陇东旱塬长期不同施肥农田土壤的氮转化特征及其分子生物学机制

Understanding the processes of soil nitrogen transformation is the base for recommending fertilizer properly and reducing the environment pollution, however, nitrogen transformation and its mechanisms are lack of explanation in the rainfed dry highland soil. Ammonia oxidation is the key point of nitrogen transformation in the soil. In dry land, ammonia oxidation is driven mainly by the coactions of the ammonia oxidizing archaea (AOA) and the ammonia oxidizing bacteria (AOB). However, contributions of AOA and AOB to ammonia oxidation as well as their mechanisms in Loess Plateau soil are still unclear by now. Based on a long term fertilization experiment started in 1978, the project is to explore the fate of fertilizer nitrogen using 15N isotope method, to investigate the temporal and spatial properties of nitrogen pool and its response to the crop changes, and to probe the characteristics of ammonia oxidation in the soils as affected by different long term fertilizations by combining laboratory incubation experiments. Taking amoA as functional genetic markers, we also examine the effects of different long term fertilizations on AOA and AOB population diversities, and detect the amoA gene abundances and their expressions on RNA level by technologies of high-throughput sequencing and qPCR in order to find out the relationships among the ammonia oxidation, the amoA genes and their expression quantities. The results will elucidate the effects of different fertilizations on nitrogen transformation, clarify the contribution of AOA and AOB to soil ammonia oxidation and its molecule mechanism in the dryland soils, and uncover the inner connection between fertilization and soil nitrogen transformation. The researches will provide theoretical basis for regulating soil nitrogen pool and increasing the nitrogen use efficiency in the rainfed agricultural area.

理解土壤氮素转化过程是合理施氮和降低环境污染的基础，但雨养下的旱塬土壤氮素转化及其机制研究不足。氨氧化作用是全球氮循环的中心环节，旱地土壤氨氧化主要由氨氧化古菌（AOA）和氨氧化细菌（AOB）共同作用完成，但AOA及AOB在旱塬农田氨氧化中的功能及机制尚缺乏深入解析。本申请依托始于1978年的定位试验，结合室内试验，采用示踪法研究长期不同施肥下肥料氮的去向；研究旱塬农田土壤氮素的时空变化、应答作物转换的特征以及硝化潜势、氨氧化调控效应等氨氧化特性；运用高通量测序、定量PCR等技术，以amoA基因为分子标记，研究长期不同施肥农田土壤AOA和AOB的多样性、amoA基因及其RNA表达特征，剖析不同调控措施对amoA基因含量、表达量的影响及其与氨氧化的关系。通过研究，明确长期施肥对旱塬土壤氮转化的影响，揭示施肥与土壤氮素转化的内在联系，为雨养农业下的土壤氮库调控、氮肥利用效率提升提供理论依据。

理解土壤氮素转化过程是合理施氮和降低环境污染的基础，但雨养下的旱塬土壤氮素转化及其机制研究不足。氨氧化作用是全球氮循环的中心环节，旱地土壤氨氧化主要由氨氧化古菌（AOA）和氨氧化细菌（AOB）共同作用完成，但AOA及AOB在旱塬农田氨氧化中的功能及机制尚缺乏深入解析。本申请依托始于1978年的定位试验，结合室内试验，采用示踪法研究长期不同施肥下肥料氮的去向，表明一个轮作周期后，不同处理对标记氮肥的利用率随着轮作期延长而显著降低，其肥料叠加利用率范围在31.4%~50.1%。四茬作物标记氮肥去向总体表现为：吸收量＞土壤残留量＞总损失量；研究旱塬农田土壤氮素的时空变化、应答作物转换的特征以及硝化潜势、氨氧化调控效应等氨氧化特性，表明从1979-2019年，各处理土壤全氮显著增加，土壤碱解氮先下降后升高趋势。长期不同施肥处理之间土壤硝化潜势差异显著（P<0.05），MNP处理硝化潜势最高。硝化潜势与TN、MBN和NO3--N 含量均呈极显著正相关关系（P<0.01）。添加DCD显著延长了不同处理的硝化时间，且抑制氨氧化过程。运用高通量测序、定量PCR等技术研究发现长期不同施肥行为和生育季节决定了土壤微生物群落结构、丰度及其表达。厚壁菌、变形菌、放线菌、酸杆菌、拟杆菌和芽单胞菌为优势菌门。0～20cm及40～60cm土层土壤古菌arch-amoA基因丰度明显大于细菌amoA丰度（40～320倍），arch-amoA丰度与全氮和碱解氮显著相关。有机肥或秸秆配施化肥处理明显增加了孕穗和收获期arch-amoA丰度和基因转录拷贝数，氨氧化古菌在旱塬土壤氨氧化中起一定主导作用。通过以上研究，明确长期施肥对旱塬土壤氮转化的影响，揭示施肥与土壤氮素转化的内在联系，为雨养农业下的土壤氮库调控、氮肥利用效率提升提供理论依据。