1、分类号 密级 UDC 硕士学位论文毛竹林土壤氨氧化和固氮微生物特征及其演变规律作 者 姓 名：指 导 教 师：学科专业名称： 土壤学研 究 方 向： 土壤生物与生物化学所 在 学 院： 环境与资源学院论文提交日期： 年 月 日浙 江 农 林 大 学 年 月 日I摘要毛竹是集经济、生态、社会效益于一体的优良竹种，近 20 年，许多阔叶林和杉木林改种毛竹，加上毛竹自然入侵周边森林形成了新的毛竹纯林，因此毛竹面积不断扩大，最近 10 年扩大了 100 万公顷，至 2013 年面积已达 443 万公顷。毛竹属喜氮植物，土壤氮素水平对毛竹生长至关重要，而生物固氮是土壤氮素的重要来源。本研究应用变性梯度凝
2、胶电泳（DGGE）和实时荧光定量PCR（Real-time PCR），结合土壤的基本理化性质以及土壤净氮矿化率和净硝化率，通过比较研究毛竹纯林和阔叶林之间、毛竹入侵阔叶林后形成的三种不同林分之间土壤氮素水平、氮素转化过程以及土壤氮循环相关微生物的差异，来揭示毛竹发展过程中土壤氮循环相关微生物变化规律以及氮素转化过程和氮素有效性的关系。分别在临安和安吉采集毛竹纯林和阔叶林表层和表下层土壤；分别在长兴煤山镇和天目山自然保护区，采集了 3 个毛竹入侵带的毛竹林、毛竹和阔叶混交林以及原阔叶林土壤样品。毛竹纯林和阔叶林对比研究结果：临安阔叶林改种毛竹后两层土壤的 pH、碱解氮和表层土壤的速效钾都显著增加
3、（P0.05），而有效磷则显著下降（P0.05）；安吉地区改种前后土壤理化性质变化较小，表层土壤的有效磷和下层土壤的有机碳有较大差异（P0.05）。除下层土壤的净硝化率外，同一土层毛竹林土壤的净氮矿化率和净硝化率显著高于（P 0.05）阔叶林。临安和安吉两地土壤毛竹林的 AOA 丰度均高于阔叶，且安吉地区两种林分间存在有明显（P0.05）差异，而临安则没有检测到显著差异；临安和安吉两地毛竹林土壤的 AOB 丰度高于阔叶，但除临安表层土壤存在显著（P0.05）差异外，均没有检测到显著差异。土壤固氮细菌 PCR-DGGE 条带数及多样性指数H（Shannon-Wiener index H）为阔叶林
4、高于毛竹林，而毛竹林土壤的固氮细菌nifH 基因丰度则高于阔叶林。基于 PCR-DGGE 条带信息的聚类分析和主成分分析（PCA ）结果表明，毛竹林和阔叶林土壤固氮细菌群落结构存在明显（P0.05）差异。毛竹入侵天然阔叶林结果：随着毛竹入侵天然阔叶林，土壤 pH 从阔叶林、混交林到毛竹林依次升高；青龙山、石门洞土壤的有机碳、青龙山土壤的碱解摘要II氮和石门洞土壤的有效磷含量显著（P0.05）增加，煤山镇土壤的有机碳、速效钾和石门洞土壤的速效钾含量显著（P0.05）降低。同一地区毛竹林土壤的净氮矿化率和硝化率均为最高，毛竹林土壤净氮矿化率和净硝化率明显高于（P0.05）其它两种林分土壤（石门洞土
5、壤净硝化率除外）。随着毛竹逐渐入侵阔叶林，土壤氨氧化古菌（AOA）和氨氧化细菌（AOB ）的基因丰度总体呈下降趋势。煤山镇阔叶林土壤固氮细菌多样性（DGGE 条带信息）最丰富，多样性指数显著（P0.05）高于其他两种林分；而天目山的青龙山土壤固氮细菌多样性毛竹林最丰富，显著（P0.05）高于混交林和阔叶林，石门洞土壤三种林分之间则没有显著差异，天目山两个样地随着毛竹入侵阔叶林土壤固氮菌多样性逐渐上升，而煤山镇土壤的固氮细菌多样性则下降，说明同样是毛竹入侵阔叶林，土壤固氮细菌多样性变化规律性不一致。通过土壤固氮菌 DGGE 信息的数字化处理后的聚类分析和主成分分析结果表明，3 个入侵带不同植被下
6、土壤固氮菌群落结构都存在显著（P0.05）差异。土壤固氮菌 nifH 基因的荧光定量 PCR 结果显示，煤山镇毛竹林土壤显著（P 0.05）大于阔叶林，石门洞和青龙山阔叶林土壤大于毛竹林，说明随着毛竹逐渐入侵阔叶林，不同土壤样地固氮基因（nifH）丰度变化不同。关键词：毛竹，土壤固氮菌，氨氧化古菌（AOA），氨氧化细菌（AOB ），PCR-DGGE，荧光定量 PCR，净氮矿化率，净硝化率。ABSTRACTIIIABSTRACTMoso bamboo is one of the excellent bamboo species with a set of economic,ecological
7、and social benefits. In recent 20 years, many broadleaf forests and Chinese fir forests were converted to moso bamboo, and moso bamboo naturally invade to surrounding forest and formed a new pure moso bamboo forest, hence, the area of moso bamboo forest had reached 4.43 million hectares to 2013. Mos
8、o bamboo is fond of nitrogen, soil nitrogen is essential to the growth of bamboo, and biological nitrogen fixation is an important source of nitrogen in the soil. Therefore, the research on characteristics and evolution of nitrogen cycle related microbes are important. In this study, denaturing grad
9、ient gel electrophoresis (DGGE) and real-time quantitative PCR (Real-time PCR) were used, combined with the physical and chemical properties and soil net nitrogen mineralization rate and net nitrogen nitrification rate, and the nitrogen levels, the conversion process of nitrogen and the difference o
