大豆连作条件下施肥对东北黑土细菌群落的影响

Effects of Fertilization on Bacterial Community Under the Condition of Continuous Soybean Monoculture in Black Soil in Northeast China

【目的】表征大豆连作条件下不同施肥处理土壤细菌的群落结构特征和组成差异,并侧重分析接种根瘤菌处理的不同之处;与土壤化学性质进行关联分析,探讨引起黑土细菌菌群变化的主效环境因子,为进一步了解连作条件下东北耕地土壤中细菌群落结构的变化以及大豆的高效种植和氮肥减施提供理论支持。【方法】依托5年大豆连作定位试验,选取不施肥(CK)、磷钾肥(PK)、氮磷钾肥(NPK)、磷钾肥+接种根瘤菌(Bradyrhizobium japonicum 5821)处理(PK+5821)共4个处理的耕层土壤为研究对象,采用高通量测序(Illumina HiSeq)和real-time PCR技术,以16S rRNA基因V4区为分子标靶,解析不同施肥处理土壤细菌的菌群变化,并对细菌群落结构与环境因子进行相关性分析。【结果】与CK相比,施肥明显增加了大豆的产量和土壤养分的含量,但单施化肥降低了土壤的pH。接种B.japonicum 5821显著增加了土壤细菌的基因拷贝数,提高了土壤细菌的丰度。细菌门水平和纲水平的群落分析发现,变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和酸杆菌门(Acidobacteria)为土壤中的3大优势菌群,占所有优势菌门的70%以上;施肥明显降低了土壤中放线菌门的相对丰度,这与细菌纲水平的分析一致。多样性分析发现,CK处理与3个施肥处理的丰富度和多样性指数不同,且主坐标分析(PCoA)显示,3个施肥处理的细菌群落结构在PC1轴上聚在一起,而与CK处理是分开的,表明施肥明显改变了土壤细菌的群落构成。冗余分析(RDA)显示,全氮(F=3.2,P=0.002)对土壤细菌群落结构的影响最大,解释了24%的群落变化,各因子的贡献率依次为全氮>有效磷>速效钾>有机质>p H;Spearman相关性分析也表明,5项土壤化学指标均与不同优势菌门存在密切的相关关系。【结论】施肥改变了大豆连作条件下土壤细菌的群落结构。全氮是影响土壤细菌群落结构变化的主效环境因子。接种根瘤菌明显提高了大豆产量,同时保持了良好土壤化学性状和土壤菌群结构,很大程度地减少了化学氮肥的施用,对大豆的高效种植和氮肥减施具有重要意义。

[ Objective ] The objective of this study is to investigate the effects of fertilization on structure and composition of bacterial community in soybean continuous cropping in Chinese Mollisols, and the main environmental factors causing changes of bacterial communities, focusing on the analysis of the differences in the treatment of inoculated nodules. This study would provide a theoretical support for the efficient planting of soybean and the reduction of nitrogen fertilizer. [ Method] Based on a 5-year fertilization experiment of soybean continuous cropping, four different fertilization treatments were sampled, including CK （no fertilizer）, PK （phosphorus and potassium inorganic fertilizer）, NPK （nitrogen, phosphorus and potassium inorganic fertilizer） and PK＋5821 （phosphorus and potassium inorganic fertilizer with Bradyrhizobiumjaponicum 5821）. High-throughput sequencing （Illumina Hiseq） and quantitative PCR of 16S rRNA gene were used to analyze bacterial communities in soil samples in different fertilization treatments. Correlation analysis of the bacterial community structure and environmental factors was followed. [Result] Compared with CK, fertilization significantly increased soybean yield and concentration of soil nutrients, it was found that single application of chemical fertilizer decreased soil pH. Inoculation with B. japonicum5821 significantly increased the gene copy number of soil bacteria, and soil bacterial abundance. The analysis of bacterial communities at phylum and class level showed that Proteobacteria, Actinobacteria and Acidobacteria were dominant bacteria, occupying more than 70% of all phyla; Fertilization significantly reduced the relative abundance of Actinobacteria in the phylum analysis, which is consistent with the level of class. Diversity analysis showed that the indexes （richness and diversity） of CK treatment were different from those in other three treatments. The principal coordinate analysis （PCoA） showed that the bacterial community structure in the 3 fertilization treatments （PK, NPK and PK＋5821） clustered together on the PC1 axis, and the CK treatment was separated, which indicated that fertilization significantly changed the soil bacterial community composition. Redundancy analysis （RDA） showed that total nitrogen （F=3.2, P=0.002） had the greatest effect on the soil bacterial community structure, explaining 24% of the community changes. The order of contribution rate was total nitrogen 〉 available phosphorus 〉 available K〉 organic matter〉 pH. Spearman correlation analysis also showed that all the five soil physiochemical characteristics were closely related with different dominant bacteria. [ Conclusion] Fertilization changed the bacterial community structure in continuous soybean cropping, and total nitrogen was the main environmental factor impacting the bacterial abundances and communities. Inoculation with B. japonicum 5821 could not only significantly improve soil quality and increase soybean yield, but also reduce the application of chemical nitrogen fertilizer. This study could provide important information for the efficient planting of soybean and the reduction of nitrogenous fertilizer.