3.5 Correlation between N2O emission and the fungal community and soil properties
Associations between N2O emission, soil properties and community structures of the fungal phyla and genera were analyzed by RDA. For the fungal phyla, axis1 and axis2 explained 60.7% and 7.5% of the total variation, respectively, in the community structure (Fig. 6a). N2O emission was positively correlated with fungalAscomycota , with the highest correlation (R = 0.726, P = 0.008). The dominant phylum of fungi in the tested soil was Ascomycota, which accounted for 21.1-36.6% of the total fungal microbial abundance. The trend of change in the abundance of Ascomycota in the four treatments was consistent with that of N2O emission, which meant that Ascomycota was an important contributor to N2O emission.
For the fungal genus community structure, RDA analysis explained 73.2% of the total variation, and axis1 and axis2, respectively, explained 46.6% and 26.6% of the total variation (Fig. 6b). N2O emission showed a positive correlation with the abundance ofPyrenochaetopsis , Myrothecium , Zopfiella ,Humicola , Bullera , and Conocybe . The generaPyrenochaetopsis , Myrothecium , Zopfiella , andHumicola belong to the phylum Ascomycota, and Bullera andConocybe belong to the phylum Basidiomycota. Further quantitative analysis of the correlation between N2O emission and the relative abundance of the fungal genera revealed that Myrothecium(R2 = 0.556, P = 0.005), Pyrenochaetopsis (R2 = 0.478, P = 0.013), and Humicola (R2 = 0.372, P = 0.035) were positively correlated with N2O emission, while Cryptococcus(R2 = 0.551, P = 0.006) was negatively correlated with N2O emission (Fig. 7). Pyrenochaetopsis ,Myrothecium , and Humicola were also found to be the main functional fungi in N2O emission. However,Zopfiella , Bullera , and Conocybe were not significantly correlated with N2O emission.
In addition, N2O emission was positively correlated with soil NO3–N and negatively correlated with soil pH and NH3+-N (Fig. 6). The influence of soil properties on fungal community structure decreased in the order of pH > NO3–N > NH3+-N > SOC > TN. N fertilizer use led to changes in soil properties and in the fungal community structure, and ultimately affected N2O emission.

4. Discussion

In this study, we specifically studied the effects of different fertilization amounts on N2O emission from lawn soil and microbes, and further analyzed the community changes within the major functional microorganisms and their correlation with N2O emissions. The results showed that N fertilizer significantly increased the N2O emission from lawn soil (Fig. 2b). This was consistent with most studies reporting that exogenous nitrogen addition significantly enhanced soil N2O emission[28-31]. The contribution of fungi to the N2O emissions in lawn soil was the highest of the three evaluated microbial communities, accounting for 45%, which was significantly higher than that of bacteria (31%) and other microorganisms (24%) (Fig. 3). Therefore, our results showed that fungi played a larger role than bacteria in N2O emission in lawn soil. This result supported that soil N2O was produced primarily by fungi and emphasized the importance of fungi on N2O emissions in the lawn soil. This result was also similar to results found by other studies. For instance, it has been found that fungi are the major contributors to N2O emission of soil in grazing grasslands in Tibet[17,41], grazing grasslands in New Zealand[35], tea in southern China[36], and croplands in China[42]. Meanwhile, our result was consistent with results from the traditional agricultural ecosystem, crop-livestock integrated ecosystem, organic agricultural ecosystem, and the artificial forest ecosystem in which fungi contributed 40-51% to N2O emission [17,42]. Therefore, it is of great significance to explore the microbial mechanism of N2O production by fungi for reducing N2O emission from lawn soil.
In addition, fungi are better adapted to the environment than bacteria. N fertilizer significantly increased the NO3–N concentration and decreased pH in lawn soil (Table. S1). Bacteria preferred ammonia oxidation at high NH3+-N concentrations [21]. This also proved that fungi contributed more to the N2O emissions in the lawn soil. In our lawn soil, the NH4+- N was low, while the NO3–N concentration was high, indicating that the lawn soil environment was more suitable for the growth of fungal communities.
