咸水滴灌棉田土壤氨氧化微生物参与硝化作用的机理

Fresh water scarcity and the deterioration of irrigation water quality have become a fundamental and chronic problem for sustainable agricultural development in arid regions. Inevitability, saline water must be used for irrigation in these areas. Saline irrigation will increase soil salinity, thereby affecting soil N transformations. Nitrification, which is primarily carried out by microorganisms, is the central process in the N cycle. The main microorganisms involved in this step, ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), have attracted the attention of many scholars. However, the microbial oxidation of ammonia is likely to be different in saline environments than in non-saline environments. The mechanism of nitrification in a saline environment is not well understood. Therefore, the objective of this long-term, fixed-position field study is to clarify the relative contribution of AOA and AOB to soil nitrification in a saline water drip-irrigated cotton field. The effects of irrigation water salinity and N application rate on ammonia-oxidizing microbial abundance, community structure and functional activity will be determined using polymerase chain reaction (PCR), fluorescence quantitative PCR, denaturing gradient gel electrophoresis, clone library and sequencing technology, high-throughput sequencing, and stable isotope tracer techniques. The results should provide important information about the mechanism by which soil microorganisms oxidize ammonia during nitrification in arid areas when saline water is used for drip irrigation. The results of this study will provide a theoretical basis for (i) the rational use of water resources in arid regions with saline soil and (ii) the effective management of N and the regulation of the N cycle.

淡水短缺和灌溉水质盐化是干旱区农业可持续发展所面临的严峻问题，干旱区应用咸水灌溉已成为必然。咸水灌溉会导致土壤盐分增加，影响土壤氮素的转化。硝化作用是由微生物驱动的氮素转化核心过程，氨氧化古菌（AOA）和氨氧化细菌（AOB）作为参与硝化作用的关键微生物，近年来备受国内外学者关注。但当前盐渍环境下氨氧化微生物研究的结果不一致，硝化作用的机理也未被很好地揭示。因此，本研究通过多年咸水灌溉田间定位试验，采用聚合酶链式反应、荧光定量PCR、变性梯度凝胶电泳、克隆文库和测序技术、以及高通量测序等研究手段，并结合稳定性同位素示踪技术，阐明AOA和AOB对于咸水滴灌棉田土壤硝化作用的相对贡献以及不同灌溉水盐度和施氮量对棉田土壤氨氧化微生物丰度、群落结构和功能活性的影响，揭示干旱区咸水滴灌条件下土壤氨氧化微生物参与硝化作用的机理，为干旱区咸水资源的合理利用以及氮素的有效管理及调控氮素循环过程提供理论依据。

淡水短缺和灌溉水质盐化是干旱区农业可持续发展所面临的严峻问题，咸水灌溉会导致土壤盐分积累进而改变土壤理化和生物学性质，这将直接影响氮素转化过程。但是，咸水灌溉对土壤硝化反硝化微生物的影响还不清楚。因此，阐明长期咸水滴灌土壤氮素转化和关键微生物之间的关系对于促进干旱区咸水资源利用和土壤氮素管理具有重要的科学意义。研究表明：微咸水、咸水灌溉显著降低土壤NO3--N含量和潜在硝化势（PNR），但显著增加土壤盐分和NH4+-N含量。微咸水、咸水处理AOA和AOB amoA基因拷贝数均显著低于淡水处理。PNR与AOA和AOB的丰度均呈显著正相关关系。与淡水处理相比，咸水、微咸水处理显著增加AOA群落的香浓指数，咸水处理显著降低AOB群落的香浓指数。AOA和AOB群落的优势类群分别为Candidatus Nitrosocaldus和Nitrosospira；咸水、微咸水处理抑制AOA群落的Betaproteobacteria生长，而咸水处理中Candidatus Nitrosocaldus显著高于淡水和微咸水处理。AOB群落中Nitrosomonas 的相对丰度随着灌溉水盐度的增加而显著降低。Lefse分析显示，AOA在咸水灌溉下仅有1个差异物种，而AOB在微咸水灌溉时有5个差异物种。AOA群落结构的改变与土壤NO3--N，pH和盐度的变化密切相关，而AOB群落结构的改变仅与NO3--N和pH显著相关。盐分是影响氨氧化微生物生长及群落结构的主导因素，AOA和AOB共同参与土壤硝化作用，淡水、微咸水灌溉条件下AOB可能是硝化作用主导微生物种群，而咸水灌溉条件下AOA可能是主导微生物种群。此外，咸水灌溉处理显著抑制N2O排放，咸水灌溉和氮肥施用改变nirK、nirS、nosZ型反硝化细菌群落结构，灌溉水盐度对于反硝化细菌群落结构的影响要大于施肥。Lefse分析显示nirK、nirS、nsoZ型反硝化细菌差异物种随着灌溉水盐度的增加而增加，咸水灌溉显著改变反硝化细菌群落结构，导致优势种群数量增加。nosZ，nirK和nirS丰度的增加会促进N2O排放，长期咸水灌溉降低土壤N2O排放，但会导致土壤盐分的持续上升。研究结果可为进一步探讨干旱区农业土壤中硝化反硝化微生物对不同灌溉水盐度的响应机制及其在氮素转化中的作用提供一定科学依据。