【目的】基于水稻田间定位试验,利用nirS功能基因研究不同氮肥水平对稻田土壤反硝化细菌群落多样性的影响。【方法】运用PCR-DGGE(聚合酶链反应-变性梯度凝胶电泳)结合DNA克隆测序和荧光定量PCR(Real-time PCR)技术对反硝化细菌nirS基因进行检测,分析田间定位试验第4年不同氮肥水平下稻田nirS型反硝化细菌群落结构和丰度的变化。【结果】依据DGGE图谱计算的群落多样性指数显示,与不施肥对照处理(CK)比较,施用氮肥处理(N1:75 kg N.hm-2,N2:150 kg N.hm-2和N3:225 kg N.hm-2)可促进稻田土壤nirS型反硝化细菌群落多样性指数提高,尤其在水稻生长的齐穗期和成熟期后者均显著高于前者(P<0.05)。但群落多样性指数在N1、N2和N3处理间的差异主要表现在水稻分蘖期和齐穗期的表层土壤中,N3可显著高于N1(P<0.05)。冗余分析结果显示水稻生育时期对稻田土壤nirS型反硝化细菌群落结构的影响较大,表层和根层土壤的群落结构都与生育时期存在显著相关性(P=0.002,0.002);而不同氮肥水平对群落结构的显著性影响仅表现在稻田表层土壤中(P=0.002)。荧光定量PCR结果显示氮肥水平提高可促进稻田土壤nirS型反硝化细菌丰度增加,在水稻分蘖期和齐穗期内表层和根层土壤的nirS基因拷贝数均存在CK
Ammonia oxidation, the first and rate-limiting step of nitrification, is carried out by both ammonia-oxidizing bacteria （AOB） and ammonia-oxidizing archaea （AOA）. However, the relative importance of AOB and AOA to nitrification in terrestrial ecosystems is not well understood. The aim of this study was to investigate the effect of the nitrogen input amount on abundance and community composition of AOB and AOA in red paddy soil. Soil samples of 10-20 cm （root layer soil） and 0-5 cm （surface soil） depths were taken from a red paddy. Rice in the paddy was fertilized with different rates of N as urea of N1 （75 kg N ha＂ yr-1）, N2 （150 kg N ha~ yrl）, N3 （225 kg N ha1 yrl） and CK （without fertilizers） in 2009, 2010 and 2011. Abundance and community composition of ammonia oxidizers was analyzed by real-time PCR and denaturing gradient gel electrophoresis （DGGE） based on amoA （the unit A of ammonia monooxygenase） gene. Archaeal amoA copies in N3 and N2 were significantly （P〈0.05） higher than those in CK and N1 in root layer soil or in surface soil under tillering and heading stages of rice, while the enhancement in bacterial amoA gene copies with increasing of N fertilizer rates only took on in root layer soil. N availability and soil NO3--N content increased but soil NH4＋-N content didn＇t change with increasing of N fertilizer rates. Otherwise, the copy numbers of archaeal amoA gene were higher （P〈0.05） than those of bacterial amoA gene in root lary soil or in surface soil. Redundancy discriminate analysis based on DGGE bands showed that there were no obvious differs in composition of AOA or AOB communities in the field among different N fertilizer rates. Results of this study suggested that the abundance of ammonia-oxidizers had active response to N fertilizer rates and the response of AOA was more obvious than that of AOB. Similarity in the community composition of AOA or AOB among different N fertilizer rates indicate that the community composition of ammonia-o
