【作者】 张文学；

【导师】 何萍； 周卫；

【作者基本信息】 中国农业科学院 ， 植物营养学， 2014， 博士

【摘要】 氮肥在稻田易于损失，其当季利用率低。施用缓/控释肥是提高稻田氮肥利用率的有效途径，然而适宜的缓/控释肥料种类少。本文以脲酶抑制剂(N-丁基硫代磷酰三胺，NBPT)和硝化抑制剂(3,4-二甲基吡唑磷酸盐，DMPP)为实验对象，采用15N示踪的微区试验研究了生化抑制剂对稻田氮素损失的影响；采用田间试验研究了添加脲酶抑制剂时稻田氮肥的减施潜力、稻田适宜的脲酶抑制剂施用比例、脲酶抑制剂与硝化抑制剂配施下的土壤氮素供应特征；采用分子生态学方法研究了脲酶抑制剂与硝化抑制剂对土壤微生物群落结构以及功能微生物多样性的影响。研究取得以下进展：（1）15N示踪微区试验表明，添加NBPT或NBPT与DMPP配施可以显著提高水稻地上部氮素回收率和土壤残留氮量。与单施尿素处理相比，添加NBPT处理的氨挥发速率峰值降低27.04%，累积氨挥发损失量降低21.65%；NBPT与DMPP配施时，氨挥发速率峰值降低12.95%，累积氨挥发损失量降低13.58%；而添加DMPP时，氨挥发速率峰值增加23.61%，累积氨挥发损失量与单施尿素的差异不显著。对于降低稻田氨挥发损失，添加NBPT或NBPT与DMPP配施的两个处理效果较为理想，硝化抑制剂不宜单独添加。（2）研究发现，N2O排放通量主要集中在施肥后的7天内，添加DMPP显著降低了N2O的排放通量，但水稻生长后期排放通量剧增，尤其是添加抑制剂的处理增加幅度更大；添加DMPP或NBPT与DMPP配施可以显著减少施肥后21天内的N2O排放量，而对总排放量无明显影响。（3）大田试验表明，施氮量为135kg N/hm2时，NBPT添加比例小于1%时，增产不显著，而在1%~1.5%时可以显著提高籽粒产量以及地上部氮素回收率；稻田添加1%的NBPT时，施氮量为135kg N/hm2的籽粒产量最高，与农民习惯施氮（单施尿素180kg N/hm2）相比，早、晚稻分别增产8.54%和12.87%，氮肥利用率分别提高6.78%和9.46%，节约氮肥25%；研究发现，添加1%的DMPP则增产不显著；而NBPT与DMPP配施时，对于增产与提高氮素回收率的效果显著。（4）与单施尿素相比，添加NBPT时显著降低了分蘖期土壤中的脲酶活性与铵态氮含量，而显著提高了孕穗期土壤中的铵态氮含量，对此时的脲酶活性则无显著影响，可见基施的NBPT主要在孕穗期之前起作用；逐步回归分析发现，水稻分蘖期与孕穗期土壤中的铵态氮含量对水稻产量影响显著，而且孕穗期的影响大于分蘖期，因此，添加NBPT可以节约氮肥、显著增产的主要原因可能是其保持孕穗期较高的土壤铵态氮含量。（5）磷脂脂肪酸（phospholipid fatty acid，PLFA）分析表明，添加NBPT或DMPP显著减少了分蘖期土壤PLFA总量以及部分饱和脂肪酸和羟基脂肪酸含量，其中包括部分细菌的标记脂肪酸；然而在孕穗期，NBPT和DMPP的这种生化抑制作用不明显，而且NBPT与DMPP配施处理的PLFA总量显著高于其余处理的，可见两种生化抑制剂对微生物的影响主要集中在孕穗期之前。（6）利用定量PCR研究发现，尽管土壤氨氧化古菌（AOA）的amoA基因拷贝数是氨氧化细菌（AOB）的6~10倍，然而DMPP主要影响AOB的amoA基因丰度，AOA在分蘖期与孕穗期均保持相对稳定，表明DMPP主要通过限制AOB的生长来抑制稻田土壤硝化过程；DGGE（Denaturing Gradient Gel Electrophoresis，变性梯度凝胶电泳）图谱分析表明，亚硝化单胞菌和亚硝化螺菌均是施肥土壤中AOB的优势菌群，施用氮肥显著提高了土壤AOB与反硝化菌的群落结构多样性，NBPT和DMPP对AOB群落结构的影响小于施氮效应。更多还原

