Effects Of Warming,Nitrogen Addition And Precipitation Reduction On Nitrifiers And Denitrifiers In A Semi-Arid Grassland On The Loess Plateau

The ongoing climate change resulting from human activities can significantly influence the structure and functions of plant and microbial communities in terrestrial ecosystems.However,our capacity to predict the impacts of climate change on ecosystem processes is critically hampered by our limited knowledge of microbial responses to climate change factors.Climate change factors such as warming,N inputs and precipitation alterations can significantly alter plant growth and subsequent carbon（C）allocation belowground for soil microorganisms.However,the underlying mechanisms by which soil microbes,in particular N-cycling microbes,respond to and feedback to climate change factors remain poorly understood.N-cycling microbes control the transformations of reactive N in soil and convert N into different forms,including gaseous forms such as nitrous oxide（N₂O）and N₂.N₂O is one of the most important greenhouse gases in the atmosphere,and nitrification and denitrification processes are major sources of N₂O emissions to the atmosphere.Nitrifiers and denitrifiers are largely responsible for these two processes,respectively.Ammonia oxidation,the first and rate-limiting step of nitrification process,has long been believed to be driven by ammonia-oxidizing microbes（mainly ammonia-oxidizing archaea,AO A,and ammonia-oxidizing bacteria,AOB）.Denitrification produces N₂O mainly by highly diverse denitrifying microbes with distinct reductases encoded by various genes,including nirK,nirS and nosZ.While nirK-and nirS-type denitrifiers produce N₂O,nosZ-type denitrifiers control the conversion of N₂O to N₂,which represents the only known sink for N₂O.Climate change factors such as warming,precipitation reduction and N deposition may directly or indirectly impact nitrification and denitrification,thereby altering N₂O production.However,climate warming will likely concur with N inputs and precipitation reduction under future climate change scenarios.Yet,few have examined effects of concurring warming,N inputs and precipitation reduction on N-cycling microbes and the underlying processes,particularly in fragile ecosystems such as semi-arid grasslands on the Loess Plateau.Semi-arid grasslands occupy 11%of the global land surface and are considered highly vulnerable to climate change,owing to its unique climatic features.It is predicted that with global warming of future global change,the area of global semi-arid grasslands will continue to expand,which will increase the uncertainty of global climate change.They are also important sources of N₂O emissions.Despite of the important role of microbial communities on N₂O production and consumption,we have limited knowledge of the biological pathways and mechanisms involved in N₂O emissions from semi-grasslands under global climate change scenarios.Taking advantage of a long-term field experiment in a semi-arid grassland on the Loess Plateau,northwestern China,we examined how climate warming,precipitation reduction（30%precipitation reduction）and N inputs affect nitrification,denitrification and N-cycling key microbes.Specific objectives of this study were to:（1）determine the effects of precipitation reduction,N addition and their combinations on nitrification and denitrification processes;（2）determine the effects of warming,N addition and their combinations on nitrification and denitrification processes;（3）examine the effects of precipitation reduction,N addition and their combinations on N-cycling microbes,focusing on AO A,AOB and nirK-,nirS-,nosZⅠ-and nosZⅡ-type denitrifiers;（4）characterize the effects of warming,N addition and their combinations on N-cycling microbes,focusing on AOA,AOB and nirK-,nirS-,nosZⅠ-and nosZⅡ-type denitrifiers.The main results of this study were as follows:1.For the precipitation reduction and N addition experiment,precipitation reduction and nitrogen addition significantly increased the content of nitrate and potential nitrification rate.There were significant interactions between precipitation reduction and nitrogen addition on potential nitrification rate and potential denitrification activity.N addition significantly increased AOB abundance by five-fold but had no effect on AOA.Precipitation reduction had no significant effect on either the abundance of AO A or AOB.Both precipitation reduction and N addition significantly affected the T-RFs in AOA and AOB.The alterations of AOB under N inputs were largely due to the enhancement of 110bp T-RFs in AOB.2.Compared with nitrifiers,precipitation reduction had no effect on nirK and nirS gene abundance,but N addition significantly increased nirS gene abundance in the absence of precipitation reduction.Interestingly,precipitation reduction and N addition reduced nirK/nirS ratio.Precipitation reduction reduced the abundance of nosZⅡ genes significantly.Significant interactions between precipitation reduction and N addition on nosZⅡ gene abundance were observed.For denitrifier community,N addition,precipitation reduction and their interaction significantly affected the nirK community structure.N addition but not precipitation reduction significantly affected the community structure of nirS.N-induced changes in nirS largely stemmed from the enhancement of 100bp T-RFs in nirS.Neither precipitation reduction nor nitrogen addition or their interactions had any significant effect on the community structure of nosZ.3.For the warming and N addition experiment,warming significantly increased soil microbial biomass,potential nitrification rate and potential denitrification activity.N addition significantly increased the abundance AOB and had no effect on AO A.Warming had no significant effect on either the abundance of AOA or AOB.N addition significantly affected the community structure of AOB.Significant interactions between N addition and warming on the community structure of AOA and AOB.4.Compared with nitrifiers,neither warming,nitrogen addition nor their interaction had any significant effect on nirK gene abundance.N addition significantly increased the abundance of nirS,leading to the reduced nirK/nirS ratio.N addition significantly affected the community structure of nirK-and nirS-type denitrifiers significantly increased the abundance of nosZⅡ.Significant interactions between N addition and warming affected the community structure of nirK-type denitrifiers.None of warming,N addition and their interactions had any significant effect on the community structure of nosZ-type denitrifiers.Taken together,our results show that:（1）AOB played a inant role in ammonia oxidation in this semi-arid grassland on the Loess Plateau,（2）AOB was more sensitive to N substrate availability than AOA under warming,（3）when the substrate was sufficient,nirK-type denitrifiers may be more sensitive to drought than nirS-type denitrifiers.nirS-type denitrifiers responded faster to substrate concentration than nirK-type denitrifiers.Compared with nosZⅠ-type denitrifiers,nirK-and nirS-type denitrifiers were more sensitive to climate change,（4）Further more,nosZⅡ-was more sensitive to environmental change than nosZⅠ-denitrifiers.Therefore,our results have the following implications:（1）it can provide data support and theoretical basis for the study of ecosystem carbon and nitrogen relationship model;（2）it has important theoretical and practical significance to predict the global climate change trend and to evaluate the role of soil microbes in climate change.