Curr Microbiol (2015) 70:282–289 DOI 10.1007/s00284-014-0714-5

The Influence of Land Use on the Abundance and Diversity of Ammonia Oxidizers Dayong Zhao • Juan Luo • Jianqun Wang • Rui Huang • Kun Guo • Yi Li • Qinglong L. Wu

Received: 10 August 2014 / Accepted: 6 September 2014 / Published online: 21 October 2014 Ó Springer Science+Business Media New York 2014

Abstract Nitrification plays a significant role in soil nitrogen cycling, a process in which the first step can be catalyzed by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). In this study, six soil samples with distinct land-use regimes (forestland soil, paddy soil, wheat-planted soil, fruit-planted soil, grassland soil, and rape-planted soil) were collected from Chuzhou city in the Anhui province to elucidate the effects of land use on the abundance and diversity of AOA and AOB. The abundance of the archaeal amoA gene ranged from 2.12 9 104 copies per gram of dry soil to 2.57 9 105 copies per gram of dry soil, while the abundance of the bacterial amoA gene ranged from 5.58 9 104 copies per gram of dry soil to 1.59 9 108 copies per gram of dry soil. The grassland and the rape-planted soil samples maintained the highest abundance of the bacterial and archaeal amoA genes, respectively. The abundance of the archaeal amoA

Dayong Zhao and Juan Luo have contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0714-5) contains supplementary material, which is available to authorized users. D. Zhao (&) State Key Laboratory of Hydrology-Water Resources and Hydraulic Engineering, Hohai University, Nanjing 210098, China e-mail: [email protected] D. Zhao  J. Luo  J. Wang  R. Huang  K. Guo  Y. Li College of Hydrology and Water Resources, Hohai University, Nanjing 210098, China Q. L. Wu State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, China

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gene was positively correlated with the pH (P \ 0.05). The ammonia concentrations exhibited a significantly positive relation with the abundance of the bacterial amoA gene (P \ 0.01) and the number of OTUs of AOB (P \ 0.05). The community composition of AOB was more sensitive to the land-use regimes than that of AOA. The data obtained in this study may be useful to better understand the nitrification process in soils with different land-use regimes.

Introduction Nitrification is a two-step process that includes the oxidation of ammonia to nitrite and its subsequent oxidation to nitrate. Ammonia oxidation, which is the first and ratelimiting step, plays a critical role in global nitrogen cycling [3, 16]. Initially, ammonia-oxidizing bacteria (AOB) were typically thought to be the only prokaryote capable of the obligatory aerobic, chemoautotrophic process [15, 24]. In recent years, the recognition that ammonia-oxidizing archaea (AOA) could also catalyze the process has changed the understanding of nitrogen cycling [11, 20, 39]. Extensive studies have focused on AOA and have shown the ubiquitous existence of AOA in various ecosystems, including marine [20], lake [18], estuary [3, 4, 28], thermal spring [43] , and soil [25] ecosystems. Most studies targeted a key functional gene, the amoA gene, which encodes the alpha subunit of the ammonia monooxygenase enzyme that catalyzes ammonia oxidization. The abundance and the diversity of AOA and AOB in soil ecosystems, including forest, grassland, and agricultural soil, have been investigated [5, 9, 17, 22, 31, 33, 35, 41]. The quantification of archaeal and bacterial amoA genes has shown a greater abundance of archaeal ammonia oxidizers than bacterial ammonia oxidizers in many soils [1, 8, 14, 25].

D. Zhao et al.: The Influence of Land Use on the Abundance and Diversity of Ammonia Oxidizers

However, some other studies found the opposite results in certain soils [10, 19]. At the same time, environmental factors also play important roles in shaping the community of ammonia oxidizer. The abundance and diversity of AOA and AOB changed with soil pH [29, 34], temperature [2], ammonium concentration [30], and fertilization practices [17]. Several studies that focused on rice field soils [6, 40] and grassland soils [23, 42] indicated that the soil type was the key factor in determining the abundance and community compositions of archaeal and bacterial amoA genes [13, 26, 27, 36]. Inversely, Chen et al. [7] collected three types of paddy soils from different cities in China that were cultured for 10 weeks with pot experiments. Only minor changes in the abundance and community structure of both AOB and AOA were observed. At present, limited information is available on more varied land-use regimes. Therefore, more investigations are needed to reveal the differences in abundance and community composition of AOA and AOB with different land-use regimes. In the present study, six soil samples were collected from different stations with distinct land-use regimes. Molecular analysis including real-time quantitative PCR and construction of the clone libraries were carried out to elucidate the abundance and diversity of AOA and AOB.