10、f nitrogen cycle related microbes in soil were studied to reveal the relationship of evolution law of nitrogen cycle related microbes, the conversion process of nitrogen and the availability of nitrogen. Topsoil and subsoil of moso bamboo and broadleaf forest were collected from Linan and Anji, soil
11、 of 3 invaded areasmoso bamboo, broadleaf and mixed forest were collected from Meishan in Changxing and National Nature Reserve of Mount Tianmu.The comparison of moso bamboo and broadleaf forest: pH, avaliable nitrogen in both soil layers and potassium in topsoil were significantly increased (P0.05)
12、, and phosphorus decreased significantly(P0.05); Physical and chemical properties of soil varies little after replanting in Anji plots. phosphorus in topsoil and organic carbon in subsoil are quite different(P0.05). In addition to the net nitrification rate of the subsoil, net nitrogen mineralizatio
13、n and net nitrification rates of moso bamboo forest soil were significantly higher (P0.05) than broadleaf forest in the same soil layer. The rich source of ammonia oxidation microorganisms can improve net nitrogen mineralization rate and net nitrogen nitrification rates effciently. Abundance of AOA
14、functional gene (amoA) of moso bamboo was higher than broadleaf in Linan and Anji, and there are significant (P0.05) differences in both types in Anji, however, there is no significant difference was detected in Linan, Abundance of AOB functional gene (amoA) of moso bamboo was higher than broadleaf
15、in Linan and Anji, and there is no significant difference in addition to the topsoil ABSTRACTIVof Linan. Results of PCR-DGGE and diversity index H of nitrogen-fixing bacteria showed that, diversity index of nitrogen-fixing bacteria in broadleaf forest soil was higher than moso bamboo forest, however
16、, abundance of nifH gene in moso bamboo forest was higher than broadleaf forest. Cluster analysis and PCA which based on PCR-DGGE showed that nitrogen-fixing bacteria community structure had a big difference in the two forest types.With the gradual invasion of moso bamboo to broadleaf forest, the ab
17、undance of ammonia-oxidizing archaea (AOA) gene and ammonia oxidizing bacteria (AOB) gene were decreased. Diversity (DGGE bands information) of nitrogen-fixing bacterial of broadleaf forest soil in Meishan was the richest, the diversity index was significantly (P0.05) than the other two stands; and
18、diversity of nitrogen-fixing bacterial in moso bamboo forest in Qinglongshan was the richest, significantly (P0.05) higher than that of mixed forest and broadleaf forest, there is no significant difference in the three soil stands, indicating that diversity change regularity of soil nitrogen-fixing
