• ISSN 1008-505X
  • CN 11-3996/S
张文学, 王少先, 夏文建, 孙刚, 刘增兵, 李祖章, 刘光荣. 脲酶抑制剂与硝化抑制剂对稻田土壤硝化、反硝化功能菌的影响[J]. 植物营养与肥料学报, 2019, 25(6): 897-909. DOI: 10.11674/zwyf.18237
引用本文: 张文学, 王少先, 夏文建, 孙刚, 刘增兵, 李祖章, 刘光荣. 脲酶抑制剂与硝化抑制剂对稻田土壤硝化、反硝化功能菌的影响[J]. 植物营养与肥料学报, 2019, 25(6): 897-909. DOI: 10.11674/zwyf.18237
ZHANG Wen-xue, WANG Shao-xian, XIA Wen-jian, SUN Gang, LIU Zeng-bing, LI Zu-zhang, LIU Guang-rong. Effects of urease inhibitor and nitrification inhibitor on functional nitrifier and denitrifier in paddy soil[J]. Journal of Plant Nutrition and Fertilizers, 2019, 25(6): 897-909. DOI: 10.11674/zwyf.18237
Citation: ZHANG Wen-xue, WANG Shao-xian, XIA Wen-jian, SUN Gang, LIU Zeng-bing, LI Zu-zhang, LIU Guang-rong. Effects of urease inhibitor and nitrification inhibitor on functional nitrifier and denitrifier in paddy soil[J]. Journal of Plant Nutrition and Fertilizers, 2019, 25(6): 897-909. DOI: 10.11674/zwyf.18237

脲酶抑制剂与硝化抑制剂对稻田土壤硝化、反硝化功能菌的影响

Effects of urease inhibitor and nitrification inhibitor on functional nitrifier and denitrifier in paddy soil

  • 摘要:
    目的 在农业生产中,脲酶抑制剂 (urease inhibitor,UI) 与硝化抑制剂 (nitrification inhibitor,NI) 常作为氮肥增效剂来提高肥料利用率。本文研究了在我国南方红壤稻田施用脲酶抑制剂与硝化抑制剂后,土壤中氨氧化细菌 (ammonia oxidizing bacteria,AOB)、氨氧化古菌 (ammonia-oxidizing archaea,AOA) 以及反硝化细菌的丰度以及群落结构的变化特征,旨在揭示抑制剂的作用机理及其对土壤环境的影响。
    方法 试验在我国南方红壤稻田进行,共设5个处理:1) 不施氮肥 (CK);2) 尿素 (U);3) 尿素 + 脲酶抑制剂 (U + UI);4) 尿素 + 硝化抑制剂 (U + NI);5) 尿素 + 脲酶抑制剂 + 硝化抑制剂 (U + UI + NI),3次重复。脲酶抑制剂与硝化抑制剂分别为NBPTN-(n-butyl) thiophosphrictriamide,N-丁基硫代磷酰三胺和DMPP (3,4-dimethylpyrazole phosphate,3,4-二甲基吡唑磷酸盐)。通过荧光定量PCR (Real-time PCR) 研究水稻分蘖期与孕穗期抑制剂对三类微生物标记基因拷贝数的影响,并分析土壤铵态氮、硝态氮与三种菌群丰度的相关性;利用变性梯度凝胶电泳 (Denaturing Gradient Gel Electrophoresis,DGGE) 分析抑制剂对土壤AOB、AOA以及反硝化细菌群落结构的影响,并对优势菌群进行系统发育分析。
    结果 1) 荧光定量PCR结果表明,施用氮肥对两个时期土壤中AOB的amoA基因与反硝化细菌nirK基因的拷贝数均有显著提高,而对AOA的amoA基因始终没有明显影响;AOB与nirK反硝化细菌的丰度与两个时期的铵态氮含量、分蘖期的硝态氮含量呈极显著正相关,与孕穗期的硝态氮含量相关性不显著;DMPP仅在分蘖期显著减少了AOB的amoA基因拷贝数,表明DMPP主要通过限制AOB的生长来抑制稻田土壤硝化过程;NBPT对三类微生物的丰度无明显影响;2) DGGE图谱表明,在分蘖期与孕穗期,施用氮肥均明显增加了图谱中AOB的条带数,而对AOA却没有明显影响;氮肥明显增加了孕穗期反硝化细菌的条带数;与氮肥的影响相比,抑制剂NBPT与DMPP对AOA、AOB以及反硝化菌的群落结构影响甚微;系统发育分析结果表明,与土壤中AOB的优势菌群序列较为接近的有亚硝化单胞菌和亚硝化螺菌。
    结论 在南方红壤稻田中,施入氮肥可显著提高AOB与反硝化细菌的丰度,明显影响两种菌群的群落结构,而AOA较为稳定;NBPT对三类微生物的群落结构丰度无明显影响;硝化抑制剂DMPP可抑制AOB的生长但仅表现在分蘖期,这可能是其缓解硝化反应的主要途径;这也说明二者对土壤生态环境均安全可靠。