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determined according to our previous study [44]. The total carbon (TC) and the total nitrogen (TN) were measured according to ISO 13878-1998 and ISO 10694-1995 using an elemental analyzer (EA3000, Euro Vector, Italy). The soil ammonia (NH4?-N), nitrate (NO3--N), and nitrite (NO2--N) concentrations were extracted from the soil samples with 2 M KCl and measured by a continuous flow analyzer (San??, SKALER, Netherlands). DNA Extraction DNA was extracted from 0.25 g soil samples with the PowerSoil DNA Isolation Kit (MoBio Laboratory, Solana Beach, CA), according to the manufacturer’s instructions. The extracted DNA was verified on a 0.8 % agarose gel. Then, the concentration of the extracted DNA was determined using a BioPhotometer (Eppendorf, Hamburg, Germany). Real-time Quantitative PCR

The study was conducted in the Huashan watershed, upstream of the Chengxi Reservoir in the city of Chuzhou (32.312N, 118.318E), Anhui province, China. The Huashan watershed has a total area of 80.1 km2 in which most soils are used for agricultural crops or forestland. Six sites with different land-use regimes [FS: forestland soil (32.26179N, 118.14592E); PS: paddy soil (32.28060N, 118.17474E); WPS: wheat-planted soil (32.24452N, 118.201 32E); FPS: fruit-planted soil (32.29761N, 118.18365E); GS: grassland soil (32.2947N, 118.19037E); and RPS: rape-planted soil (32.29138N, 118.19935E)] were selected within the watershed in April 2012 for sampling. A 20 by 20 m square was chosen from an area of each of the sample site. Within each square, 10 random soil samples were taken from the top 15 cm of the soil. These samples were pooled together to reduce any spatial variability. The samples were transferred to sterile tubes immediately, stored on dry ice, and transported to the laboratory.

Real-time quantitative PCR was conducted to investigate the archaeal and bacterial amoA gene copy numbers and was performed on the basis of SYBR Green I using an IQ5 Thermocycler (RG65HD, Corbett, Australia). The primers for the archaeal amoA PCR amplification were Arch-amoAF/Arch-amoAR [12], and the primers for the bacterial amoA PCR amplification were amoA-1F/amoA-2R [32]. The standard curves were constructed using tenfold serial dilutions of plasmid DNA of known concentrations covering 3.40 9 102–3.40 9 108 archaeal amoA gene copies per ll for AOA and 1.59 9 102–1.59 9 108 bacterial amoA gene copies per ll for AOB. The reaction mixtures for the archaeal and bacterial amoA PCR amplification were 20 ll, including 5 ng of the DNA template, 19SYBR Premix Ex TaqTM buffer (Takara, Japan), and 0.2 lM of each archaeal and bacterial amoA primer. For the thermal cycles, the following protocol for the archaeal amoA PCR amplification was used: 3 min at 95 °C, 45 cycles of 30 s at 95 °C, 1 min at 53 °C, 20 s at 72 °C, and finally 7 min at 72 °C. The following protocol for the bacterial amoA PCR amplification was used: 3 min at 95 °C, 45 cycles of 30 s at 95 °C, 1 min at 50 °C, 20 s at 72 °C, and finally 7 min at 72 °C. Melting curve and 2 % agarose gel electrophoresis were employed to check the specificity of the PCR products. Data analysis was carried out with the Rotor-Gene 6000 software package. The amplification efficiencies were 0.94–1.06 (R2 = 0.991–0.995) for archaeal amoA gene and 0.95–1.04 (R2 = 0.994–0.996) for bacterial amoA gene.

Chemical Analysis of the Soil Samples

Clone Library and Phylogenetic Analysis

All of the soil samples were dried with a freeze dryer (ALPHA1-2, CHRIST, Germany). The soil pH was

Clone libraries were constructed with the DNA extracted from the six soil samples. PCR reactions were carried out

Materials and Methods Study Sites and Sample Collection

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D. Zhao et al.: The Influence of Land Use on the Abundance and Diversity of Ammonia Oxidizers

