 min at 56 °C, and 1 min at 72 °C. The second amplication concerned the bacterial anammox 16S rRNA gene and involved the use of the diluted PCR products (500 times) and the set Amx368f-820r primer (Schmid et al. 2000, 2003); we used the following PCR conditions: 96 °C for 10 min, 25 cycles of 30 s at 96 °C, 1 min at 58 °C, and a final extension at 72 °C for 1 min. The PCR products were examined with electrophoresis on 2.0% agarose gel and purified by a DNA gel extraction kit (Promega, WI, USA). Highthroughput sequencing was proceeded by the Illumina MiSeq platform by Personal Biotech., Shanghai, China. Sequencing analysis was processed as described previously with some modifications (Scholer et al. 2017; Vestergaard et al. 2017). Paired-end reads were assigned to a sample by the

Real-time q-PCR was performed to evaluate the abundance of hzsB gene by using hzsB_396F and hzsB_742R primers (Wang et al. 2012a). Thermal cycling and data analysis were performed by the ABI 7500 (Applied Biosystems, CA, USA). The q-PCR reaction contained 10 μL 2× Master Mix (Amersham Pharmacia Biotech, Freiburg, Germany), 0.5 μL each primer (10 μM), 2 μL DNA as a template, and 7 μL ddH2O to give a final volumes of 20 μL. The standard curve was performed with 101- to 1010-fold serial dilutions of plasmid DNA with the hzsB gene. Three replicates and three nontemplate controls were conducted for each quantitative assay. The thermal profile started at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 40 s, and 82 °C for 10 s. The amplification efficiencies ranged from 90 to 110%, and correlation coefficients higher than 0.99 were accepted. The melt curve assays were evaluated to further confirm the specificity of PCR amplifications.

Statistical analysis The basic management and arrangement of data were conducted with Microsoft Excel 2016 and the data were the mean of replicates ± standard deviation (S.D.). One-way ANOVA (Duncan or Dunnett’s T3 test, p < 0.05) was performed to analyze differences between groups. General statistical analyses were assessed by the IBM SPSS statistics 23.0. The data

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below the detection limit were considered as zero by the statistical analysis. The figures were drawn with Sigmaplot 14.0.

Results The main chemical properties of the different fertilized soils are shown in Table 1. Soil moisture in the CK treatment was significantly higher than that in the NPK and NPKS treatments. Soil pH decreased significantly in the NPK treatment. The concentration of exchangeable NH4+ increased significantly in both fertilizer treatments compared to the CK treatment, while no significant difference of nitrate concentration was observed in three treatments. There was no difference in total C and total N contents between the CK and NPK treatments, but these values were significantly lower than those in the NPKS treatment. Soil EC increased significantly after longterm fertilization and could be ranked as NPKS>NPK>CK. Therefore, NPK and NPKS fertilizer applications have different effects on soil properties. The abundance of the hzsB gene ranged from 4.9 to 6.1 × 106 copies g−1 dry soil in the three treatments (Fig. 1a). Significant differences in hzsB gene numbers were observed between three soils and according to these values treatments ranked as NPK>NPKS>CK. The abundance of 16S rRNA gene in the NPK treatment was significantly lower than that in the CK and NPKS treatments. However, the proportion of anammox cell numbers to total bacteria copy numbers was at a higher level of 3.1 permillage in the NPK treatment than in the CK (1.0 permillage) and NPKS (0.8 permillage) treatments. Rates of anammox and denitrification are shown in Fig. 1b. Overall, the rates of denitrification were much higher than anammox rates in all treatments. Only about 6.2–7.8% of total N loss was potentially produced by anammox, while the remainder was released by denitrification (92.2–93.8% of the N2 production). Higher anammox rate occurred in the NPKS treatment (2.4 nmol N g−1 soil h−1) than in the CK and NPKS treatments (1.7 and 1.8 nmol N g−1 soil h−1, respectively). The denitrification activity of soil was significantly higher in the NPKS treatment (36.1 nmol N g−1 soil h−1) than in the CK Table 1 Effects of long-term different fertilization on soil chemical properties