19、bacterial in the process of bamboo invasion broadleaf forest is inconsistent. Cluster analysis and principal component analysis (PCA) showed that nitrogen-fixing bacteria community structure is quite different under different vegetation in three invasion areas. Quantitative PCR results of abundance
20、of soil nitrogen-fixing bacteria nifH genes showed that moso bamboo forest soil is significant (P0.05) greater than the broadleaf forest, broadleaf forest soil is greater than the bamboo forest in Shimendong and Qinglongshan, it described that as the gradual invasion of moso bamboo to broadleaf fore
21、st, the changes of abundance of nitrogenase gene (nifH) is different in different.soil plots.Key words: Moso bamboo, nitrogen-fixing bacteria, ammonia oxidizing archaea (AOA), ammonia oxidizing bacteria (AOB), PCR-DGGE, fluorescence quantitative PCR, net nitrogen mineralization rate, net nitrificati
22、on rate.目录V目 录摘要 .IABSTRACT .III目 录 .V1 文献综述 .11.1 毛竹发展现状 .11.2 毛竹林土壤氮素及氮素循环 .11.3 土壤微生物作用对土壤氮素的影响 .21.3.1 土壤固氮微生物与土壤氮素的关系 .21.3.2 土壤硝化细菌与土壤氮素的关系 .21.3.2.1 土壤硝化细菌与氮素的转化 .21.3.2.2 土壤硝化细菌与氮素的损失 .31.4 不同土地利用方式对土壤氮素循环主要微生物的影响 .41.5 不同植被类型对土壤氮素循环主要微生物的影响 .51.5.1 不同植被类型对土壤固氮微生物的影响 .61.5.2 不同植被类型对土壤硝化反硝化微生
23、物的影响 .62 研究背景与研究内容 .72.1 研究背景与意义 .72.2 研究内容 .72.2.1 毛竹和阔叶林土壤氨氧化和固氮微生物特征比较 .72.2.2 毛竹入侵阔叶林过程中土壤氨氧化微生物和固氮细菌群落结构的变化 .72.3 技术路线 .73 毛竹和阔叶林土壤氨氧化和固氮微生物特征比较 .83.1 研究地区与材料方法 .83.1.1 研究区概况 .83.1.2 样品采集与处理 .83.1.3 主要分析方法 .93.1.3.1 土壤理化性质分析 .93.1.3.2 土壤净氮矿化率和净硝化率测定 .93.1.3.3 土壤总 DNA 的提取 .93.1.3.4 土壤固氮细菌的 PCR 扩
24、增 .93.1.3.5 固氮细菌 nifH 基因变性梯度凝胶电泳（DGGE） .103.1.3.6 固氮细菌 nifH 基因荧光定量 PCR .103.1.3.7 氨氧化古菌（AOA）、氨氧化细菌（AOB）定量 PCR .103.1.3.8 数据处理 .113.2 结果与分析 .113.2.1 土壤理化性质 .113.2.2 土壤氮的矿化和硝化作用 .123.2.3 氨氧化微生物功能基因（amoA）丰度 .133.2.3.1 氨氧化古菌（AOA）功能基因（amoA）丰度分析 .133.2.3.2 氨氧化细菌（AOB）功能基因（amoA）丰度分析 .14目录VI3.2.4 土壤固氮细菌群落特征
25、.153.2.4.1 土壤固氮细菌群落结构（DGGE） .153.2.4.2 土壤固氮细菌群落结构多样性 .163.2.4.3 土壤固氮细菌的聚类分析 .163.2.4.4 土壤固氮细菌的主成分分析（PCA） .173.2.5 固氮基因（nifH）丰度分析 .183.3 讨论与结论 .193.2.1 土壤氮素水平、氮转化过程及其与微生物的关系 .193.3.2 毛竹林和阔叶林土壤固氮细菌多样性和 nifH 基因丰度结果分析 .204 毛竹入侵阔叶林过程中土壤氨氧化微生物和固氮细菌群落结构的变化 .224.1 研究地区与材料方法 .224.1.1 研究区概况 .224.1.2 样品采集与处理 .
26、224.1.3 主要分析方法 .234.1.3.1 土壤理化性质 .234.1.3.2 土壤净氮矿化率和净硝化率测定 .234.1.3.3 土壤总 DNA 提取 .234.1.3.4 固氮菌 nifHPCR 扩增 .234.1.3.5 固氮细菌 nifH 基因变性梯度凝胶电泳（DGGE） .234.1.3.6 固氮细菌 nifH 荧光定量 PCR .234.1.3.7 氨氧化古菌（AOA）、氨氧化细菌（AOB）定量 PCR .234.1.3.8 数据处理 .234.2 结果与分析 .234.2.1 土壤理化性质 .234.2.2 土壤氮的矿化和硝化作用 .244.2.2.1 土壤矿化态-N 和
27、 NO3-N 含量变化 .244.2.2.2 土壤净氮矿化率和净硝化率 .264.2.3 氨氧化微生物功能基因（amoA）丰度分析 .264.2.3.1 氨氧化古菌（AOA）功能基因（amoA）丰度分析 .264.2.3.2 氨氧化细菌（AOB）功能基因（amoA）丰度分析 .294.2.4 土壤固氮细菌群落特征 .294.2.4.1 土壤固氮细菌群落结构（DGGE） .294.2.4.2 土壤固氮细菌群落结构多样性 .304.2.4.3 土壤固氮细菌的聚类分析 .314.2.4.4 土壤固氮细菌的主成分分析（PCA） .334.2.5 固氮基因（nifH）丰度分析 .334.3 讨论与结论 .354.3.1 土壤氮素水平、氮转化过程及其与微生物的关系 .354.3.2 土壤固氮细菌多样性与 nifH 丰度关系分析 .365 结论与展望 .365.1 主要结论 .365.2 展望 .37参考文献 .38个人简介 .44目录VII致谢 .45