with the primers described above. Triplicate PCR products were pooled, gel purified by the Axygen PCR cleanup purification kit, and cloned by the pGEM-T vector (Promega, Madison, WI, USA). The ligation products were used to transform to Escherichia coli competent cells (DH5a, Takara, Japan). Picked transformants were grown overnight on LB agar plates containing 100 lg/ml ampicillin, 40 lg/ml X-Gal, and 24 lg/ml IPTG. The clones were checked by PCR amplification using vector primers (T7 and SP6) and sent to Shanghai Majorbio Biotechnology Co., Ltd. for DNA sequencing. The vector sequences were removed by the DNAStar software package. All of the amoA sequences were compared with GenBank database sequences using BLAST. Operational taxonomic units (OTUs) were defined as sequence groups in which sequences differed by B5 % nucleotide differences. Multiple sequence alignments of the most related environmental sequences and clone sequences were carried out by ClustalX [38]. Indices of diversity, library coverage, and nonparametric richness estimations were performed using the software program DOTUR. Neighbor-joining phylogenetic trees (based on Jukes-Cantor distances) were constructed based on alignments of the nucleic acid sequences using MEGA4.0 [37]. Statistical Analysis Significant differences of the soil properties and the archaeal or bacterial amoA gene abundance among the different samples were evaluated by one-way ANOVA and post hoc comparisons with the SPSS 13.0 package (SPSS, Chicago, IL). Correlation analysis (the two-tailed Pearson correlation coefficients) between the abundance or number of OTUs of the amoA gene and environmental factors was also performed. All statistical significance was acceptable at P \ 0.05. Nucleotide Sequence Accession Numbers The amoA gene sequences obtained in this study have been deposited in the GenBank database under the accession numbers JX862207–JX862358 for the archaeal amoA gene sequences and JX862359–JX862527 for the bacterial amoA gene sequences.

Results The Chemical Properties of the Soils The pH values of the different soil samples ranged from 3.76 to 6.99 (Fig. S1). The pH of the WPS sample was the highest followed by the PS (6.67) and the FS (6.43). The

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Fig. 1 The copy numbers of the archaeal and bacterial amoA genes in different soil samples. Data are presented as mean ± standard deviation (n = 3). Different superscript letters refer to significant differences between the samples (P \ 0.05). FS forestland soil, PS paddy soil, WPS wheat-planted soil, FPS fruit-planted soil, GS grassland soil, and RPS rape-planted soil

pH values of these three samples were all near 7.0. The pH values of the fruit-planted and GS samples were almost the same. The nitrate concentrations of the grassland, PS, and RPS samples (21.04, 19.86, and 16.00 mg/kg, respectively) were significantly higher than the concentrations of the other samples. Only slight differences were observed in the nitrite concentrations of the six soil samples. The highest content of NH4?-N appeared in the GS (9.12 mg/kg), which was nearly twice that of all of the other samples. The FS sample maintained the highest levels of TC and TN (Fig. S1). The Abundance of the Archaeal and Bacterial amoA Genes The abundance of the archaeal amoA gene ranged from 2.12 9 104 copies per gram of dry soil to 2.57 9 105 copies per gram of dry soil, while the abundance of the bacterial amoA gene ranged from 5.58 9 104 copies per gram of dry soil to 1.59 9 108 copies per gram of dry soil (Fig. 1). The bacterial amoA gene abundance was higher than that of the archaeal amoA gene abundance in all of the soil samples. No significant difference in the archaeal amoA gene abundance was observed among the different samples, and all of the values were around 105 copies per gram of dry soil, except for the fruit-planted soil sample (2.12 9 104 copies per gram of dry soil). However, significant variations in the abundance of the bacterial amoA gene were observed in the different soil samples. The samples collected from the GS maintained the highest bacterial amoA gene abundance of 1.59 9 108 copies per

D. Zhao et al.: The Influence of Land Use on the Abundance and Diversity of Ammonia Oxidizers Table 1 Diversity of the archaeal and bacterial amoA gene sequences from different soil samples Clone libraries

No. of OTUs

C (%)

H0

Schaol

FS PS

4

100.00

1.111

4

6

86.96

1.480

9

WPS

10

86.11

1.821

13.3

FPS

6

88.00

1.435

9

GS RPS Total

Table 2 Pearson correlation coefficients of environmental variables and relative abundances or numbers of detected OTUs of ammoniaoxidizing prokaryotes in soil samples with different land-use regimes Environmental variables

AOA

6 12 31

95.83 74.91 92.07

1.665 2.128 2.911

6 19 50.5

AOB FS

2

100.00

0.460

2

PS

4

93.33

1.085

5.5

WPS

6

89.66

1.216

7.5

FPS

3

96.67

0.769

3

GS

10

82.14

2.054

20

RPS

2

100.00

0.264

2

Total

17

95.40

1.756

31

FS forestland soil, PS paddy soil, WPS wheat-planted soil, FPS fruitplanted soil, GS grassland soil, RPS rape-planted soil, C coverage of the clone libraries, H0 shannon-weaver index, Schaol Chao1 index

