关键词: 尿素施用量, 氨氧化古菌, 土壤酸化, 冗余分析, 454测序

Abstract: 【Objective】 This paper aimed to investigate the characteristics of ammonia oxidizing archaeal (AOA) communities in black soil under long-term fertilization in Northeast China to identify the effects of different doses of urea on the abundance and structure of the AOA community. Combined with the soil physiochemical characteristics, the main driving factors would be revealed. This study provides evidence for further understanding of the nitrification process and mechanism in black soil and enhanced fertilization method.【Method】Based on a 34 year fertilization experiment in Heilongjiang Academy of Agricultural Sciences, three groups of samples were selected with different fertilization including samples without urea (without fertilizer CK and phosphorus-potassium PK), samples with one-time urea (one time nitrogen N₁, nitrogen-phosphorus NP, nitrogen-phosphorus- potassium NPK and nitrogen-potassium NK) and samples with two-time urea (two time nitrogen N₂). We applied 454 pyrosequencing and quantitative PCR of the Arch-amoA gene to analyze the effects of urea on the AOA communities in black soil. A correlation analysis was carried out to reveal the main important factors for determining the AOA community composition. 【Result】It was found that AOA Arch-amoA copy numbers in black soil were obviously reduced from 2.64×10⁷ to 8.34×10⁵ /g soil along with the increase of the urea amount, and pH in black soil was the direct reason for the decrease of the Arch-amoA gene. Both clustering and Nonmetric Multidimensional Scaling (NMDS) analyzed result proved that the AOA community structure in each fertilizer treatment with the same urea inputs was similar with each other, while the AOA community structure in fertilizer treatments with different urea addition was significantly different with each other. And a redundancy analysis indicated that soil pH, concentration of water soluble organic carbon and nitrate were the main environmental factors (P < 0.05) affecting the AOA community variation. What’s more, phylogenetic analysis demonstrated that the AOA in black soil in the Northeast of China belonged two categories Nitrososphaera and Nitrosotalea, 99.3% of Arch-amoA sequences in fertilizer treatment without urea and 90.1% of Arch-amoA sequences in fertilizer treatment with one-time urea addition belonged to Nitrososphaera, and 67.9% of Arch-amoA sequences with two-time urea addition belonged to Nitrosotalea.【Conclusion】Our research demonstrated the effects of long-term different fertilization on the ammonia oxidizing archaea community, and found out important factors for determining bacterial structures. AOA Arch-amoA copy numbers and communities in black soil with long term urea addition were strongly affected by different doses of urea. AOA diversity in one-time urea treatments increased while that in two-time urea treatment decreased. It is concluded that soil pH, water soluble organic carbon and nitrate nitrogen concentration were the main factors affecting AOA community variation. The results may be greatly meaningful to the rational utilization of fertilizer and agricultural sustainable development.

Key words: doses of urea addition, AOA, soil acidification, RDA, 454 pyrosequencing