Our results showed that the N2O emissions of the N300 treatment which represented the highest level of N fertilizer addition, did not produce the highest emission levels, and they were instead significantly lower than that of the N225 treatment (Fig. 2b). The accumulation of N2O began to decline after the highest N225 treatment, indicating that high N fertilizer application could effectively promote N2O emission, but it was not the case that the higher the nitrogen application, the higher the N2O emission. We speculated that it might be related to soil microorganisms with N transformation function. Thus, increasing the number of microorganisms involved should result in increased production of N2O emissions. Further analysis revealed that the dominant fungi in the lawn soil accounted for the top three fungal communities, namely Ascomycota, Basidiomycota, and Mucoromycota. Nitrogen fertilizer significantly increased the relative abundance of Ascomycota, while it significantly decreased the relative abundance of Basidiomycota (Fig. 4a). We found a positive correlation between N2O emission and Ascomycota through RDA analysis (Fig. 6a). Moreover, the growing trend of Ascomycota during the four nitrogen fertilizer treatments was consistent with the N2O emission trend in lawn soil. N2O emissions reached their highest levels in the N225 treatment, rather than the N300 treatment. We hypothesized that this might be related to the microbial biomass and nitrogen transformation in lawn soil. We found that the relative abundance of Ascomycota in the N225 treatment (36.6%) was higher than that of the N300 treatment (35.1%). This result not only explained why N2O emission in the N225 treatment was higher than that in the N300 treatment, but also indicated that Ascomycota played an important role in the N2O emissions in lawn soil. It has been reported that among fungi, Ascomycota and Basidiomycota preferred to use soil nitrate for denitrification and released N2O[32]. 90% percent of the fungi reported to produce N2O belong to the phylum Ascomycota, followed by fungi in the Basidiomycota and Mucoromycota which account for 7% and 3%, respectively. Representative N2O-producing Ascomycota [33,34]. Ascomycota preferred to grow with nitrogen than Basidiomycota. Meanwhile, high nitrogen could inhibit N2O reductase activity [35]. These results supported our conjecture.
At the fungal genus level, the relative abundance ofPyrenochaetopsis , Myrothecium , Zopfiella , andHumicola increased significantly after nitrogen fertilizer treatment, while the relative abundance of Chaetomium ,Simplicillium , Cryptococcus , Mortierella andPhoma significantly decreased (Fig. 4b). Myrothecium ,Zopfiella , Pyrenochaetopsis , Humicola of Ascomycota and Bullera and Conocybe of Basidiomycota were positively correlated with N2O emission (Fig. 6b). Regression analysis showed Pyrenochaetopsis , Myrothecium , andHumicola of Ascomycota were positively correlated with N2O emission, while Bullera and Conocybeof Basidiomycota were not significantly correlated with N2O emission. These results also indicated that Ascomycota might be the key microbial population driving nitrogen transformation in lawn soil. Moreover, we found that Myrotheciumwas the fungal genus with the highest correlation coefficient for N2O emission in lawn soil through correlation analysis (Fig. 7). According to the results of previous studies,Myrothecium had a strong ability to produce N2O. The N2O production capacity of Myrothecium was 21.4 nmol N2O mL-1d-1, and the efficiency was far higher than that ofPyrenochaetopsis and Humicola , respectively 3.5 and 5.1 nmol N2O mL-1d-1[34]. Meanwhile, the relative abundance ofMyrothecium in Ascomycota was the highest in the N225 treatment (1.88%) and significantly higher than that in the other three treatments (0.37%–0.82%). We found that the adaptability ofMyrothecium was stronger under the condition of nitrogen addition, but the condition of high nitrogen decreased, which was consistent with the previous research results[32,34]. We concluded that Myrotheciumplayed an important role in the increased N2O emission in lawn soil. Therefore, it is of strategic significance to study the mechanisms related to Myrothecium and resultant N2O emissions in order to reduce increases in N2O emissions from the urban lawn soil in future.
During the incubation period, we found that N fertilizer increased N2O flux in the lawn soil (Fig. 2a). Further, the N2O flux of the N225 and N300 treatment showed the highest emission peaks on the 5th day, while N0 and N150 treatments did not show emission peaks, indicating that high nitrogen fertilizer input could significantly stimulate the N2O emission of lawn soil, which was consistent with the results of previous studies[36-37]. This indicated that N fertilizer increased the N2O emissions in lawn soil, and also strengthened the soil nitrogen stimulation effect. Meanwhile, some laboratory experiments showed that N2O could rapidly reach the emission peak within a short period after N fertilizer addition, and the cumulative emissions accounted for more than half of the total emissions and then rapidly declined[8,38]. Mate analysis showed that the proportion of nitrogen addition was linearly correlated to N2O emissions [39]. However, the low N fertilizer application (Urea) did not significantly enhance N2O emission, which was because the fact that the lawn was irrigated after low urea application, leading to urea hydrolysis into inorganic nitrogen which was directly used by plants [7,40].

