长期施肥和温度对酸性旱地土壤硝化微生物和总细菌群落的影响

发布时间：2018-08-06 19:21

【摘要】：我国酸性土壤总面积高达2×108hm2,约占全国土壤面积的23%,其中一半以上土壤为农业土壤或具有潜在的农业价值,随着人类对土地利用效率和农业经济效益的需求增加,农业生产伴随着大量肥料的投入,但经济作物对氮肥的利用率却普遍低于40%,氮肥的过量投入导致土壤氮循环失衡,加剧了土壤酸化,降低了农业经济效益。硝化作用是土壤氮循环的重要过程,由硝化微生物驱动,其中氨氧化过程是硝化作用的限速步骤。然而,氨氧化微生物在酸性条件下对土壤硝化作用的影响尚存争议,土壤硝化作用强度及氨氧化微生物对其的相对贡献受多个环境因子的综合调控,长期施肥酸性旱地土壤的土壤硝化作用及其功能微生物的季节演变尚不明确,此外,长期施用无机肥和有机肥对酸性旱地土壤总微生物群落多样性影响也不尽相同。因此本研究选用江西进贤红壤研究所长期定位施肥30年酸性旱地土壤为研究材料,选用(1)不施肥(CK)、(2)施氮磷钾无机肥(NPK)、(3)施腐熟猪粪有机肥(OM)三种处理,构建实验室微域培养实验与原位土壤分析实验,通过添加硝化抑制剂的短期培养并联用定量PCR (Quantitative PCR,qPCR)、变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis,DGGE)及 Illumina MiSeq 高通量测序等分子生态学技术,分析培养过程中发挥硝化活性的、且占据活性优势的硝化微生物类群,探究自养硝化微生物群落及活性特征并认知其与土壤理化因子间的关系,明确长期施肥酸性旱地土壤硝化活性的季节演变特征以及酸性旱地土壤中氨氧化古菌(Ammonia-oxidizing archaea,AO A)和细菌(Ammonia-oxidizing bacteria, AOB)在不同培养温度下对氨氧化过程的相对贡献,研究长期施无机肥和有机肥对酸性旱地土壤中总微生物群落多样性的影响,比较二者影响的异同,分析影响群落变化的主要环境因子。结果显示,施肥对土壤理化性质有显著影响,供试土壤自养硝化作用占总硝化速率的73.60%～85.32%,施肥显著提升土壤自养硝化活性,其中施有机肥提升效果更为明显;OM 土壤氨AOA和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与总氮含量显著正相关,AOA和AOB amoA基因丰度分别受有机碳含量和pH的显著影响,Nitrososphaera相对丰度与NO3--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。在4 ℃～23 ℃ 土壤硝化活性较低,CK、NPK和OM土壤的最高土壤硝化速率分别为 2.29 mg·(kg· d)-1、6.00 mg·(kg· d)-1 和 11.58 mg·(kg· d)-1,而在夏季23 ℃～35 ℃ 土壤硝化活性最高,三种土壤的最高土壤硝化速率分别为5.19 mg·(kg·d)-1、7.62 mg·(kg·d)-1 和 14.25 mg·(kg·d)-1。CK、NPK 和 OM 土壤中 AOB贡献的自养硝化作用分别在28 ℃、18 ℃和18 ℃达到峰值,AOA贡献的自养硝化作用均在35 ℃达到峰值。随着温度的上升,AOA对自养硝化作用的贡献逐渐上升并占据主导,施无机肥和有机肥未改变土壤AOA的对温度的响应,但AOB活性最高温度显著降低。C8H14作为土壤硝化抑制剂其抑制效果与施加浓度正相关,选用0.5%的添加浓度作为区分CK和NPK 土壤中AOA和AOB对土壤硝化作用的相对贡献时效果最佳。相较于长期施化肥,长期施有机肥显著改变了酸性红壤的细菌群落组成和多样性。ABT (Aggregated Boosted Tree)分析发现土壤pH是细菌群落的总重要的环境因子。此外,施有机肥显著改变了例如代谢和遗传基因信息处理等代谢途径和功能基因的相对丰度。结果显示施有机肥对土壤酸碱度调节有着重要影响,进而改变了长期施肥酸性旱地土壤的微生物群落组成和多样性。本文对施肥驱动下的酸性旱地土壤硝化微生物群落演变规律的研究,为认识自养硝化微生物生理代谢多样性及生态功能提供了实验数据,也为酸性旱地土壤施肥管理提供参考。
[Abstract]:The total area of acid soil in China is up to 2 x 108hm2, accounting for about 23% of the country's soil area, of which more than half of the soil is agricultural soil or has potential agricultural value. With the increasing demand for land use efficiency and agricultural economic benefit, agricultural production is accompanied by a large amount of fertilizer input, but the utilization rate of economic crops to nitrogen fertilizer is common. Under 40%, the excessive input of nitrogen fertilizer leads to the imbalance of soil nitrogen cycle, aggravates soil acidification and reduces agricultural economic benefit. Nitrification is an important process of soil nitrogen cycle, which is driven by nitrification microorganism, and the process of ammonia oxidation is the rate limiting step of nitrification. However, the nitrification of ammonia oxidizing microorganisms to soil under acid conditions The influence of soil nitrification and the relative contribution of ammonia oxidizing microorganism on it was regulated by multiple environmental factors. The nitrification of soil in acid Dryland Soil and the seasonal evolution of its functional microorganisms were not clear. In addition, the long-term application of organic fertilizer and organic manure to the total microorganism of acid Dryland Soil The effect of community diversity is not the same. Therefore, this study selects 30 years of acid Dryland Soil in Jinxian Red Soil Institute of Jiangxi as research materials, selected (1) non fertilization (CK), (2) nitrogen, phosphorus and potassium inorganic fertilizer (NPK), (3) three treatments of pig manure organic manure (OM), and constructed laboratory microfield culture experiment and in situ soil analysis experiment The molecular ecology techniques such as the short-term culture of PCR (Quantitative PCR, qPCR), denatured gradient gel electrophoresis (Denaturing Gradient Gel Electrophoresis, DGGE) and Illumina MiSeq high-throughput sequencing were used to analyze the nitrification activity in the culture process and occupy the active nitrifying microorganism. Groups, explore the characteristics of autotrophic nitrification microorganism community and activity