長(zhǎng)期施肥和溫度對(duì)酸性旱地土壤硝化微生物和總細(xì)菌群落的影響
發(fā)布時(shí)間：2018-08-06 19:21
【摘要】：我國(guó)酸性土壤總面積高達(dá)2×108hm2,約占全國(guó)土壤面積的23%,其中一半以上土壤為農(nóng)業(yè)土壤或具有潛在的農(nóng)業(yè)價(jià)值,隨著人類對(duì)土地利用效率和農(nóng)業(yè)經(jīng)濟(jì)效益的需求增加,農(nóng)業(yè)生產(chǎn)伴隨著大量肥料的投入,但經(jīng)濟(jì)作物對(duì)氮肥的利用率卻普遍低于40%,氮肥的過量投入導(dǎo)致土壤氮循環(huán)失衡,加劇了土壤酸化,降低了農(nóng)業(yè)經(jīng)濟(jì)效益。硝化作用是土壤氮循環(huán)的重要過程,由硝化微生物驅(qū)動(dòng),其中氨氧化過程是硝化作用的限速步驟。然而,氨氧化微生物在酸性條件下對(duì)土壤硝化作用的影響尚存爭(zhēng)議,土壤硝化作用強(qiáng)度及氨氧化微生物對(duì)其的相對(duì)貢獻(xiàn)受多個(gè)環(huán)境因子的綜合調(diào)控,長(zhǎng)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節(jié)演變尚不明確,此外,長(zhǎng)期施用無機(jī)肥和有機(jī)肥對(duì)酸性旱地土壤總微生物群落多樣性影響也不盡相同。因此本研究選用江西進(jìn)賢紅壤研究所長(zhǎng)期定位施肥30年酸性旱地土壤為研究材料,選用(1)不施肥(CK)、(2)施氮磷鉀無機(jī)肥(NPK)、(3)施腐熟豬糞有機(jī)肥(OM)三種處理,構(gòu)建實(shí)驗(yàn)室微域培養(yǎng)實(shí)驗(yàn)與原位土壤分析實(shí)驗(yàn),通過添加硝化抑制劑的短期培養(yǎng)并聯(lián)用定量PCR (Quantitative PCR,qPCR)、變性梯度凝膠電泳(Denaturing Gradient Gel Electrophoresis,DGGE)及 Illumina MiSeq 高通量測(cè)序等分子生態(tài)學(xué)技術(shù),分析培養(yǎng)過程中發(fā)揮硝化活性的、且占據(jù)活性優(yōu)勢(shì)的硝化微生物類群,探究自養(yǎng)硝化微生物群落及活性特征并認(rèn)知其與土壤理化因子間的關(guān)系,明確長(zhǎng)期施肥酸性旱地土壤硝化活性的季節(jié)演變特征以及酸性旱地土壤中氨氧化古菌(Ammonia-oxidizing archaea,AO A)和細(xì)菌(Ammonia-oxidizing bacteria, AOB)在不同培養(yǎng)溫度下對(duì)氨氧化過程的相對(duì)貢獻(xiàn),研究長(zhǎng)期施無機(jī)肥和有機(jī)肥對(duì)酸性旱地土壤中總微生物群落多樣性的影響,比較二者影響的異同,分析影響群落變化的主要環(huán)境因子。結(jié)果顯示,施肥對(duì)土壤理化性質(zhì)有顯著影響,供試土壤自養(yǎng)硝化作用占總硝化速率的73.60%～85.32%,施肥顯著提升土壤自養(yǎng)硝化活性,其中施有機(jī)肥提升效果更為明顯;OM 土壤氨AOA和AOB amoA基因絕對(duì)豐度及16S rRNA基因相對(duì)豐度顯著上升,而CK和NPK 土壤僅AOA相對(duì)豐度顯著上升,即3種土壤AOA均有明顯活性(主要類群為Nitrososphaera,99.30%),而AOB僅在OM 土壤有活性(主要類群為Nitrosospira,99.99%),另外還發(fā)現(xiàn)OM 土壤中亞硝酸鹽氧化細(xì)菌(NOB)有較強(qiáng)活性(主要類群為Nitrospira,96.69%);逐步回歸分析顯示自養(yǎng)硝化活性與總氮含量顯著正相關(guān),AOA和AOB amoA基因豐度分別受有機(jī)碳含量和pH的顯著影響,Nitrososphaera相對(duì)豐度與NO3--N含量顯著正相關(guān),而Nitrosospira和Nitrospira相對(duì)豐度則與C/N顯著負(fù)相關(guān)。在4 ℃～23 ℃ 土壤硝化活性較低,CK、NPK和OM土壤的最高土壤硝化速率分別為 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,三種土壤的最高土壤硝化速率分別為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貢獻(xiàn)的自養(yǎng)硝化作用分別在28 ℃、18 ℃和18 ℃達(dá)到峰值,AOA貢獻(xiàn)的自養(yǎng)硝化作用均在35 ℃達(dá)到峰值。隨著溫度的上升,AOA對(duì)自養(yǎng)硝化作用的貢獻(xiàn)逐漸上升并占據(jù)主導(dǎo),施無機(jī)肥和有機(jī)肥未改變土壤AOA的對(duì)溫度的響應(yīng),但AOB活性最高溫度顯著降低。C8H14作為土壤硝化抑制劑其抑制效果與施加濃度正相關(guān),選用0.5%的添加濃度作為區(qū)分CK和NPK 土壤中AOA和AOB對(duì)土壤硝化作用的相對(duì)貢獻(xiàn)時(shí)效果最佳。相較于長(zhǎng)期施化肥,長(zhǎng)期施有機(jī)肥顯著改變了酸性紅壤的細(xì)菌群落組成和多樣性。ABT (Aggregated Boosted Tree)分析發(fā)現(xiàn)土壤pH是細(xì)菌群落的總重要的環(huán)境因子。此外,施有機(jī)肥顯著改變了例如代謝和遺傳基因信息處理等代謝途徑和功能基因的相對(duì)豐度。結(jié)果顯示施有機(jī)肥對(duì)土壤酸堿度調(diào)節(jié)有著重要影響,進(jìn)而改變了長(zhǎng)期施肥酸性旱地土壤的微生物群落組成和多樣性。本文對(duì)施肥驅(qū)動(dòng)下的酸性旱地土壤硝化微生物群落演變規(guī)律的研究,為認(rèn)識(shí)自養(yǎng)硝化微生物生理代謝多樣性及生態(tài)功能提供了實(shí)驗(yàn)數(shù)據(jù),也為酸性旱地土壤施肥管理提供參考。
[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.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S154.3
本文編號(hào)：2168738
[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.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S154.3
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