     

    Abstract:
    Objectives Urease inhibitor (UI) and nitrification inhibitor (NI), as nitrogen synergist, are used to improve the efficiency of nitrogen (N) fertilizer in agriculture. The aim of this study was to investigate the effect of UI and NI on the abundance and community structure of ammonia oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and denitrifying bacteria in paddy soil, and reveal the synergy mechanism of the inhibitors and their effects on soil environment.
    Methods A field experiment was conducted in a paddy field of red soil in southern China. A randomized block design with five treatments and three replicates was as follows: 1) CK (no N control), 2) U (Urea), 3) U + UI, 4) U + NI, 5) U + UI + NI. The urease inhibitor and nitrification inhibitor were NBPT N-(n-butyl) thiophosphrictriamide and DMPP (3,4-dimethylpyrazole phosphate) respectively. The abundance and composition of AOB, AOA and denitrifying bacteria were investigated by fluorescent quantitative real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches based on amoA and nirK genes, at tillering stage and booting stage, respectively. Then, dominant populations was sequenced and phylogenetic analyzed.
    Results 1) Real-time PCR analysis showed that N fertilizer remarkably increased the gene copy numbers of amoA in AOB and nirK in denitrifying bacteria in soil at both stages, respectively. But this effect was not observed in the abundance of AOA all the time. Moreover, the abundance of AOB and denitrifying bacteria was very significantly (P < 0.01) related to the content of NH4+-N at both stages and NO3--N at tillering stage. DMPP mainly decreased the abundance of AOB but not AOA at tillering stage, which revealed that DMPP inhibited the soil nitrification by limiting the growth of AOB. Little changes in the abundance of AOB, AOA and denitrifying bacteria were detected among the treatments added with NBPT. 2) The profile of DGGE fingerprint indicated that, N fertilizer notably increased the band numbers in fingerprint of AOB but not affected that of AOA at both stages. In addition, N fertilizer obviously enhanced the band number in fingerprint of denitrifying bacteria at booting stage. The effects of NBPT and DMPP on the community structures of AOB, AOA and denitrifying bacteria were too little to be observed. Phylogenetic analysis indicated that the sequences of Nitrosomonas and Nitrosospira were more similar to dominant microflora of AOB in paddy field.
    Conclusions The abundance of AOB and denitrifying bacteria was distinctly improved by N fertilizer, and the community structures of both bacteria were sensitive to N fertilizer in paddy field, but AOA was stable, in red soil area of Southern China. The effect of urease inhibitor NBPT on the abundance and community structure of AOA, AOB and denitrifying bacteria was little. The nitrification inhibitor DMPP limited the growth of AOB at tillering stage, while the effect was negligible at booting stage. It may be the main way to alleviate nitrification. It is estimated that both NBPT and DMPP are harmless to the soil environment.

     

/

返回文章
返回