gram of dry soil. The fruit-planted soil samples exhibited the lowest bacterial amoA gene abundance (5.58 9 104 copies per gram of dry soil), which was lower by nearly four orders of magnitude. At the same time, the lowest abundance of the archaeal amoA gene (2.12 9 104 copies per gram of dry soil) was also found in the FPS sample (Fig. 1). Diversity Analysis of the Archaeal and Bacterial amoA Gene In total, 164 archaeal and 174 bacterial amoA gene sequences were identified in the present study. For the archaeal amoA gene, OTUs were defined as a 5 % difference in nucleic acid sequence alignment and calculated using the DOTUR software package [3]. There were 31 OTUs found for the archaeal amoA clone library, whereas the number of OTUs found for the bacterial amoA clone library was 17 in total. For the individual clone library, the number of OTUs ranged from 4 to 12 for the archaeal amoA gene and from 2 to 10 for the bacterial amoA gene (Table 1). For the archaeal amoA gene, the FS samples maintained 4 OTUs, whereas the RPS maintained 12 OTUs. For the bacterial amoA gene, the GS samples contained 10 OTUs, whereas the FS and the RPS contained only 2 OTUs. The coverage of these clone libraries varied from 74.91 to 100.00 % for the AOA and from 82.14 to 100.00 % for the AOB. The diversity indices of Shannon-Weiner (H0 ) and SChao1

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pH

Pearson correlation coefficients Archaeal amoA gene copies/g soil

No. of OTUs of AOA

No. of OTUs of AOB -0.261

-0.553

0.036

NO3--N

-0.215

0.598

-0.063

0.389

NO2--N

0.368

-0.688

0.614

-0.690

NH4?-N

0.041

TC

0.152

-0.488

-0.214

-0.600

-0.176 0.571

-0.352 -0.320

-0.550 0.506

-0.668 0.005

TN TC:TN

0.813*

Bacterial amoA gene copies/g soil

0.986**

-0.153

0.890*

* P \ 0.05; ** P \ 0.01

estimators elucidated that the RPS sample had the highest diversity in the archaeal amoA gene, and the GS sample maintained the highest diversity in the bacterial amoA gene. The Relationships Between Environmental Factors and the Abundance and Diversity of the Archaeal and Bacterial amoA Gene Pearson’s correlation coefficients were used to investigate the relationships between environmental factors and the abundance/richness of the archaeal and bacterial amoA genes. The abundance of the archaeal amoA gene was positively correlated with the pH (P \ 0.05), whereas there was no significant correlation found between the abundance of the bacterial amoA gene and the pH (Table 2). The ammonia concentrations exhibited a significantly positive correlation with the abundance of the bacterial amoA gene (P \ 0.01) and the number of OTUs of the AOB (P \ 0.05). However, the ammonia concentrations showed no significant relation with the abundance and richness of the archaeal amoA gene (Table 2). Phylogenetic Analysis of the AOA and AOB In the present study, three major clusters, Nitrososphaera, Nitrosopumilus, and Nitrosotalea, were found in the archaeal amoA gene sequences obtained from the different land-use regimes (Fig. S2A). Overall, 79.80 % of all the sequences fell into the Nitrososphaera cluster, while the others fell into Nitrosopumilus and Nitrosotalea clusters. All of the soil samples contained the subcluster S1, except for the FPS and the GS (Fig. 2a). The archaeal amoA sequences isolated from the FS and the PS contained only the Nitrososphaera cluster, with 3 and 2 subclusters,

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D. Zhao et al.: The Influence of Land Use on the Abundance and Diversity of Ammonia Oxidizers

Fig. 2 Relative proportions of archaeal (a) and bacterial (b) amoA gene representing different clusters from different soil types. FS forestland soil, PS paddy soil, WPS wheat-planted soil, FPS fruit-planted soil, GS grassland soil, and RPS rapeplanted soil

respectively. The subcluster S1 was predominant with a percentage C50 % in all soil samples except for the GS (12 %) and the FPS (0 %). The WPS sample contained all five subclusters in the Nitrososphaera cluster, whereas the RPS sample contained 4 subclusters without subcluster S5. Both of the two soil samples contained the Nitrosopumilus cluster (9 and 23 %, respectively), suggesting that the diversities of the archaeal amoA gene in the WPS and the RPS samples were highest. The bacterial amoA sequences obtained in this study were divided into Nitrosomonas oligotropha, Nitrosomonas europaea, Nitrosospira, and undefined Nitrosospira

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lineages (Fig. S2B). The Nitrosomonas oligotropha lineage contained bacterial amoA sequences isolated from all of the land-use regimes with distinct proportions that were 17 % (FS), 36 % (PS), 62 % (WPS), 37 % (FPS), 31 % (GS), and 7 % (RPS) (Fig. 2b). In addition to the Nitrosomonas oligotropha lineage, an undefined Nitrosospira lineage was the only detected bacterial amoA gene group isolated from the FPS sample and the RPS sample, with the relative percentages of 63 and 93 %, respectively. The Nitrosospira lineage isolated from the FS had a relative percentage of 83 %. The second highest component of the Nitrosospira lineage (61 %) was observed in the bacterial amoA

D. Zhao et al.: The Influence of Land Use on the Abundance and Diversity of Ammonia Oxidizers

sequences isolated from the PS. The other two soil samples (WPS and GS) contained all four of the bacterial amoA lineages.