5. Conclusions

Nitrogen fertilizer significantly promoted N2O emissions in lawn soil, although this result was not linearly related to the amount of fertilizer applied. When the amount of fertilizer applied was 225 kg·ha·yr-1, N2O emission was the highest, but it decreased when the amount of fertilizer applied increased to 300 kg·ha·yr-1. N fertilizer significantly altered the soil microbial community structure. Through biological inhibitor treatment, we found that fungi were the main contributors to N2O emission in lawn soil, accounting for 45% of the total N2O emissions. Pyrenochaetopsis , Myrothecium , andHumicola of Ascomycota were significantly positively correlated with N2O emission and were the predominant contributors to N2O emissions within lawn soil. These findings will help to draw up appropriate measures for mitigation of N2O emissions in lawn soil.
Funding information This research was supported by the Liaoning Province Grassland Plant Resources Special Investigation Fund (1103-01044415001) in China.
Author Contributions:Z.X. and L.B. conceived and designed the experiments; Z.X. and W.Y. performed the experiments; Z.X., J.L. and B.R. analyzed the data; Z.X. wrote the paper.
Acknowledgements: We gratefully acknowledge Dr. Feng Zhou (Shenyang Institute of Applied Ecology, Chinese Academy of Sciences) for providing valuable comments and suggestions. We acknowledge Bowen Duan and Yifan Lin (Shenyang Agricultural University) for their assistance during our experiments. We gratefully acknowledge the time and expertise devoted to reviewing this manuscript by the reviewers and the members of the editorial board.
Conflicts of interest: The authors declare no conflict of interest.
Data Availability Statement
- Relative abundances of the main fungal phyla and genus in the lawn soil of all treatments, and soil properties at 0 - 20 cm soil depth sampling: Dryad Doi https://doi.org/10.5061/dryad.nvx0k6dnv.
-All data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Chen H, Xia Q, Yang T, Bowman D, Shi W. The soil microbial community of turf: linear and nonlinear changes of taxa and N-cycling gene abundances over a century-long turf development. FEMS Microbiol. Ecol. 2018, 95(2): fiy224.
  2. IPCC. 2007. Changes in atmospheric constituents and in radiative forcing. Pp. 2.9.2–2.9.3 in S. Solomon, D. Qin, and M. Manning, et al. eds. Climate change 2007: the physical science basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge Univ. Press, Cambridge, U.K.
  3. Kanter D, McDermid S, Nazarenko L. Nitrous oxide’s ozone destructiveness under different climate scenarios//Proceedings of the 2016 International Nitrogen Initiative Conference,” Solutions to improve nitrogen use efficiency for the world”. 2016.
  4. Zhang, Z.; Zhu, B.; Xiang, H. Effect of Nitrogen Fertilizer for Wheat on N2O Emission and Denitrification in Purple Soil.J. Agro-Environ. Sci. 2010, 29, 2033–2040.
  5. Wang, J.; Xiong, Z.; Yan, X. Fertilizer-induced emission factors and background emissions of N2O from vegetable fields in China. Atmos Environ . 2011, 45, 6923–6929.
  6. Yang, W.; Weber, K.; Silver, W. Nitrogen loss from soil through anaerobic ammonium oxidation coupled to iron reduction. Nat. Geosci. 2012, 5, 538–541.
  7. Cardenas, L.; Thorman, R.; Ashlee, N.; Butler, M.; Chadwick, D.; Chambers, B. Quantifying annual N2O emission fluxes from grazed grassland under a range of inorganic fertiliser nitrogen inputs. Agr. Ecosyst. Environ. 2010, 136, 218–226.
  8. Lin, Y.; Ding, W.; Liu, D.; He, T.; Yoo, G.; Yuan, J.; Chen, Z.; Fan, J. Wheat straw-derived biochar amendment stimulated N2O emissions from rice paddy soils by regulating the amoA genes of ammonia-oxidizing bacteria. Soil Biol. Biochem.2017, 113, 89–98.