(20.1 nmol N g−1 soil h−1) and NPK (21.2 nmol N g−1 soil h−1) treatments. The anammox rates of per functional gene were calculated by considering the q-PCR results of Fig. 1a and the rates of anammox (the right column of Fig. 1b), and ranged from 7.0 to 11.4 fmol day−1, with values being higher in the NPKS treatment (11.4 fmol day−1) than in the CK and NPK treatments (8.3 and 7.0 fmol day−1, respectively). This indicates that rice straw combined with chemical fertilizer can be an important treatment responsible for N loss in paddy soils. A total of 42 OTUs (97% cutoff) were obtained from three treatments (24 OTUs from CK, 20 OTUs from NPK, and 27 OTUs from NPKS, respectively) and 969 effective anammox bacterial sequences were analyzed by using the Gene database. All sequences from the soil samples were mostly affiliated to the genera of Candidatus Brocadia, Candidatus Anammoxoglobus, Candidatus Scalindua, or as unidentified Planctomycetaceae (Fig. 2). Candidatus Brocadia was the most abundant anammox bacterial genus (75–89%) and showed no significant difference among the three treatments. The Candidatus Anammoxoglobus-like sequences were also identified in all treatments, accounting for a small fraction (1.7% of the anammox 16S rRNA gene sequences) in the NPK treatment, significantly lower than that in the CK (5.5%) and NPKS (4.7%) treatments. Very low relative abundances of Candidatus Scalindua (0–1.1%) were detected in the three treatments and ranked as NPKS>NPK>CK. The fraction of unclassified genera of anammox bacteria also differed among three paddy soils. The Shannon, Simpson, Chao 1, and ACE diversity indices of anammox bacterial communities showed lower values in the NPK soils than in the CK and NPKS soils, indicating that the anammox bacterial community showed the lowest diversity in the long-term mineral fertilizer (NPK)-treated soil (Table 2).

Discussion Anammox bacteria prefer neutral to slightly alkaline environments (pH 7–8.5) (Chen et al. 2009; Jetten et al. 2001; Mulder

Treatment

CK

NPK

NPKS

Moisture (%) pH Exchangeable NH4+-N (mg kg−1) NO3−-N (mg kg−1) Total C (g kg−1) Total N (g kg−1) EC (μS cm−1)

27.31 ± 0.04a 5.18 ± 0.03a 9.93 ± 0.89b 1.72 ± 0.09a 11.37 ± 0.10b 0.94 ± 0.01b 35.02 ± 1.12c

25.52 ± 0.04b 4.99 ± 0.01b 20.49 ± 1.56a 1.55 ± 0.34a 11.30 ± 0.24b 0.92 ± 0.02b 46.47 ± 0.64b

25.77 ± 0.03b 5.17 ± 0.02a 24.76 ± 2.18a 1.50 ± 0.16a 13.82 ± 0.17a 1.19 ± 0.00a 60.63 ± 0.33a

Different letters in the same row indicate significant difference between treatments (p < 0.05)

Biol Fertil Soils Fig. 1 The abundance of anammox bacteria targeting the hzsB gene and total bacteria targeting the 16S rRNA gene (a), and anammox, denitrification activity, and their contributions to total N2 production (b) in different fertilized paddy soils. Different letters indicate significant differences between treatments (p < 0.05)

et al. 1995; Strous et al. 1997; Yamamoto et al. 2008). In the present study, we used acid soils (pH 4.99–5.18). Active anammox bacteria were also observed in a low pH (3.9) freshwater (Zhu et al. 2015b), an acid paddy soil (pH 4.8) (Shan et al. 2018), and an acid sediment (pH 5) (Wang and Gu 2014). Furthermore, the functional gene abundance (4.9 to 5.1 × 106 copies g−1 soil) was higher than those detected in some other paddy soils (Gu et al. 2017; Shen et al. 2014; Wang et al. 2012b; Yang et al. 2015). These results extended the pH range where anammox can be detected, indicating anammox bacteria can also be adapted to acid paddy soils. The potential anammox rates ranged from 1.7 to 2.4 nmol N g−1 h−1 and these values were similar to those of a typical paddy soil (0.27–5.25 nmol N g−1 h−1) (Yang et al. 2015), a paddy soil with high load of slurry manure (2.3–2.9 nmol N g−1 h−1) (Zhu et al. 2011), and a rice-wheat rotation soil with different fertilization regimes (0.68–2.08 nmol N g−1 h−1) (Gu et al. 2017). The detected anammox activity and rate of per