Discussion In the present study, the bacterial amoA gene abundance was higher than that of the archaeal amoA gene abundance in all of the soil samples. A previous study showed a consistent result in which bacteria rather than archaea dominated microbial ammonia oxidation in an agricultural soil [19]. However, other studies obtained contradictory results. Adair and Schwartz [1] found that ammonia-oxidizing archaea are more abundant than ammonia-oxidizing bacteria in semiarid soils of northern Arizona, USA, and that the AOA and AOB occupied different niches. Leininger et al. [25] also demonstrated that crenarchaeota was the most abundant ammonia-oxidizing organism in soil ecosystems on earth. The discrepancy may be caused by the complexity of soil environments, which are affected by numerous environmental factors. A significantly positive correlation was found between the pH and the abundance of the archaeal amoA gene. There have been several previous studies that showed similar results [17]. However, Nicol et al. [29] found that the archaeal amoA gene and transcript abundance decreased with an elevated soil pH. The inconsistency in this relationship could be attributed to the complicated relationships between the pH and other environmental factors. The pH may merely reflect changes in other environmental factors, such as the availability of ions and trace metals [21], which can have both inhibitory and stimulating effects on archaeal amoA gene abundance. In the present study, there was no significant correlation found between the pH and the bacterial amoA gene abundance. Previous studies found a positive relationship between the bacterial amoA gene abundance and the pH [17, 29]. In addition, the ammonia concentrations exhibited a significantly positive relationship with the abundance of the bacterial amoA gene and the number of OTUs of the AOB, which was consistent with a previous study [40]. As the substrate for ammonia oxidizers, ammonia availability is likely to affect the abundance and community composition of the AOB and AOA [1]. Previous studies in agricultural soils indicated that AOB populations exhibit longterm, rather than short-term responses to the variations in the concentration of ammonia nitrogen [1]. The abundance of the AOA and AOB varied among the different land-use regimes. No significant difference in the archaeal amoA gene abundance among the different samples was observed. However, significant variations with four orders of magnitude in the abundance of the bacterial

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amoA gene were observed in the different soil samples. The bacterial amoA gene abundance showed a more pronounced variation than that of the AOA. The AOA sequences obtained from the different soil samples included three major clusters, Nitrososphaera, Nitrosopumilus, and Nitrosotalea. Subcluster S1 was predominant in all of the soil samples other than the FPS and the GS, which were dominated by the Nitrosotalea cluster (48 %) or subcluster S3 (29 %). The maximum diversity of the AOA community composition was detected in the WPS followed by the RPS. The different land-use regimes exhibited an obvious influence on the community composition of the AOA. The bacterial amoA sequences obtained in this study were also influenced by the land-use regimes. The WPS and GS contained all four bacterial amoA lineages. The Nitrosomonas oligotropha lineage contained bacterial amoA sequences isolated from all of the soil samples with different percentages. The Nitrosospira lineage accounted for a large portion of the sequences isolated from the FS and PS samples. A study showed that the community composition of AOB showed significant differences between the primary soil and agricultural soil, while the AOA community composition showed no significant correlation with the soil developmental stage [26]. In conclusion, the influence of land-use regimes on the abundance and community structure of AOA and AOB was investigated. The results demonstrated that a distinct effect of land-use regimes was observed. The abundance of the bacterial amoA gene was higher than that of the archaeal amoA gene in all six soil samples with different land-use regimes. The community structures of both the AOA and AOB varied with the land-use regimes. The community composition of the AOB was more sensitive to the land-use regimes than that of the AOA. Further investigations are need to elucidate the mechanism about how the land-use regimes affecting the abundance and community composition of AOA and AOB to better understand the nitrogen cycling process in soils. Acknowledgments This work was supported by the Ministry of Water Resources’ Special Funds for Scientific Research on Public Causes (201201026), National Natural Science Foundation of China (41371098), China Postdoctoral Science Foundation (2014T70470, 2014M561568) and Jiangsu Planned Projects for Postdoctoral Research Funds (1401093C).

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