Responses Of N₂O Emission And Reduction As Well As Associated Microbes To Temperature And Different Fertilization Regimes In Arable Soils

Nitrous oxide?N₂O?is an important greenhouse gas,and owns the ability to delete the ozone layer.Agricultural soils accounted for the majority of anthropogenic N₂O,which is driven by the excessive input of chemical synthetic nitrogen fertilizer and animal manure.Therefore,researches on the production and consumption of N₂O in arable soils are earning more and more attention.The effects of temperature on N₂O emission in arable soils are increasingly earning more and more attention in the context of global climate change.Many researches suggest that the sensitivity of N₂O emission to temperature fluctuation varied among soils,this is reflected in the high variability of N₂O emission temperature sensitivity index(Q₁₀)among different soils.However,the associated microbial mechanisms are still unclear.Meanwhile,the conventional emission of N₂O is net outcome of production and consumption process;therefore,to reveal the mechanisms leading to the variation of Q₁₀among soils necessitates the consideration of both production and consumption process.In order to explore possible microbial mechanisms leading to the varation of Q₁₀ among different arable soils and fertilization regimes,a series of microcosm experiments was established to mintor the net N₂O emission of three different soil types subject to different fertilization regimes under three incubation temperature?15,25,and 35??;Meanwhile,the C₂H₂ inhibition method was used to discriminate the production and consumption processes in combination of the T-RFLP and real-time quantitative PCR?Q-PCR?to analyze the community structure and abundance of associated functional guilds.This study is comprised of three experiments.In experiment I,three arable soils receiving different fertilizer treatments were selectedd including a paddy soil derived from red soil in Fujian?no fertilizer control,conventional fertilization,optimized fertilization,incremental nitrogen fertilization,and optimized fertilization plus incorporation of crop straw?,a cultivated loessial soil in Gansu?no fertilizer control,low input of synthetic nitrogen fertilizer,optimized input of synthetic nitrogen fertilizer,and hight input of synthetic nitrogen fertilizer?,and a paddy soil derived from Xiashu Loess in Jiangsu?no fertilizer control,inorganic fertilization,combination of inorganic and organic fertilization,combination of inorganic fertilization and incorporation of crop straw?.Before the measurement of cumulative net N₂O emission of these soils under 15,25,and 35?,a pre-incubation procedure was adopted to minimize the effect of differences in substrate availability on Q₁₀.The results indicated that,both the effect of fertilization and temperature,as well as the interaction between them significantly influenced net N₂O emission.The estimated Q₁₀s were distributed in a narrow range?1.07-1.67?and lower than the theoretical Q₁₀ values?=2?,thus highlighting that the availability of substrate was the key factor regulating Q₁₀ in this study.The effect of fertilization on Q₁₀ was dependent on the temperature range,fertilization regime,and soil type,which corresponded to the finding that only Q₁₀s?25-35??of these treatments with the input of chemical nitrogen fertilizer exceeding 300 kg ha^-1 year^-1 were significantly higher than that of CK.In experiment II,the destructively collected soil samples from experiment I were subjected to molecular microbal eocological analysis.The CK and sole application of chemical fertilizer with the input rate of synthetic nitrogen above 300 kg N ha^-1 year^-1 were selected.The T-RFLP and Q-PCR were used to explore the response pattern of community traits of N₂O emission-related microbial functional groups including ammonia oxidizing archaea?AOA?and bacteria?AOB?as well as nirS-and nirK-denitrifiers.The T-RFLP analysis indicated that,except the AOA in Gansu cultivated loessial soil,the application of inorganic fertilizer significantly shifted the community structures of ammonia oxizers?AOA and AOB?and denitrifiers?nirS-and nirK-denitrifiers?in tested soils,the elevation of temperature only shifted the community structure of AOA and nirS-denitrifiers in Fujian paddy soil,as well as the community structure of nirS-denitrifiers in Gansu cultivated loessial soil.Q-PCR analysis indicated that the abundance of AOA significantly increased in response to the elevation of temperature in all tested soils,while the inorganic fertilization regimes lead to siginificantly higher abundance of AOB compared with CK.In contrast,there was no any consistent response pattern to temperature for nirS-and nirK-denitrifiers among soils in terms of guild abundance.The correlation analysis indicated that,the AOAs were significantly and positively correlated with the cumulative net N₂O emission in terms of either abundance?Fujiang and Gansu?or community structure?Jiangsu?,highlighting an important role of AOA played in N₂O emission in all tested soils.In contrast with Fujian paddy soil and Gansu cultivated loessial soil soil,the cumulative net N₂O emission was highly positively correlated with DON,DOC,and nitrate in Jiangsu paddy soil,thus implying that significant amount of N₂O in this soil derived from heterotrophic nitrification.In the third experiment,the paddy soil derivred from Xiashu Loess in Jiangsu was further selected to investigate the effect of temperature on the production and reduction rate of N₂O in soils receiving different fertilizer treatments,namely no fertilization control?CK?,inorganic fertilization?NPK?,and combination of inorganic fertilizer and pig manure?MNPK?.The C₂H₂ inhibition method was used to discriminate the production and consumption process;meanwhile,the Q-PCR was used to monitor the abundance of nosZI-and nosZII-type N₂O reducers.The results indicated a minor part of N₂O production stemmed from autotrophic nitrification;instead,the majority of N₂O production came from heterotrophic nitrification.Both the production and consumption rate of N₂O were significantly influenced by fertilization regimes and temperature.Linear regression analysis indicated that,different from CK and MNPK,the production rate of N₂O showed a trend of increase while the consumption rate of N₂O showed a trend of decrease in response to the elevation of temperature in NPK treatment.This was presumably responsible for the higher emission sensitivity of N₂O emission in NPK treatment.The Q-PCR indicated that the abundance of nosZII-type N₂O reducers was significantly lower in NPK than in CK and MNPK,and more sensitive to fertilization than nosZI-type reducers.Multivariate analysis of variance indicated that the temperature significantly impacted the abundance of nosZI-N₂O reducers,while the the abundance of nosZII-N₂O reducers were siginificantly impacted by the interaction between temperature and time,as well as between temperature and fertilization treatment.The linear regression suggested that only the abundance of nosZII-N₂O was significantly correlated with the production and reduction rate of N₂O.In conclusion,the decrease of abundance of nosZII-type N₂O reducers caused by inorganic fertilization was mainly responsible for the higher emission temperature of N₂O in this treatment in Jiangsu paddy soil.