长期施肥酸性旱地土壤硝化活性及自养硝化微生物特征

构建微域培养结合梯度凝胶电泳(DGGE)、Illumina MiSeq高通量测序、生物信息学分析等分子生态学技术,以不施肥土壤为对照(CK),研究长期施化肥(NPK)和有机肥(OM)对酸性旱地土壤硝化活性及自养硝化微生物群落的影响,并认知其与土壤理化因子间的关系.结果表明,施化肥和有机肥显著提高土壤有机碳和无机氮含量,施有机肥提高土壤pH和总氮含量、降低C/N;供试土壤自养硝化作用占据主导(73.60%~85.32%),施肥显著提升土壤自养硝化活性,且施有机肥提升效果更为明显;微域培养后,OM土壤氨氧化古菌(AOA)和细菌(AOB)amoA基因绝对丰度及16S rRNA基因相对丰度显著上升,而CK和NPK土壤仅AOA相对丰度显著上升,即3种土壤AOA均有明显活性(主要类群为Nitrososphaera,99.30%),而AOB仅在OM土壤有活性(主要类群为Nitrosospira,99.99%),另外还发现OM土壤中亚硝酸盐氧化细菌(NOB)有较强活性(主要类群为Nitrospira,96.69%);逐步回归分析显示自养硝化活性显著受总氮含量影响,AOA和AOB amoA基因丰度分别受有机碳含量和pH影响,Nitrososphaera相对丰度与NO_3~--N含量显著正相关,而Nitrosospira和Nitrospira相对丰度则与C/N显著负相关.可见,长期施肥后土壤总氮含量的提升显著刺激自养硝化活性;以Nitrososphaera为主的AOA在酸性旱地土壤硝化作用中发挥了重要作用,施有机肥土壤pH上升及C/N下降刺激了Nitrosospira(AOB)生长,从而改变了酸性旱地土壤中活跃的自养硝化微生物类群.

Nitrification Activity and Autotrophic Nitrifiers in Long-term Fertilized Acidic Upland Soils

Soil microcosm incubation, molecular ecology techniques including denaturing gradient gel electrophoresis and Illumina MiSeq high-throughput sequencing, and bioinformatics analysis were carried out to investigate the effect of long-term fertilization with chemical fertilizers (NPK) and organic manure (OM) on soil nitrification activity and the autotrophic nitrifying communities in acidic upland soils. No fertilization soil (CK) was the control. Relationships between soil nitrification activities, autotrophic nitrifying communities, and soil characteristics were further evaluated. Long-term fertilization significantly increased the soil organic carbon and inorganic nitrogen contents. Fertilization with organic manure significantly increased soil pH and total nitrogen contents, but decreased soil C/N. Autotrophic nitrification dominated soil nitrification, and accounted for 73.60%-85.32% of total nitrification. Fertilization significantly increased soil autotrophic nitrification activity and the highest value was observed in the OM soil. During the microcosm incubation, the absolute abundances of amoA genes and the relative abundances of 16S rRNA genes of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the OM soil significantly increased. The relative abundances of 16S rRNA genes of the AOA greatly increased in both CK and NPK soils. These results suggested the remarkable activity of AOA in the three soils (the predominant population was Nitrososphaera,>99.30%) and implied that AOB was active in the OM soil (the predominant population was Nitrosospira,>99.99%). We also found the activity of nitrite-oxidizing bacteria (NOB) in the OM soil, and the predominant population was Nitrospira (>96.69%). Stepwise regression analysis demonstrated that soil autotrophic nitrification activity was significantly affected by soil total nitrogen content, whereas the abundances of archaeal and bacterial amoA genes were significantly affected by soil organic carbon content and soil pH, respectively. We also found significant positive correlation between the relative abundance of Nitrososphaera and soil nitrate content and a negative correlation between the relative abundance of Nitrosospira and Nitrospira with soil C/N. Overall, our results showed that long-term fertilization greatly increased soil nitrification activity and altered the autotrophic nitrifying communities in acidic upland soils. Soil autotrophic nitrification activity was significantly stimulated by soil total nitrogen content. The Nitrososphaera group played a critical role in nitrification of acidic upland soils. The increased soil pH and decreased soil C/N stimulated the growth of Nitrosospira.