施肥与土壤改良对农田土壤中全程氨氧化细菌种群及功能的影响

Complete ammonia oxidizing bacteria (Comammox) was firstly reported in Nature Journal in the year 2015, which is a major breakthrough in the global nitrogen cycle research. However, the ecological function of Comammox in the farmland soil was still unclear. This project based on the hypothesis that the farmland management can influence the nitrification microorganism population and its relative contribution to the nitrification process. Stable isotope nucleic acid probe (DNA-SIP), quantitative PCR and high-throughput sequencing methods were used and the key functional genes of nitrification were chosen as the molecular targets. The research contents focused on the following three aspects: (1) the study on the effect of fertilization regime on the population and functional activity of Comammox, which will reveal the community response and controlled mechanism of Comammox to the changes of soil physical and chemical properties; (2) the study on clarifying the relative contribution rate of different nitrification microorganisms to N2O, which will reveal the coexistence relationship between Comammox and traditional nitrifying microorganism; (3) the community evolution characteristics of nitrifying microorganisms in the transformation process of the red soil nutrient condition form infertile to fertile will be explained by the long-term fertilizing experiment. It is expected that the results of the study will elucidate the community adaptability and ecological function of Comammox under the influence of fertilization regimes and soil improvement measures and provide theoretical support for regulating greenhouse gas emission and realizing sustainable agricultural development.

2015年，Nature上首次报道了全程氨氧化细菌（Comammox）的发现，是全球氮循环研究的重大突破。然而，农田土壤中Comammox的生态学功能仍不清楚。围绕农田管理措施影响硝化微生物种群及其对硝化过程的相对贡献这一科学问题，采用稳定性同位素核酸探针（DNA-SIP）、定量PCR、高通量测序等技术手段，以硝化过程关键功能基因为分子标靶，重点研究（1）施肥模式对Comammox种群和功能的影响，揭示其对土壤理化性质变化的群落响应和受控机制；（2）明确不同硝化微生物对N2O产生的相对贡献率，揭示Comammox与传统硝化微生物的共存关系；（3）利用红壤改良试验，阐释土壤养分由贫瘠向良好转化过程中硝化微生物的群落演变特征。预期研究结果将从种内与种间层面阐明Comammox在农田施肥模式和改良措施影响下的群落适应性和生态功能，为调控农田温室气体排放及实现农业可持续发展提供理论支撑。

Comammox同时具有氨氧化和亚硝酸盐氧化功能，且不同种属具有特异的生态位分化特性，因此研究其与传统硝化微生物在农田管理措施影响下的共存关系及其对温室气体N2O排放的相对贡献十分必要。项目首先系统地分析了长期施肥对不同类型、不同土层中AOA、AOB、NOB和Comammox等功能微生物群落丰度、组成结构和功能活性的影响；然后采用抑制剂与15N稳定同位素示踪技术相结合，定量化区分硝化过程、硝化细菌反硝化、异养反硝化和联合反硝化等过程对N2O产生的相对贡献及相关微生物的群落响应特征；最后重点关注Comammox在贫瘠红壤改良下的群落组成及分布，并利用DNA-SIP揭示氮素添加对贫瘠红壤Comammox及硝化微生物的功能活性的影响。主要研究结果如下：（1）施肥模式和土层可显著影响土壤中AOA、AOB、NOB和Comammox等功能微生物群落丰度、组成结构和功能活性，但在潮棕壤、红壤和黑土中影响程度不同；（2）土壤pH值、NH4+-N和有机质含量是影响土壤Comammox群落组成的关键因素；（3）土壤中的Comammox Nitrospira主要包括cladeA和cladeB两类，其具有不同的生理代谢特性，在不同施肥处理和土层中占据不同生态位，并在土壤氨氧化过程中发挥重要作用；（4）异养反硝化是潮棕壤土壤N2O排放的主要贡献途径，有机肥M处理中最高贡献可达98.7%以上，与土壤中nirK、nirS和nosZ等基因丰度和群落组成相关，且受土壤温度、C/N等环境要素显著影响；（5）施用化肥加剧土壤酸化，提高了真菌反硝化在N2O排放中的相对贡献；（6）土壤改良措施显著影响土壤理化性质及酶活性。与AOA不同，AOB和Comammox群落组成受改良措施影响发生明显聚类，其中化肥加石灰的NPKL处理显著不同于其它改良措施；（7）DNA-SIP结果显示在氮素缺乏的贫瘠红壤中AOA在氨氧化作用中起关键作用。而当补充氮素后，土壤中AOB的活性被激发，发挥重要作用。当氮素和乙炔同时添加后，AOB受到抑制，但是Comammox cladeA的活性被激活。本研究拓宽了对农田生态系统中Comammox分布特性及生态功能的认识，为农田N2O减排及合理施肥研究奠定理论基础。