长期施用氮肥对土壤细菌硝化基因多样性及组成的影响

Effect of Long-Term Application of Nitrogen Fertilizer on the Diversity of Nitrifying Genes (amoA and hao) in Paddy Soil

以中国科学院桃源农业生态试验站水稻长期定位试验为平台(28°55′49.8″N,111°26′25.7″E),运用PCR、克隆文库构建等分子生物学技术研究长期单施氮肥(尿素)对亚硝化基因(amoA和hao)多样性及其群落结构的影响.结果表明,长期单施氮肥使amoA基因多样性降低(Shannon指数减少了11%),而hao基因多样性几乎不受影响,对amoA优势基因型的影响大于对hao基因的影响;长期施用氮肥使amoA和hao基因的菌群组成分别发生了显著性(p=0.02)和极显著性变化(p=0.002).系统发育分析表明amoA基因主要与未经培养的氨氧化细菌基因相似,相似率主要集中在86%～99%,可能主要来自亚硝化螺菌属;而hao基因主要与Silicibacteria、亚硝化螺菌属(Nitrosospira)和甲基球菌属(Methylococcus)相似,相似率在66%～80%,长期施用氮肥使hao基因主要集中在α-Proteobacteria纲且与Silicibacteria有较近的亲缘关系.总体来说,长期单施氮肥使水稻土中亚硝化基因amoA的多样性降低,使amoA与hao的群落组成发生显著变化.

The aim of this study was to determine the effect of long-term（16 years） application of nitrogen fertilizer on the diversity of nitrifying genes（amoA and hao） in paddy soil on the basis of long-term paddy field experimental station（started in 1990） located in Taoyuan,with the molecular approaches of PCR,constructing libraries and sequencing.The fertilizer was urea and no fertilizer was as control.The Shannon index showed that long-term application of nitrogen fertilizer made the diversity of amoA gene descend while no effect on the diversity of hao gene.The LIBSHUFF statistical analyses demonstrated that both amoA and hao libraries of CK and N treatments were significantly different from each other and the rarefaction curves of libraries failed to meet the plateaus indicating that there were lots kinds of genes haven＇t been detected.The results of blasting with GenBank and the phylogenetic tree showed that the amoA genes detected in our study had a similarity with the uncultured gene of amoA,which showed some similar to Nitrosospira.Otherwise,the hao genes cloned showed a relationship to the genes of cultured bacteria such as Silicibacteria,Nitrosospira and Methylococcus,and the hao genes found in the N treatment dominated in α-Proteobacteria.These results suggest that long-term fertilization of nitrogen had significant impacts on the diversity or community of amoA and hao genes.