and recognize its relationship with soil physical and chemical factors, clarify the seasonal evolution characteristics of nitrification activity in acid dryland soil as well as Ammonia-oxidizing archaea, AO A, and bacteria (Ammonia-oxidizing bacteria, AOB) in acid Dryland Soil in different cultures. The relative contribution of temperature to ammonia oxidation process was studied. The effects of long-term application of inorganic fertilizer and organic fertilizer on the diversity of total microbial community in acid dryland soil were studied. The differences and similarities of the two factors were compared and the main environmental factors affecting the community change were analyzed. The effect was 73.60% ~ 85.32% of the total nitrification rate, and fertilization significantly increased the soil autotrophic nitrification activity, and the effect of application of organic fertilizer was more obvious. The absolute abundance of AOA and AOB amoA gene in OM soil and the relative abundance of 16S rRNA gene increased significantly, while the AOA relative abundance of CK and NPK soil increased significantly, that is, 3 kinds of soil AOA have obvious activity ( The main groups were Nitrososphaera, 99.30%), while AOB was only active in OM soil (the main group was Nitrosospira, 99.99%). In addition, the nitrite oxidizing bacteria (NOB) in OM soil had strong activity (the main group was Nitrospira, 96.69%). The stepwise regression analysis showed that the autotrophic nitrification activity was positively correlated with the total nitrogen content, AOA and AOB amoA. The gene abundance was significantly affected by the content of organic carbon and pH, and the relative abundance of Nitrososphaera was positively correlated with the content of NO3--N, while the relative abundance of Nitrosospira and Nitrospira was negatively correlated with C/N. The soil nitrification activity at 4 ~ 23 C was lower, and the highest soil nitrification rate in CK, NPK and OM soils was 2.29 mg. (kg d) respectively. 0 mg. (kg. D) -1 and 11.58 mg. (kg d) -1, and the highest nitrification activity in the soil at 23 ~ 35 C in summer. The maximum soil nitrification rate of the three soils was 5.19 Mg (kg. D) -1,7.62. The contribution of AOA to the autotrophic nitrification reached a peak at 35. As the temperature increased, the contribution of AOA to autotrophic nitrification increased gradually and dominated. The application of inorganic and organic fertilizer did not change the response of soil AOA to the temperature, but the maximum temperature of AOB significantly reduced the inhibition effect and application of.C8H14 as a soil nitrification inhibitor. Concentration is positive correlation, and the best effect of using 0.5% added concentration as the relative contribution of AOA and AOB to soil nitrification in CK and NPK soils is best. Compared with long-term fertilizer application, long-term application of organic fertilizer significantly changes the composition and diversity of bacterial community in acid red soil and diversity.ABT (Aggregated Boosted Tree) analysis found that soil pH is a bacterial group In addition, the application of organic manure significantly changed the relative abundance of metabolic pathways and functional genes, such as metabolic and genetic information processing. The results showed that organic fertilizer had an important influence on the regulation of soil acidity and alkalinity, and then changed the microbial community composition and diversity of long-term fertilized acid dryland soil. In this paper, the study on the evolution of nitrifying microorganism community in acid dryland soil under fertilization has provided experimental data for understanding the physiological metabolic diversity and ecological function of autotrophic nitrification microorganisms, and also provides reference for fertilizer management in acid dryland soil.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S154.3

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