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

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.