摘要
摘要:为了研究长期定位施肥对棕壤中氨氧化细菌（ammonia-oxidizing bacteria，AOB）种群结构多样性和垂直分布特征的影响，本研究采用化学分析、荧光定量PCR（qPCR）和变性梯度凝胶电泳（PCR-DGGE）技术，针对沈阳农业大学试验区不同施肥方式（不施肥、低量无机氮肥、高量无机氮肥、无机氮肥与有机肥配施）下不同土壤深度（0~20 cm、20~40 cm、40~60 cm）的土壤理化性质、AOB丰度及种群多样性进行分析，比较不同施肥方式对土壤AOB种群的影响。结果显示，与不施肥相比，施肥会降低土壤pH，增加土壤铵态氮（70.5%~939.21%）和硝态氮（253.20%~625.48%）含量。随土壤深度增加，土壤pH升高，铵态氮和硝态氮含量除低量无机氮肥处理外，多呈降低趋势。土壤增施氮肥可提高AOB丰度，降低总细菌丰度。其中，0~20 cm土层中AOB丰度较高，且高量无机氮肥处理的AOB数量最高，为9.65×10⁵拷贝数·g^-1（干土）。DGGE图谱分析显示，不同处理下，AOB群落结构多样性指数存在明显差异（P < 0.05），各多样性指数均在表层（0~20 cm）最高，增施氮肥则显著降低AOB的多样性。聚类分析表明，4个施肥处理中，高量无机氮肥处理聚为一类，其他处理则因土壤深度不同而异；3个土壤深度中，除不施肥处理外，所有施肥处理均表现为0~20 cm、20~40 cm土层发生聚类，40~60 cm则明显与其他两层分开。冗余梯度分析（RDA）显示，硝态氮（P=0.027）是造成影响AOB群落结构差异的主要原因。上述研究结果表明，长期定位施肥土壤AOB的数量和群落结构多样性受施肥方式显著影响，并表现出明显的垂直分布特征。与无机氮肥相比，有机无机配施处理有助于改善土壤pH，维持不同土壤深度下AOB群落结构多样性。
关键词:棕壤/
施肥方式/
氮肥/
氨氧化细菌(AOB)/
群落结构/
土壤深度
Abstract:Studies about ammonia oxidizing bacteria (AOB) have mainly focused on the topsoil and little has remained known about community distribution in the subsoil. There therefore has remained the need to understand the impact of long-term fertilization on AOB abundance, community structure and vertical distribution in order to deepen the exploration of microbial mechanisms of nitrogen (N) transformation and to develop sound fertilization regimes for sustainable soil quality in the study area and beyond. Thus, a long-term (1987-2015) fertilization experiment was set up in the brown earth in Shenyang Agriculture University, Liaoning Province, China. Four treatments were set, no fertilizer (CK), low N fertilizer (N₂), high N fertilizer (N₄) and low N fertilizer plus organic mature (M₂N₂). Soil samples were collected at three different depths (0-20 cm, 20-40 cm and 40-60 cm). The soil physico-chemical properties, 16S rDNA and AOB-amoA gene abundance (real-time PCR, qPCR) and AOB community structure and diversity (denaturing gradient gel electrophoresis, PCR-DGGE) were investigated. While soil pH decreased, the content of soil ammonium N (NH₄⁺-N) increased by 70.5%-939.21% and that of nitrate N (NO₃^--N) by 253.20%-625.48% in the fertilization treatments over CK treatment. Also while soil pH increased, the contents of soil NH₄⁺-N and NO₃^--N decreased with increasing soil depth, except for N₂ treatment. The results of qPCR showed that fertilization treatments increased AOB abundance, but decreased total bacterial abundance compared to CK treatment. AOB amoA gene abundance was generally higher at the 0-20 cm than at the 20-40 cm and 40-60 cm soil layers. AOB abundance peaked in the N₄ treatment, with 9.65×10⁵ copies per g dry soil. The Shannon diversity (H), evenness (E_H) and richness (S) indexes of AOB from DGGE fingerprints responded increasingly significantly (P < 0.05) to fertilization regimes and soil-fertilization interactions with increasing soil depth. Although the tested diversity indexes were highest in the surface soil (0-20 cm), N fertilizer treatments (N₂, N₄ and M₂N₂) significantly reduced AOB diversity indexes. Based on cluster analysis of the DGGE fingerprints, AOB community structure in the soil varied with fertilization treatments and soil depth. Three soil depths of high N fertilizer (N₄) treatment was grouped together clearly. For other treatments, it was grouped according to soil depth with no discernible difference in AOB community structure among CK, N₂ and M₂N₂ treatments. The 0-20 cm and 20-40 cm deep soils under fertilizer treatments formed single cluster with no less than 57% similarity, while 40-60 cm soil layer formed another cluster. Redundant gradient analysis (RDA) further showed that NO₃^--N (P=0.027) was the key factor that shaped AOB community under different fertilization treatments. The results indicated that AOB number and community structure diversity after long-term fertilization significantly varied with fertilization treatment, and showed obvious vertical distribution characteristics. Compared with chemical fertilizer (N₂ and N₄) application, organic manure plus chemical fertilizer (M₂N₂) more favorably improved soil pH and maintained AOB community diversity in the subsoil.
Key words:Brown soil/
Fertilization regime/
Nitrogen fertilizer/
Ammonia oxidizing bacteria (AOB)/
Community structure/
Soil depth



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