【Abstract】 Nitrogen (N) fertilizer is easily lost in paddy fields, leading to a low efficiency. The application ofslow/controlled release fertilizer is considered an effective measure to solve the problem. At present,however, the types of slow/controlled release fertilizer are scarce for paddy fields. Taking the ureaseinhibitor (N-(n-butyl) thiophosphric triamide, NBPT) and nitrification inhibitor (3,4-dimethylpyrazolePhosphate, DMPP) as the experimental materials, the effects of biological inhibitors on Ntransformation in paddy field was investigated using15N tracer method. We also studied the potential ofreducing N fertilizer application when urease inhibitor was present, and the reasonable proportion ofurease inhibitor to N fertilizer, and the characteristics of soil N supply when combined application ofthe urease inhibitor and nitrification inhibitor. In addition, the influences of NBPT and DMPP on soilmicrobial community and the diversity of the functional organisms involving N cycling are alsoexplored by using molecular ecology methods and techniques. The main findings are as follows:(1) The results of stable isotope15N-traced urea showed that the addition of NBPT or combinedaddition of NBPT and DMPP can significantly increased the recovery of applied N in the above-groundparts and soil residual N. Compared to the urea treatment, the peak of volatilization and cumulative lossof NH3from the treatment added with NBPT were significantly reduced by27.04%and21.65%,respectively, and those from the treatment added with combination of NBPT and DMPP were markedlydecreased by12.95%and13.58%, respectively. On the contrast，adding DMPP alone enhanced thepeak of NH3volatilization by23.61%, the NH3losses did not increase significantly. In summary, theeffects of urea added with urease inhibitor or combination of urease and nitrification inhibitors are betterthan urea added with nitrification inhibitor alone, in view of reduction of NH3volatilization in paddyfields.(2) The results showed that N2O flux was higher in the first7days after fertilization, and theapplication of DMPP markedly reduced the N2O flux. However, the flux sharply increased after63dayspost-fertilization at mature stage of rice, especially in the treatment where inhibitor was present. Thetwo treatments added with DMPP can significantly reduce the N2O emission in the first21days afterfertilization, but cannot change total emission.(3) The results of field experiment reveal that urea of135kg N/ha added with <1%NBPT did notmarkedly increase the yield, but the addition of NBPT ranged from1%to1.5%could significantlyimprove the rice yield and recovery of applied N in the above-ground parts. Compared with the normalurea rate of180kg N/ha, the grain yields are increased by8.54%and12.87%in early and late rice, therecovery of applied N improved by6.78%and9.46%, respectively, and25%of N fertilizer can besaved as a result. Our results showed that addition of DMPP (1%) did not markedly increase the yield.But combined application of NBPT and DMPP could significantly improve rice yield and nitrogenrecovery efficiency. (4) Compared to urea treatment, the addition of NBPT notably reduced urease activity and NH+4-Ncontent at tillering stage, but enhanced the NH+4-N content and maintained the a similar activity ofurease at booting stage, revealing that the role of urease inhibitor NBPT was active before booting stage.Stepwise regressions analysis revealed that grain yield of rice was significantly associated with soilNH+4-N content at tillering and booting stage, especially the latter. Therefore, the remarkableimprovement of soil NH+4-N content at booting stage seemed to a key factor which leaded to decreaseof N fertilizer by25%and a significant yield increase in the treatment added NBPT.(5) The PLFA analysis showed that addition of NBPT or DMPP significantly decreased the totalPLFA and content of saturated fatty acids and hydroxy fatty acids, some of which were bacteria markers.However, this inhibiting effect of biological inhibitors was not found at booting stage, and a higher totalPLFA was obversed when both NBPT and DMPP were added to urea. Thus, the influence of biologicalinhibitors on microbial community mainly focused on the period before booting stage.(6) The result of quantitative PCR showed that archaeal amoA gene copy numbers were moreabundant than AOB by6~10times. However, nitrification inhibitor (DMPP) mainly affected theabundance of AOB. The abundance and community of AOA were not affected by N fertilizer at bothtillering and booting stage. These data showed that DMPP inhibited the soil nitrification throughlimiting the growth of AOB. The profile of DGGE indicated that Nitrosomonas and Nitrosospira wasdominant flora of AOB in paddy soil. N fertilizer increased the diversity of AOB and denitrifyingbacteria. The effects of NBPT and DMPP on the community of AOB were smaller than that of Nfertilizer.更多还原