氯甲基吡啶对滴灌农田N2O排放的影响及硝化-反硝化关键微生物对其响应的分子生态机制

Elucidating the influence of nitrapyrin on soil N2O emmision flux and soil nitrification-denitrification process microbial community diversity will play a effective role for improving nitrogen resource utility efficiency as well as decreasing N2O emission. In the present research, soil simulation experiment and filed plot trail will be carried out. the effect of nitrapyrin on soil N2O emission would be study through assimilation box - GC and microelectrode. By means of Biolog method and soil enzymatic technique, the response of soil microbial functional diversity and the activities of soil Urease, nitrate reductase (NR), nitrite reductase（NiR）, Hydroxylamine reductase (HyR) on nitrapyrin would be measured. Using 15N-labeling and acetylene inhibition methods, the amount of N2O emission between soil nitrification and denitrification contribution would be quantified. Employing soil microbial molecular ecological technique of 16S rDNA/RNA-PCR-DGGE、qPCR、15N-SIP probe, the influence of nitrapyrin on soil microbial such as ammonia-oxidizing bacteria（AOB）and ammonia-oxidizing archaea (AOA) community component structure diversity could be analyzed. Meanwhile, the quantity relationship between soil N2O emission flux and soil nitrification-denitrification microbial community diversity would be studied. Our research results could exert a major role on using nitrapyrin to regulate soil nitrification-denitrification microbial community diversity in theory. Furthermore, its also could provide an effective way for increasing nitrogen resource utility efficiency and decreasing soil N2O emission technically in drip irrigation agriculture.

揭示氯甲基吡啶对土壤N2O排放通量和硝化-反硝化微生物多样性的影响是提高滴灌农田氮素养分资源高效利用和减少温室气体排放的有效措施。本研究通过土壤模拟和滴灌小区试验，采用静态箱-GC法与N2O微电极结合研究氯甲基吡啶对滴灌农田土壤N2O排放的影响；采用Biolog和酶学方法揭示土壤硝化-反硝化过程微生物代谢功能多样性以及与氮素转化相关酶活性对氯甲基吡啶的响应；采用乙炔-氧抑制法量化硝化和反硝化过程对N2O排放的贡献；利用16S rDNA/RNA-PCR-DGGE、qPCR及稳定性同位素探针技术等分子生态手段，分析氯甲基吡啶对AOB、AOA等关键微生物群落结构多态性作用的分子生态机制；厘清土壤硝化-反硝化微生物多样性与土壤N2O排放间的数量关系。研究结果可为今后滴灌农田运用氯甲基吡啶调控土壤硝化-反硝化微生物群落多态性，提高氮素养分利用效率和减少N2O排放提供理论参考与技术支撑。

为降低干旱区滴灌农田氮肥损失，减少温室气体排放，提高氮肥利用率，项目采用田间滴灌与模拟培养试验相结合的研究方式，旨在探明硝化抑制剂氯甲基吡啶应用于滴灌农田对土壤氮转化及其N2O排放的影响，以及对相关微生物的作用机制。主要研究结果如下：.1. 在干旱区滴灌农田，尿素随水滴施后7天内完成硝化，激发N2O排放。尿素添加氯甲基吡啶随水滴施在35天内能使硝化得到有效抑制，第7天硝化抑制率最高，平均硝化抑制率可达37.83%。氯甲基吡啶的添加使农田施肥季N2O累积排放量和排放系数分别降低14.28%和32.38%，氮肥利用率提高10.70%。在覆膜滴灌条件下，硝态氮对N2O排放的贡献要大于铵态氮。.2. 尿素的随水滴施提高了土壤微生物的代谢能力和强度、多样性和丰富度以及对各类碳源的利用，氯甲基吡啶的添加则提高了土壤微生物的代谢能力和强度、多样性和丰富度以及对大多数碳源的利用但影响不显著。尿素的随水滴施提高了土壤脲酶、羟胺还原酶、亚硝酸还原酶以及硝酸还原酶活性，氯甲基吡啶的添加虽然降低了脲酶和羟胺还原酶活性，增加了亚硝酸还原酶和硝酸还原酶活性但未产生显著影响。.3. 尿素的随水滴施提高了土壤氨氧化细菌(AOB)和氨氧化古菌(AOA)以及反硝化细菌的功能基因丰度，改变了其群落结构。氯甲基吡啶的添加抑制了AOB、AOA和反硝化细菌的基因丰度，改变了群落结构，但对AOB的基因丰度和群落结构的影响最为显著。在滴灌农田土壤中，硝化、反硝化微生物数量的增加均能促进N2O排放，NO3--N对氨氧化和反硝化微生物基因丰度的综合影响最为显著。.综上，在干旱区滴灌农田，尿素添加1%施氮量的氯甲基吡啶随水滴施能有效抑制硝化，显著降低N2O排放，对土壤总微生物群和氮转化关键酶活性并未产生显著影响。氯甲基吡啶主要通过抑制AOB的丰度和改变其群落结构来抑制硝化进而减低N2O排放，但不可低估其对反硝化微生物及其调控的N2O排放的影响。以上研究结果可为促进滴灌农田氮素养分资源高效利用、N2O减排与环境保护双重目标的实现提供理论依据与技术途径。