Effects Of Biochar Application On Soil N₂O Emissions And Nitrogen Uptake And Utilization Of Winter Wheat And Its Microbial Mechanism

The extensive emissions of N₂O from dryland not only cause environmental pollution but also negatively impact crop nitrogen uptake and utilization,thus severely impeding sustainable agricultural development.Biochar has potential advantages in reducing N₂O emissions and increasing crop nitrogen uptake by directly or indirectly modifying the soil microenvironment because of its unique physical and chemical properties.Microbes are particularly sensitive to changes in the soil environment,which play a crucial role in the chemical cycle of soil components.This sensitivity can have significant effects on N₂O emissions and crop nitrogen uptake and utilization.However,the microbial mechanism of soil N₂O emissions and crop N uptake and utilization in the presence of biochar has been unknown.Therefore,based on long-term biochar application in winter wheat fields（biochar was applied in October 2011 and October 2016,respectively）,this study established four biochar application rates(B0:0 t·ha^-1,B5:5 t·ha^-1,B10:10 t·ha^-1,B20:20 t·ha^-1)and investigated the characteristics of soil N₂O emission,as well as changes in soil ammonia oxidation and denitrification microbial community abundance in the winter wheat season from 2018 to 2021 to clarify the roles of soil ammonia oxidation and denitrification microbes in the process of soil N2O production under biochar application.Meanwhile,fluorescence quantitative PCR and high-throughput sequencing technologies were used to investigate the change of nitrogen cycle-related microbe abundance,rhizosphere soil bacterial community composition,and nitrogen uptake and utilization of winter wheat.Combined with soil physical and chemical properties,the association between nitrogen cycle-related microbes,rhizosphere soil bacteria and winter wheat growth under biochar application was elucidated.The main findings of this study are as follows:（1）Biochar application reduced cumulative N₂O emissions in three winter wheat seasons,with reductions ranging from 7.69%to 41.75%compared to the control.The inhibitory effect of the B5 and B10 treatments on N₂O emissions was stronger than that of the B20 treatment.N₂O emissions mainly occurred within a certain period after fertilization.The cumulative emissions during the peak period accounted for 45.30%to 74.91%of the total cumulative emissions.This emphasized the need for researching the N₂O emissions during the peak period.During the peak period,biochar addition improved the soil moisture content with the increased biochar application rate.Although biochar reduced the content of soil ammonium and nitrate nitrogen,there were only small differences in the numerical values among different treatments.Furthermore,biochar addition significantly（P<0.05）increased the ratio of N₂O consumption genes（nos ZI+nos ZII）to N₂O production genes（nir S+nir K+Fnir K）in denitrification,as well as the abundance of N₂O production genes AOAamo A and AOBamo A in ammonia oxidation during the peak period of N₂O emissions.The increase in the ratio of（nos ZI+nos ZII）to（nir S+nir K+Fnir K）was stronger for the B5 and B10 treatments than for the B20 treatment,while the effects on AOAamo A and AOBamo A abundance were weaker than for the B20 treatment.N₂O emission flux was positively correlated with soil moisture,nitrate nitrogen content,and the abundance of AOAamo A,AOBamo A,nir S,nos ZI,and nos ZII genes（P<0.05）.These results indicated that,low-ammount biochar had a superior inhibitory impact on N₂O emissions compared to high-amount biochar,which may be due to higher soil nitrogen and water content in high-rate biochar addition.Biochar application could reduce N₂O emissions by increasing the ratio of（nos ZI+nos ZII）to（nir S+nir K+Fnir K）.However,biochar may also increase N₂O emissions during the ammonia oxidation process by improving the abundance of AOAamo A and AOBamo A.The overall effect of biochar on N₂O emissions might be related to the dominant processes of nitrification and denitrification under different moisture conditions.（2）The application of medium and high amounts of biochar tended to increase soil moisture and inorganic nitrogen at different growth stages and significantly（P<0.05）increased organic carbon and total nitrogen content,water-stable aggregate stability(R_0.25,MWD,GMD)and soil porosity,but had no significant effect on soil p H.Compared to control,biochar treatments tended to increase the gene abundance of bacteria 16S r RNA in the tillering stage,with the highest value under B10 or B20 treatment.Meanwhile,biochar addition significantly（P<0.05）improved the gene abundance of ammonia-oxidizing bacteria AOAamo A and AOBamo A in the tillering stage,which increased by 7.03%-114.08%and 1.49%-71.22%,respectively,compared with the control.However,for denitrifying bacteria with nir S,nir K,and nos Z gene abundance,although biochar application showed an increasing trend,there was