高寒草甸N2O排放对放牧强度的响应及微生物学机制

For the current lack of the responsibel microbial mechanism of soil N2O emission flux from different grazing intensity alpine meadow,this study would be carried out in Haibei grassland system station.It had fatal research significance on N2O mitigation and greenhouse effect. Alpine meadows effected by anthropogenic activity in the Tibetan plateau are an important source of atmosphere N2O.This study adopt the method of spatial sequence instead of time successional sequence.Four degradation stages grassland were seleted under long-term different grazing intensity ie Gramineae-Kobresi humilis,K.humilis,K.pygmaea community and herb-black beach.The high frequency observation of soil N2O emissions will be monitored during the freeze-thaw period,early growing season,the peak of growing season and the end of growing season, and dormancy period.These all aimed to illustrate how the N2O flux were affected by grazing intensity. The key functional genes amoA of ammonia oxidizing archaea,ammonia oxidizing bacteria that participated in the nitrification, and genes of nirK, nirS and nosZ which participated the denitrification bacteria were analysed as target genes. Richness and diversity character of nitrification and denitrification bacteria were studied by fluorescence quantitative PCR and Terminal-restriction fragment length polymorphism(T-RFLP). Therefore,the couping relation were determined among grazing intensity, soil nitrous oxide emission flux, and affected factors(grassland production，biodiversity, ammonia nitrogen and nitrate nitrogen,soil organic carbon, and pH), and soil microbe. This would help to explore the microbial mechanism of soil N2O emission in alpine meadow of different grazing intensity. Orientation regulation on nitrogen transformation, and finding mitigation measure were concered by this study. This research would provide scientic foundation for these research.

受人类活动影响的草地是大气N2O排放源，开展放牧胁迫高寒草地N2O排放特征及微生物机理研究，对于准确估算区域N2O排放量和探讨其发生机理具有重要意义。本研究选择牧户长期稳定放牧所形成的"禾草矮嵩草、矮嵩草、小嵩草群落和杂类草黑土滩次生裸地"四个退化演替阶段草地为研究对象。在冻融期、生长季初期、盛期、末期和休眠季高频观测草地生态系统N2O排放特征，阐明放牧强度对N2O通量的影响；以参与硝化过程(氨氧化细菌和古菌)关键功能基因amoA和参与反硝化作用的关键基因nirK、nirS及nosZ为靶标基因，通过T-RFLP和荧光定量PCR技术，阐明硝化和反硝化细菌丰度和多样性特征，揭示优势种群变化规律；解析放牧、土壤N2O排放及其关键影响因子(草地生产力、多样性、铵态氮和硝态氮、有机碳、pH)和土壤微生物特征之间的耦合关系，明晰放牧高寒草甸土壤N2O发生的微生物学机制，制定适宜的减排措施提供理论依据。

青藏高原高寒草甸为N2O排放源，放牧是其重要利用方式，长期超载放牧已造成草地大面积退化。开展放牧胁迫高寒草甸N2O排放特征及其微生物机理研究，对于准确估算区域N2O排放量和探讨其发生机理具有重要意义。本研究选择青海海北国家站地区，牧户长期稳定放牧所形成的"禾草矮嵩草、矮嵩草、小嵩草群落和杂类草黑土滩次生裸地"不同演替状态草地为研究对象。观测生态系统N2O排放特征，阐明放牧强度对N2O通量的影响；以参与硝化过程关键功能基因amoA 和参与反硝化作用的关键基因nirK、nirS及nosZ为靶标基因，通过荧光定量PCR和16S rDNA技术，阐明硝化和反硝化细菌丰度和多样性特征。结果表明：高寒草甸土壤N2O排放速率表现为脉冲式变化特征，生长季盛期草地N2O排放速率>生长季末期>生长季初期>冻融期>休眠季。中度放牧高寒草甸土壤N2O排放速率>重度>轻度>极重度放牧样地，但不同年份间会存在较大差异波动。长期放牧显著降低了高寒草地土壤有机碳，从6.67%到3.83%。中度放牧增加土壤全氮含量，重度和极重度放牧降低全氮。中度和重度放牧降低生物量。各因素对高寒草甸N2O的影响强度顺序依次为全氮含量>容重>pH>有机碳含量>生物量。多元逐步回归方程揭示土壤全氮、容重、有机碳含量是综合影响N2O排放的重要因素。高寒草甸N2O排放速率与氨氧化古菌和氨氧化细菌amoA丰度、亚硝酸还原酶的nirK+nirS丰度间存在正相关关系，与氧化亚氮还原酶nosZ基因丰度存在负相关关系。AOA的amoA基因丰度是影响N2O产生和排放的最重要因素。随放牧强度增加，nosZ丰度呈现出先降低后增加趋势，在极重度放牧时期达到峰值。N2O逐渐被还原为N2趋势增强，降低N2O排放。土壤OUTs丰度均表现为休眠季重度放牧>休眠季极重度放牧>生长季中度>生长季轻度放牧>生长季重度放牧>休眠季中度放牧>休眠季轻度放牧>生长季极重度放牧。各环境因子对细菌群落特征的影响顺序依次为：土壤铵态氮>硝态氮>生物量>容重>pH>SOC>全氮。明晰放牧高寒草甸土壤N2O发生的微生物学机制，为制定适宜的减排措施提供理论依据。