氮素肥料诱导黑土脱氮过程及其微生物群落结构和功能研究

The microbial denitrification induced by nitrogen fertilizers is one of the most important reason of N loss in black soil of Northeast China, in which N2O as a greenhouse gas involved in stratospheric ozone depletion was produced. Thus, there is growing concern about the study on the microbial denirification in cropland with black soil of Northeast China. As a popular strategy for exploring the ecological traits of denitrifying microorganism is, Polymerase Chain Reaction (PCR) method using genomic DNA as the template was widely used to target the nirK and nirS gene encoding the Nitrite Reductase. However, all of recent results have underestimated the abundance, diversity and functional importance of the denitrifying microorganism in cropland soil, because of the unexpressed nirK and nirS gene in genomic DNA and the insufficient coverage of the widely used primers. In this study, we established a soil microcosm for simulating the denitrification process in cropland field. Through the real-time PCR and Miseq high-throughput sequencing technique, we focused the variety of the seven phylogenetic nir clusters as described in previous study. Based on these results, we accurately and completely claried the the ecological traits of denitrifying microorganism inducing the denitrification in black soil applied with nitrogen fertilizers. It will be a valuable research which clarifying the N loss casued by denitrification and the related effects in black soil of Northeast China.

氮素肥料诱发的微生物脱氮过程产生的温室气体氧化亚氮（N2O），是东北黑土农田氮素流失的主要原因。因此，东北黑土农田微生物脱氮过程的研究日益受到关注。目前解析脱氮微生物生态特征的主要手段是以DNA为模板，通过PCR方法研究脱氮微生物编码亚硝酸还原酶的nirK和nirS基因。然而，由于基因组DNA可包含不表达的nirK和nirS基因，以及已有特异性引物对脱氮微生物种群覆盖率不足等原因，造成已有研究结果低估环境中脱氮微生物种群的丰度、多样性和功能性。本项目构建土壤微生态模拟体系模拟黑土农田中氮肥引起的脱氮过程。基于该体系，以基因组RNA和DNA为模板，结合定量PCR和Miseq高通量测序技术，以及申请人最新报道的8个nir基因系统发育类群的特异性引物，更全面和准确地解析氮肥施入后，黑土农田中主导脱氮过程的脱氮微生物种群特征。该研究对揭示黑土农田脱氮引起的氮素流失过程及其影响因素具有重要学术价值。

中国黑土区农田土壤肥沃，富含有机质和营养物质，是中国大豆、玉米和小麦等作物的主产区。然而，迫于中国粮食安全的压力，大量施用化学肥料已经成为黑土农田一种常态的农业措施。长时间的施用化学肥料造成了黑土农田耕作层土壤有机质含量下降、团粒结构破坏、蓄水保肥能力下降、最终导致土壤肥效作用降低。为了黑土农田的可持续利用，在施用化学肥料的基础上额外配施有机质，比如粪肥，已经逐渐成为一种较为流行的、被鼓励的施肥措施。近年来，人们开始认识到这种化肥配施有机质的方式尽管有效的增加了作物的产量、保护了土壤肥力，但却附带产生了大量的氧化亚氮。为了解析明确产生这些响应化肥配施有机肥措施以及有机肥配施措施释放的N2O的微生物代谢途径和类群，我们通过构建实验室级的soil microcosms来进行模拟黑土农田氮素肥料施入后的N2O排放，并利用抗生素抑制和基于亚硝酸盐还原酶基因（nirK）的克隆文库方法进行了微生物代谢途径和类群的解析。soil microcosm模拟实验结果表明黑土在施入氮素肥料后N2O出现峰值的时期较为集中，其中有机肥的配施会加速土壤N2O的释放。抗生素抑制试验结果表明微生物反硝化作用主导了黑土区农田响应氮素化肥或化肥配施有机质措施的N2O排放。其中，反硝化细菌、尤其是具有nirK-II型功能基因的细菌是响应氮素肥料N2O释放的主要微生物类群。而有机质在农田土壤表面的配施可以促进真菌反硝化作用并加大N2O的释放量。克隆文库实验结果表明供试黑土农田土壤中具有nirK基因的镰孢菌属真菌，尤其是尖镰孢菌响应了土壤配施有机质措施的N2O产生者。研究结果有助于解析中国黑土农田N2O形成和释放机制，为合理进行施肥管理提供参考。