little difference among the treatments.Correlation analysis showed that AOAamo A,AOBamo A,and nos Z were significantly positively correlated with soil moisture,inorganic nitrogen,organic carbon,C/N,soil porosity,and aggregate stability（P<0.05）.In addition,biochar applied at 10 t ha^-1 and 20 t ha^-1significantly（P<0.05）increased above-ground and root biomass,nitrogen uptake at different growth stages of winter wheat,as well as the relative growth rate（RGR）and nitrogen uptake rate（RNU）at tillering stage.The winter wheat growth characteristics were positively correlated with soil physical properties(soil porosity,R_0.25,MWD,GMD)and organic carbon content.Linear regression found the abundance of AOAamo A,AOBamo A,and nos Z genes showed an extremely significant positive correlation with RGR and RNU（P<0.001）.These results indicated that the application of medium and high amounts of biochar increased the abundance of AOAamo A,AOBamo A,and nos Z genes through enhancing soil moisture,nitrogen availability,organic carbon content,and soil physical structure,which promoted the growth and nitrogen uptake of winter wheat.（3）The analysis of the rhizosphere soil bacterial community and growth characteristics of winter wheat at different growth stages showed that the rhizosphere bacterial communityβ-diversity was significantly（P<0.05）affected by growth stage and biochar application rates.The growth stage explained the biggest variation of rhizosphere community（R²=0.196,P<0.001）,followed by the interaction between biochar and crop growth stage（R²=0.170,P<0.001）and different biochar addition rates（R²=0.114,P<0.001）.Soil moisture,ammonium nitrogen,nitrate nitrogen content,and sucrase activity in the rhizosphere explained the variation at the genus level of the soil dominant bacteria.Compared with B20 treatment,there was an obvious crossover between B5 and B10 treatments in the types of biomarkers.Similarly,co-occurrence network analysis found that,when compared to the control,the proportion of modularity and positive correlation was higher in the B5 and B10 treatments,but lower in the B20 treatment.The biomarkers enriched under biochar treatment belonged to the Cyanobacteria,Gemmatimonadetes,Nitrospirae,and Chloroflexi.The Chloroflexales,Thermobioceraceae and Parviterribacter were positively correlated（P<0.05）with the relative nitrogen uptake rate（RNU）and carbon uptake rate（RCU）of winter wheat.Partial least squares structural equation modeling also indicated that variations in the rhizosphere bacterial community under biochar treatment could partially explain the growth traits of winter wheat.The above results indicated that biochar addition could change the community structure of rhizosphere bacteria through increasing soil moisture,inorganic nitrogen content,and enzyme activity.The application of low and medium amounts of biochar could maintain the stability of the community structure of bacteria.Biomarkers enriched under biochar treatment were closely related to soil carbon and nitrogen cycling,which may ultimately promote the growth of winter wheat.（4）Biochar application increased the yield of winter wheat by 2.23%-18.29%compared with the control.The data on yield components indicated that the application of medium and high amounts of biochar significantly improved the spike number of winter wheat（P<0.05）,but had no significant influence on the number of grains per spike or the 1000-grain weight.Moreover,biochar significantly（P<0.05）enhanced the nitrogen fertilizer productivity（Nf P）,nitrogen fertilizer utilization efficiency（NUE）,and nitrogen fertilizer agronomy utilization efficiency（NAE）,increasing by 2.23%-16.76%,10.21%-79.35%,and 4.36%-40.50%,respectively,with the highest values observed under the B10 or B20treatment.Further analysis revealed that the nitrogen utilization index（Nf P,NUE,NAE）was positively correlated with SOC,TN,soil water-stable aggregate stability index(R_0.25,MWD,GMD),rhizosphere soil NO₃^--N,and catalase activity（P<0.05）.The PLS-PM analysis showed that the diversity of rhizosphere bacteria and the abundance of nitrogen cycling functional microbes had a positive effect on nitrogen utilization of winter wheat.However,N₂O emissions had a negative effect on the nitrogen utilization of winter wheat.This study clarified the role of ammonia oxidation and denitrification microorganisms in N₂O emissions under biochar application,but the overall effect of biochar application on N₂O emissions may be affected by soil moisture.The study also revealed the relationship between the abundance of nitrogen cycling related microbes and rhizosphere bacterial community and nitrogen uptake of winter wheat.Overall,long-term application of 10 t ha^-1 biochar improved soil physical properties and nitrogen availability,as well as promoted the abundance of nitrogen cycling-related microbes and changed the bacterial composition of the rhizosphere,which enhanced nitrogen uptake and utilization in winter wheat and promoted the sustainable development of dryland farmland.