Responses of Soil Hydrolytic Enzymes, Ammonia-Oxidizing Bacteria and Archaea to Nitrogen Applications in a Temperate Grassland in Inner Mongolia

www.nature.com/scientificreports OPEN Responses of soil hydrolytic enzymes, ammonia-oxidizing bacteria and archaea to nitrogen Received: 16 March 2016 Accepted: 11 August 2016 applications in a temperate Published: 06 September 2016 grassland in Inner Mongolia Xinyu Zhang1, Yuqian Tang1, Yao Shi2, Nianpeng He1, Xuefa Wen1, Qiang Yu3, Chunyu Zheng1, Xiaomin Sun1 & Weiwen Qiu4 We used a seven-year urea gradient applied field experiment to investigate the effects of nitrogen (N) applications on soil N hydrolytic enzyme activity and ammonia-oxidizing microbial abundance in a typical steppe ecosystem in Inner Mongolia. The results showed that N additions inhibited the soil N-related hydrolytic enzyme activities, especially in 392 kg N ha−1 yr−1 treatment. As N additions increased, the amoA gene copy ratios of ammonia-oxidizing archaea (AOA) to ammonia-oxidizing bacteria (AOB) decreased from 1.13 to 0.65. Pearson correlation analysis showed that the AOA gene + copies were negatively related with NH4 -N content. However, the AOB gene copies were positively − −1 −1 correlated with NO3 -N content. Moderate N application rates (56–224 kg N ha yr ) accompanied by P additions are beneficial to maintaining the abundance of AOB, as opposed to the inhibition of highest N application rate (392 kg N ha−1 yr−1) on the abundance of AOB. This study suggests that the abundance of AOB and AOA would not decrease unless N applications exceed 224 kg N ha−1 yr−1 in temperate grasslands in Inner Mongolia. Soil fertility decline is considered as a primary cause for low productivity of grasslands in northern China (approximately 150 million ha)1. Nitrogen (N) has been identified as the main nutrient that limits biomass and above-ground net primary production2. Therefore, grassland ecosystems are likely to be highly sensitive to N additions, and N may improve plant productivity and soil organic carbon stocks in this area1,3. N fertilization practices can also markedly suppress community species richness, and community stability of grassland ecosystems in N-limited environments4–6. Excess N has been widely recognized as one of major drivers of biodiversity loss in agro-ecosystems. Bai et al.3 reported that N-induced species loss occurred in mature Eurasian grasslands when N applications exceeded 17.5 kg ha−1 yr−1. Changes in above-ground biomass and species richness were observed in both mature and degraded ecosystems when N applications exceeded 105 kg ha−1 yr−1. It is important to understand the response of nitrification to N applications so as to predict the potentials of nitrate leaching and nitrous oxide (N2O) emissions. In addition, effects of enhanced N additions on the activities of soil enzymes and microbial communities that are involved in N transformations need to be understood. However, to date, there is no information about the N loading thresholds of the important functional microbes that mediate N transformations (e.g., ammonia oxidizers) below-ground in this area. Microbial N acquisition activity can be highly sensitive to N additions to grassland7,8. Activities of β -1, 4-N-acetylglucosaminidase (NAG) and leucine aminopeptidase (LAP) control the release of plant available N from organic compounds9. The responses of soil enzyme activity to nitrogen fertilization in arid soils are distinct 1Key Laboratory of Ecosystem Network Observation and Modeling, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China. 2School of Geographic Sciences, Northeast Normal University, Changchun 130024, China. 3State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China. 4The New Zealand Institute for Plant and Food Research Limited, Private Bag 4704, Christchurch, New Zealand. Correspondence and requests for materials should be addressed to X.Z. (email: [email protected]) or X.S. (email: [email protected]) SCIENTIFIC REPORTS | 6:32791 | DOI: 10.1038/srep32791 1 www.nature.com/scientificreports/ from those in other soils. Wang et al.10 suggested that β-glucosidase and acid phosphomonoesterase in temperate grasslands did not change when N was applied, whereas Zhou et al.11 found that N additions over just a two year period in the Gutbantunggut Desert led to significant decreases in urease activity. To date, there is little information about how NAG and LAP activities respond to N application in steppe ecosystems, and how these activities relate to soil N availability over the long term. Ammonia oxidation is thought to be the primary step of nitrification, in which ammonia oxidizers are respon- sible to limit the subsequent nitrification rate, regulating the balance between ammonium, nitrate, and nitrite. So this process can affect soil nitrogen availability; and is therefore vital to the nitrogen cycle in terrestrial ecosys- tems12. The ammonia monooxygenase (AMO), which is a periplasm-associated enzyme, catalyzes the reaction of oxidizing ammonia to hydroxylamine in the first step of nitrification13. Ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), which have the ammonia monooxygenase alpha subunit gene (amoA), are thought to play important roles in catalyzing this step14,15. However, due to the differences between AOA and AOB in cellular, genomic and physiological levels, AOA is more energy-effective16 and has relatively higher affinity to ammonia than AOB17. As reported, AOA prefer ammonia-poor and acidic conditions; while AOB favor N-rich and alkaline environments18. Given the background of N-limit in the grassland in Inner Mongolia, it is necessary to assess the competition mechanism of AOA and AOB in ammonia oxidation. Previous studies have examined the effects of N additions on the abundance of AOA and AOB in grassland ecosystems13,19,20. Results showed that urea-N additions of up to 150 kg N ha−1 yr−1 led to significant increases in AOB, but not in AOA, which suggested that AOB was more sensitive to N addition than AOA19. Nitrogen enrichment may cause phosphorus (P) being the main limiting nutrient and P fertilization could influence AOA and AOB abundance20. Phosphorus limitation could change the effect of N application on AOA and AOB, however, it is not clear how N application rates influence AOA and AOB abundance in semiarid temperate grasslands in the presence of P. In view of the central role played by soil microorganisms in soil N cycling, it is therefore necessary to deter- mine the effects and thresholds of long-term N applications on soil microbial activity and N cycling biochemical processes in grassland ecosystems. In this study, we used a seven-year field experiment that comprised five levels of urea gradient additions combined with P addition in the Xilinguole grassland area, Inner Mongolia. The main objectives were to (1) explore the effects of N applications on soil N hydrolytic enzyme activities; (2) quantify the abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) at different N application levels without limiting P; and (3) find a threshold for N applications that the enzyme activities, AOA and AOB will be changed if the N application is above the threshold. Results Soil physico-chemical properties and net nitrification rates. Nitrogen additions resulted in significant decreases in soil pH, and increased soil organic carbon (SOC) and inorganic N contents (Fig. 1). Soil pH decreased from 7.3 to 6.3 due to the N additions. SOC content increased from 21.6 to 24.3 g kg−1, with significant −1 + − difference (P < 0.05) between the treatments of below and above 112 kg ha N application. NH4 -N and NO3 -N were greater in the N treatments than in the control treatment (N0). Maximum NH4-N level was observed in the treatment with the highest rate of applied fertilizer, whereas the NO3-N level peaked at the medial N amount −1 − −1 −1 adding treatment of 112 kg ha rate. The net nitrification rate increased from 0.6 to 2.8 mg NO3 -N kg soil d − −1 −1 after N additions, i.e. from 0.03 to 0.11 mg NO3 -N g SOC d after N additions, with significant increases of net nitrification adjusted to SOC observed in all the N addition treatments. Soil N related hydrolytic enzyme activities. Nitrogen-related hydrolytic enzyme activities decreased as a result of high N application rates (Fig. 2). The NAG enzyme activities decreased by between 4–42% in response to N additions, and a significant decrease was observed for the N392 treatment (Fig. 2). The LAP enzyme activities decreased by between 13–52% owing to the N additions, and significant decreases were observed for the 224N and N392 treatments (Fig. 2). Regression analysis showed that both LAP and NAG activities were decreased linearly with the amount of N additions (Fig. 2, P < 0.05). Both LAP and NAG activities were positively related with pH, + and negatively correlated with NH4 -N, SOC content and nitrification rates (Table 1). There were no significant − correlations between soil NO3 -N content and NAG and LAP activities (Table 1). Ammonia-oxidizing archaea and bacteria. Gene copy numbers were used to estimate AOB and AOA abundances. The AOB gene copy numbers ranged from 1.45 × 109 to 1.06 × 1010 g−1 soil, and increased significantly in the N56, N112 and N224 treatments relative to the N0 treatment (Fig. 3). However, AOA gene copy numbers 8 9 −1 in the N56, N112 and N224 treatments, ranging from 8.52 × 10 to 1.5 × 10 g soil, were not significantly different from the control. The AOA gene copy numbers were lower in the N392 treatment than in all other treatments. The ratio of AOA to AOB gene copy numbers decreased from 1.13 in N0 to 0.65 in N392 (Fig.

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