  9. Ozlu, E.; Kumar, S. Response of surface GHG fluxes to long-term manure and inorganic fertilizer application in corn and soybean rotation.Sci. Total Environ. 2018, 626, 817–825.
  10. Gu, X.; Wang, Y.; Laanbroek, J.; Xu, X.; Song, B.; Huo, Y.; Chen, S.; Li, L.; Zhang, L. Saturated N2O emission rates occur above the nitrogen deposition level predicted for the semi-arid grasslands of Inner Mongolia, China. Geoderma . 2019, 341, 18–25.
  11. Raciti S, Burgin A, Groffman P. Denitrification in suburban lawn soils. J.Environ. Qual. 2011, 40(6): 1932-1940.
  12. Hall S, Huber D, Grimm N. Soil N2O and NO emissions from an arid, urban ecosystem. J. Geophys. Res-Biogeo. 2008, 113(G1).
  13. Case, S.; McNamara, N.; Reay, D.; Whitaker, J. The effect of biochar addition on N2O and CO2 emissions from a sandy loam soil–the role of soil aeration. Soil Biol. Biochem. 2012, 51, 125–134.
  14. Xu, C.; Han, X.; Ru, S.; Cardenas, L.; Rees, R.; Wu, D.; Wu, W.; Meng, F. Crop straw incorporation interacts with N fertilizer on N2O emissions in an intensively cropped farmland.Geoderma . 2019, 341, 18–25.
  15. Hayatsu M, Tago K, Saito M. Various players in the nitrogen cycle: diversity and functions of the microorganisms involved in nitrification and denitrification. J. Soil Sci. Plant Nut. 2008, 54(1): 33-45.
  16. Marusenko, Y.; Huber, D.; Hall, S. Fungi mediate nitrous oxide production but not ammonia oxidation in aridland soils of the southwestern US. Soil Biol. Biochem. 2013, 63, 24–36.
  17. Zhong, L.; Wang, S.; Xu, X.; Wang, F.; Rui, Y.; Zhou, X.; Shen, Q.; Wang, J.; Jiang, L.; Luo, C.; Gu, T.; Ma, W.; Chen, G. Fungi regulate response of N2O production to warming and grazing in a Tibetan grassland. Biogeosciences. 2018, 1–27.
  18. Bru, D.; Ramette, A.; Saby, N.; Dequiedt, S.; Ranjard, L.; Jolivet, C.; Philippot, L. Determinants of the distribution of nitrogen-cycling microbial communities at the landscape-scale. Isme. J. 2011, 5, 532–542.
  19. Müller, C.; Laughlin, R.; Spott, O.; Tobias-Rütting, T. Quantification of N2O emission pathways via a 15N tracing model.Soil Biol. Biochem. 2014, 72, 44–54.
  20. Laughlin, R.; Stevens, R. Evidence for fungal dominance of denitrification and codenitrification in a grassland soil. Soil Sci. Soc Am. J. 2002, 66, 1540–1548.
  21. Yang, L.; Zhang, X.; Ju, X. Linkage between N2O emission and functional gene abundance in an intensively managed calcareous fluvo-aquic soil. Sci. Rep. 2017, 7, 43283.
  22. Shan J, Li M, Sun Y, Zhou H. Recent development of turf grass industry in China. Acta Agrestia Sinica (in Chinese ). 2013, 21, 222–229.
  23. Bai, L.; Wang, Y.; Liu, Y.; Wang, X.; Tan, D. Effects in Responses of Turfgrass Soil on Nitrous Oxide Emission Processes to N Application.Acta Horticulturae Sinica (in Chinese ). 2016, 43, 1971–1979.
  24. Weisblum, B.; Davies, J. Antibiotic inhibitors of the bacterial ribosome. Bacteriological Reviews. 1968,32, 493–528.
  25. Castaldi, S.; Smith, K. Effect of cycloheximide an N2O and NO3- production in a forest and an agricultural soil. Biol. Fert. Soils . 1998, 27, 27–34.
  26. Chen, H.; Mothapo, N.; Shi, W. Fungal and bacterial N2O production regulated by soil amendments of simple and complex substrates. Soil Biol. Biochem . 2015, 84, 116–126.
