Community structure of ammonia-oxidizing bacteria under long-term application of mineral fertilizer and organic manure in a sandy loam soil

Abstract

The effects of mineral fertilizer (NPK) and organic manure on the community structure of soil ammonia-oxidizing bacteria (AOB) was investigated in a long-term (16-year) fertilizer experiment. The experiment included seven treatments: organic manure, half organic manure N plus half fertilizer N, fertilizer NPK, fertilizer NP, fertilizer NK, fertilizer PK, and the control (without fertilization). N fertilization greatly increased soil nitrification potential, and mineral N fertilizer had a greater impact than organic manure, while N deficiency treatment (PK) had no significant effect. AOB community structure was analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of the amoA gene, which encodes the alpha subunit of ammonia monooxygenase. DGGE profiles showed that the AOB community was more diverse in N-fertilized treatments than in the PK-fertilized treatment or the control, while one dominant band observed in the control could not be detected in any of the fertilized treatments. Phylogenetic analysis showed that the DGGE bands derived from N-fertilized treatments belonged to Nitrosospira cluster 3, indicating that N fertilization resulted in the dominance of Nitrosospira cluster 3 in soil. These results demonstrate that long-term application of N fertilizers could result in increased soil nitrification potential and the AOB community shifts in soil. Our results also showed the different effects of mineral fertilizer N versus organic manure N; the effects of P and K on the soil AOB community; and the importance of balanced fertilization with N, P, and K in promoting nitrification functions in arable soils.

FIG. 1.

Soil nitrification potentials under long-term application of mineral fertilizer and organic manure. Vertical T bars indicate standard deviations. Bars topped by the same letter indicate a significant difference in values (P < 0.05).

FIG. 2.

Comparison of DGGE banding patterns obtained using five different reverse primers: TC, amoA-2R-TC; TG, amoA-2R-TG; GC, amoA-2R-GC; GG, amoA-2R-GG; and 2R, amoA-2R. See the text for explanation of primer names.

FIG. 3.

DGGE analysis of amoA fragments retrieved from soils after long-term application of mineral fertilizer and organic manure. The excised bands are numbered.

FIG. 4.

Phylogenetic tree based on partial amoA sequences (150 amino acids) retrieved from the DGGE bands (shown in bold) in this study and the DNA Data Bank of Japan. The accession number for each sequence is enclosed in brackets. The scale bar indicates two changes per 100 amino acid positions.