functional gene in the NPKS treatment were significantly higher than those in the CK and NPK treatments (Fig. 1b), suggesting that combined treatment of chemical fertilizer with rice straw can stimulate the anammox process and thus the N2 loss. One possible reason might be the higher concentration of exchangeable NH4+ (Table 1). Indeed, the anammox activities are positively correlated with NH4+ concentration (Shen et al. 2014; Zhu et al. 2011, 2015a). Additionally, the total C and N, which could be the potential C and N sources for anammox bacteria, were increased after long-term application of rice straw and mineral fertilizer compared to the control and NPK treatments. Furthermore, higher EC values in the NPKS treatment (Table 1) may also suggest higher concentration of exchangeable ammonium and nitrite in soil with stimulation of anammox bacteria (Rysgaard et al. 1999). The N produced from denitrification was also higher in NPKS than in CK and NPK (Fig. 2b), and it accounted for 92.3–93.8% of the total produced N2. Therefore, the potential contribution to N2 production by anammox was very low (6.2–7.8%). In this study, the anammox activities were significantly and positively correlated with denitrification activities (p < 0.01; Fig. S3). In oxygen-limited paddy soil, where nitrification is inhibited, denitrification could provide NO2− for anammox by the reduction of NO3− (Koop-Jakobsen and Giblin 2009; Nie et al. 2015). Denitrification might be the main factor determining anammox activity in the studied paddy soil. Table 2 α-Diversity index of anammox bacteria in different fertilized paddy soils

Fig. 2 The community compositions of anammox bacteria in different fertilized paddy soils. Different letters indicate significant differences between treatments (p < 0.05)

Treatment

CK

NPK

NPKS

Shannon index Simpson index Chao 1 index ACE index

4.76 ± 0.14a 0.93 ± 0.01a 119.72 ± 11.22a 120.92 ± 18.07a

2.95 ± 0.29b 0.55 ± 0.03b 65.69 ± 12.59b 66.46 ± 13.81b

4.88 ± 0.15a 0.94 ± 0.04a 124.77 ± 14.25a 135.03 ± 14.90a

Different letters indicate significant differences between treatments (p < 0.05)

Biol Fertil Soils

The application of only chemical fertilizer decreased the diversity of anammox bacteria. Generally, the long-term mineral fertilization can decrease microbial biodiversity with potential threats to soil productivity (Shen et al. 2010), while combined application of organic and inorganic fertilizers can has a positive impact (Bronick and Lal 2005) due to positive effect on some soil properties, such as the stabilization of soil structure and the increase in the organic matter content both affecting microbial communities positively (Bronick and Lal 2005; Munkholm et al. 2002; Nie et al. 2018). A recent study showed that the combined addition of mineral N fertilizer and straw benefited both biotic and abiotic processes of soil (Pan et al. 2017). Additionally, bacteria cell numbers were significantly lower in the NPK treatment than in the other two treatments (Fig. 1a). It should be noted that higher abundance, but lower rate of anammox bacteria (or rate of per functional gene), was detected in the NPK compared to NPKS (Fig. 1). The chemical fertilization of paddy soil may provide a habitat for anammox bacteria for their growth but not for their activity. Most importantly, higher abundance but lower diversity of anammox bacteria in the NPK than in the CK and NPKS treatments implied that both the functional gene and biodiversity could be the key factors influencing anammox activities. However, the factors driving anammox activity require to be further explored. High-throughput sequencing analysis of anammox bacteria showed the presence of three genera, Candidatus Brocadia, Candidatus Anammoxoglobus, and Candidatus Jettenia, with the dominance of Candidatus Brocadia (Fig. 2), confirming what already reported (Bai et al. 2015; Sato et al. 2012; Shen et al. 2013, 2014, 2015, 2017; Wang et al. 2012b; Zhu et al. 2011). However, Candidatus Jettenia, Candidatus Anammoxoglobus, and Candidatus Scalindua were only detected in some soil types (Long et al. 2013; Wang and Gu 2013). The genus Candidatus Anammoxoglobus was the second abundant anammox genus in the selected soil (1.7–5.5%), and the percentage of the total bacteria abundance was similar to that found in upland soils (averaged 3%) (Shen et al. 2013). The abundance of Candidatus Anammoxoglobus was significantly decreased by the NPK treatment. A very low proportion of Candidatus Scalindua was detected in the paddy soils (Fig. 2). Generally, this genus is associated with salinity which occurs in estuarine and river sediments (Amano et al. 2007; Dale et al. 2009). Indeed, the relative abundance of Candidatus Scalindua in three treatments was related to the EC value (Table 1; Fig. 2). In conclusion, anammox bacteria responded differently to fertilizer in paddy soils, because the activity, abundance, and diversity of anammox community varied with different fertilization. The combined inorganic fertilizer and rice straw treatment increased the activity of anammox bacteria and their diversity, while the sole application of chemical fertilizer provided a favorable habitat for the anammox bacterial cell but

not for their activity. Future studies will need to address the effect of different agricultural soils subjected to different regimes of fertilization on anammox process. Funding information This study was financially supported by the National Natural Science Foundation of China (4170010194) and the Natural Science Foundation of Fujian Province, China (2017J0101612).

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