| Paddy field is considered as one of the most important sources responsible for greenhouse gases(GHG)emissions,e.g.methane(CH4)and nitrous oxide(N2O).Amendment of biochar has a significant impact on CH4 and N2O emission in paddy field with long-term nitrogen(N)fertilizer application.Both the CH4 and N2O emissions are produced by microorganisms in soil.CH4 emissions are the comprehensive results of methanogens and methanotrophs,and the formation of N2O is mainly by the nitrification and denitrification processes.Therefore,the investigation of N fertilizer and biochar influences on CH4 and N2O related microbial gene copy numbers and their potential activities in paddy soil might be helpful to clarify the microbial mechanisms of N fertilizer and biochar on CH4 and N2O emissions from paddy soil.In this study,five treatments with three replicates were installed as NOBO,NOB1,N1B0,N1B1,and N1B2(B0,B1,and B2 designated as biochar at 0,20,and 40 t hm-2,respectively,amended at 2012,while NO and N1 as N doses at 0 and 250 kg N hm-2 crop-1,respectively).We quantified the methanogen(mcrA),methanotrophy(pmoA),ammonia-oxidizing archaea(amoA-AOA)and ammonia-oxidizing bacteria(amoA-AOB),nitrite reductase(nirS,nirK),and N2O reductase(nosZ)gene abundance in China paddy soil by using extracting soil DNA and Real-time q-PCR technology at the mature stage of wheat and the seedling,tillering,elongation,heading,mature stages of rice.In addition,the monitoring of CH4 and N2O emissions was performed using a static chamber method.And we measured the potential of methane oxidation and production,nitrification potential and the denitrification enzyme activity simultaneously.In an effert to investigate the variation of gene copy numbers abundance and potential activity of CH4 and N2O related functional microbes in different crop growth period in paddy soil,the changes of biochar and N fertilization have been analyzed as well.Results show that N fertilizer application can significantly stimulate the soil CH4 emission fluxes during rice season.Compared with the N1B0 treatment,cumulative CH4 emissions were significantly reduced 8.8%in the N fertilizer with high dose(40 t hm-2)biochar amendment rate.Biochar amended at three years ago still has the significant reduction effect on soil CH4 emissions.Compared with N1B0 treatment,biochar amendment significantly increased soil organic carbon,microbial biomass carbon,C/N(p<0.05)and soil pH as well.Relative to N0B0 treatment,the methane oxidation potential was significantly increased from 52.4%to 269.4%in N0B1 treatment.Besides,methane oxidation potential and methane production potential were positively related to biochar application rates in the presence of N fertilizer(p<0.05).To some extent,N fertilization inhibited the growth of pmoA.Amendment of single or low rate biochar can significantly increase the pmoA gene abundance.Statistical analysis shows that there was a significant positive correlation between soil methane production potential and the change of the mcrA gene abundance.N fertilization decreased pmoA/mcrA ratio,however,biochar amendment increased pmoA/mcrA ratio in the same does N-treated treatments(p<0.05).All these findings demonstrate that biochar amendment promoted methanotrophs and methanogens in abundane,and consequently on methane oxidation capacity than on methane production potential in our study.Therefore,biochar amendment can be a helpful practice to mitigate CH4 emissions in the paddy field.The N2O emission fluxes in the rice seasonhad obvious ebb and flow relationship with CH4 emission fluxes,whichwas especially striking in mid-season drainage period.The N2O emission fluxes during the rice growing stages ranged from-16.33-534.57 ?g N m-2 h-1.In addition,N fertilizer significantly enhancedthecumulative N2O emissions in rice season,while biochar did not show significant reduction effect on that.The potential of nitrification rates in rice season were lower than those in wheat mature stages.The potential of nitrification rates promoted with the increase of biochar amendment rate.There were an different trend between ammonia-oxidizing archaea(amoA-AO A)and ammonia-oxidizing bacteria(amoA-AOB)gene abundance during different crop growing seasons,but their gene abundance in rice growing stages were all significant lower than those in the mature stage of wheat.Soil nitrification is controled by AOA and AOB,but AOB played a more important role in the nitrification process in paddy soil.However,there was not a significant correlation between N2O emission fluxes with the copy numbers of ammonia-oxidizing microorganisms and nitrification potential in the rice season.Therefore,nitrification might not be the mainly process of N2O production in rice season.N fertilization can significantly increase the abundance of the nirS and nirK genes of nitrite reductase,while N fertilization combined with biochar amendment can significantly increase the soil N2O reductase nosZ abundance,and reduce the nitrite reductase nirS and nirK genes abundance at the same time.Moreover,biochar amendment reduced the denitrification enzyme activity at a certain degree,which indicated that biochar amendment might be helpful to mitigate N2O emissions in the paddy field,but it did not show significant reduction effectin the rice season.Over all,N fertilization combined with biochar amendment effectively improved the soil acidification and soil properties in paddy soil.Biochar amended at three years ago has the significant reduction effect on soil cumulative CH4 emissions while increased the gene abundance of methanogen,but the level was lower than the improved effect on methanotrophy.Although,in this study neither single nor combined with biochar amendment show significant reduction effect on soil cumulative N2O emissions of rice season,N fertilization combined with biochar amendment can significantly increase the abundance of N2O reductase gene(nosZ)and further reduce the abundance and activity of soil nitrite reductase(nirS,nirK)in paddy soil. |
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