  27. Zhou, J.; Guan, D.; Zhou, B.; Ma, M.; Qin, J.; Jiang, X.; Chen, S.; Cao, F.; Shen, D.; Li, J. (2015) Influence of 34-years of fertilization on bacterial communities in an intensively cultivated black soil in northeast China. Soil Biol. Biochem. 2015, 90, 42– 51.
  28. Wrage, N.; Velthof, G.; Oenema, O.; Laanbroek, H. Acetylene and oxygen as inhibitors of nitrous oxide production in Nitrosomonas europaea and Nitrosospira briensis : a cautionary tale.FEMS Microbiol . Ecol. 2004, 47, 13–18.
  29. Zhang, J. Han, X. N2O emission from the semi-arid ecosystem under mineral fertilizer (urea and superphosphate) and increased precipitation in northern China. Atmos. Environ.2008, 42, 291–302.
  30. Xue, D.; Gao, Y.; Yao, H.; Huang, C. Nitrification potentials of Chinese tea orchard soils and their adjacent wasteland and forest soils. J Environ. Sci. 2009, 21, 1225–1229.
  31. Duan, P.; Zhang, X.; Zhang, Q.; Wu, Z.; Xiong, Z. Field-aged biochar stimulated N2O production from greenhouse vegetable production soils by nitrification and denitrification. Sci. Total Environ. 2018, 642,1303–1310.
  32. Prendergast-Miller, M.; Baggs, E.; Johnson, D. Nitrous oxide production by the ectomycorrhizal fungi Paxillus involutus andTylospora fibrillosa . FEMS Microbiol Lett. 2011, 316, 31–35.
  33. Shoun, H.; Fushinobu, S.; Jiang, L.; Kim, S.; Wakagi, T. Fungal denitrification and nitric oxide reductase cytochrome P450nor.Philos. T. R. Soc. B . 2012, 367, 1186–1194.
  34. Mothapo N, Chen H, Cubeta M, Grossman J, Fuller F, Shi W. Phylogenetic, taxonomic and functional diversity of fungal denitrifiers and associated N2O production efficacy.Soil Biol. Biochem. 2015, 83: 160-175.
  35. Rex, D.; Clough, T.; Richards, K.; Klein, C.; Morales, S.; Samad, M.; Grant, J.; Lanigan, G. Fungal and bacterial contributions to codenitrification emissions of N2O and N2 following urea deposition to soil. Nutr. Cycl. Agroecosys. 2018, 110, 135–149.
  36. Huang, Y.; Long, X.; Chapman, S.; Yao, H. Acidophilic denitrifiers dominate the N2O production in a 100-year-old tea orchard soil. Environ. Sci. Pollut. Res. 2015, 22, 4173–4182.
  37. Ma, L.; Shan, J.; Yan, X. Nitrite behavior accounts for the nitrous oxide peaks following fertilization in a fluvo-aquic soil. Biol. Fert. Soils. 2015, 51, 563–572.
  38. Duan, P.; Zhang, X.; Zhang, Q.; Wu, Z.; Xiong, Z. Field-aged biochar stimulated N2O production from greenhouse vegetable production soils by nitrification and denitrification. Sci. Total Environ. 2018, 642,1303–1310.
  39. Shcherbak, I.; Millar, N.; Robertson, G. Global meta-analysis of the nonlinear response of soil nitrous oxide (N2O) emissions to fertilizer nitrogen. Proc. Natl. Acad. Sci. 2014, 111, 9199–9204.
  40. Jiang, C.; Yu, G.; Fang, H.; Cao, G.; Li, Y. Short-term effect of increasing nitrogen deposition on CO2, CH4 and N2O fluxes in an alpine meadow on the Qinghai-Tibetan Plateau, China. Atmos. Environ. 2010, 44, 2920–2926.
  41. Zhong, L.; Bowatte, S.; Newton, P.; Hoogendoorn, C.; Luo, D. An increased ratio of fungi to bacteria indicates greater potential for N2O production in a grazed grassland exposed to elevated CO2. Agr. Ecol. Environ. 2018, 254, 111–116.
  42. Chen, H.; Mothapo, N.; Shi, W. The significant contribution of fungi to soil N2O production across diverse ecosystems.Appl. Soil Ecol . 2014, 73, 70–77.