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题名: 三种森林土壤氨氧化微生物对全球变化因子的响应及其机制
作者: 龙锡恩
学位类别: 博士
答辩日期: 2012
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 贺纪正
关键词: 氮沉降 ; Nitrogen deposition ; CO2升高 ; elevated CO2 ; 土壤增温 ; soil warming ; 森林火烧 ; prescribed burning ; 氨氧化微生物 ; ammonia-oxidizer
其他题名: Responses and mechanisms of three forest soils ammonia-oxidizer to global change factors
中文摘要:
      全球气候和环境变化已引起人们的广泛关注。海洋上,温室气体释放增加使得海洋逐步酸化,从而威胁海洋生态系统;陆地上,全球变暖加速了冰川融雪,会造成海平面上升,引发海侵而危及沿海生态和人们生活。全球气候变化引起的灾害天气频发也影响着人们的正常生产和生活。长期以来,对陆地生态系统在全球变化过程中的响应研究主要关注于地上部植物以及土壤碳库等的变化情况,而对地下部微生物生态系统的关注不够。地上地下生态系统相互关联,研究地下微生物生态系统对全球变化因子的响应,对于全面认识全球气候和环境变化的后果,制定适应和应对策略具有重要科学意义。 本论文以与氮循环密切相关的氨氧化微生物为指示生物,利用分子生物学手段(包括定量PCR、克隆文库和限制性酶切多态性)来研究氨氧化细菌和古菌对未来全球变化的响应,主要包括三个长期定位试验点不同全球变化因子影响下土壤氨氧化细菌和古菌amoA 基因的种群丰度和结构特征。主要研究结果如下: 1)美国杜克大学长期CO2 升高试验(4 个处理:对照(AU),施氮肥(AN),增加CO2(EU)和增加CO2 施氮肥(EN))。增加CO2 时间为10 年,施氮肥时间为2 年。施氮肥(AN)显著增加氨氧化细菌amoA 基因拷贝数;而 δ13C 与氨氧化古菌amoA 基因拷贝数的相关分析表明CO2 升高促进氨氧化古菌的生长;但同时增加CO2 和施氮肥(EN),它们对氨氧化细菌或古菌的促进作用不显著。限制性酶切分型和克隆测序表明氨氧化古菌在AN 处理中Soil_cluster_5/2 的比值显著高于其余处理;氨氧化细菌在EU 和EN 处理中包括有Cluster_1、Cluster_2、Cluster_9、Cluster_11 和Cluster_3a,并且在EU 处理中,Cluster_2 所占比例下降,而Cluster_9 和Cluster_11 所占比例上升;AU处理中有Cluster_1、Cluster_2 和Cluster_9; AN 处理中有Cluster_1、Cluster_2、Cluster_4 和Cluster_9,其中Cluster_2 都所占比例均超过50%。以上结果表明,CO2 升高会改变土壤氨氧化细菌的种群结构,但对氨氧化古菌种群结构的影响不大。环境因子与amoA 基因型的CCA 分析结果显示,土壤pH 是影响氨氧化细菌和古菌分布的主要环境因子。 2)瑞典Flakaliden 长期土壤增温试验(4 个处理:对照(IC),施肥(ILC),增温(Ih)和增温施肥(ILh))。其中施肥为完全配方施肥处理,时间为19 年,土壤增温时间为12 年。定量PCR 结果表明氨氧化细菌amoA 基因拷贝数比氨氧化古菌amoA 基因拷贝数高2-3 个数量级。氨氧化细菌amoA 基因拷贝数和细菌16S rRNA 基因拷贝数的比值显示施肥比增温对氨氧化细菌丰度的影响大,而氨氧化古菌amoA 基因拷贝数与古菌16S rRNA 拷贝数的比值显示增温对氨氧化古菌丰度的影响大。ILC 和ILh 处理中,氨氧化细菌amoA 基因型只有Cluster_2,而氨氧化古菌的Soil_cluster_5 与Soil_cluster_2 的比值要高于IC 和Ih 处理中相应的比值。CCA 的分析结果也表明施肥是影响氨氧化细菌和古菌分布的主要环境因子。 3)澳大利亚昆士兰州Peachester 森林长期计划火烧试验(3 个处理:对照组(B0),2 年烧组(B2)),4 年烧组(B4),分表层土(0-10cm)和亚表层土(10-20cm)采样。火烧试验时间为33 年。2 年烧处理显著改变土壤理化和生物学性质,pH 显著升高,土壤总碳、总氮和溶解性有机碳(DOC)和氮(DON)的含量显著降低,微生物生物量碳含量显著降低;4 年烧处理对土壤理化性质和生物性质的影响较小,只有pH 显著升高。火烧对亚表层土壤的影响小。amoA 基因拷贝数与土壤理化和生物性质的相关分析表明,氨氧化细菌丰度与pH 呈显著正相关,而与DOC/DON 比值呈显著负相关;氨氧化古菌丰度与pH 呈显著负相关,而与土壤C/N 比值呈显著正相关,与微生物生物量碳和氮均呈显著负相关。在表层土壤中,2 年烧处理中的氨氧化细菌amoA 基因拷贝数显著高于对照组。火烧显著改变氨氧化古菌的群落结构,表层土壤中,Soil_cluster_2/5 比值由B0 组的249 分别降至B2 组的3.7 和B4 组的6.9;亚表层土壤中,Soil_cluster_2/5 比值由B0 组的9.8 分别降至B2 组的4.1 和B4 组的3.0。火烧对氨氧化细菌群落结构的影响也可以从Cluster_2/11 的比值看出,表层土壤中,Cluster_2/11 的比值由B2 组的10.5 降至B4 组的2.6 和B0 组的3.2,下表层土壤中,Cluster_2/11 的比值用B2 组的5.2 降至B4 组的3.9 和B0 组的2.4。而且,从amoA 基因的分子方差分析也发现火烧对氨氧化细菌amoA 基因有显著影响。CCA 分析结果表明,在不同土层中,对氨氧化细菌和古菌amoA基因分布的影响因子不完全相同,在表层土壤中,DOC 显著影响氨氧化细菌amoA 基因的分布,硝氮、微生物生物量氮和DOC 显著影响氨氧化古菌amoA基因的分布;在下表层土壤中,土壤pH 和总碳显著影响氨氧化细菌amoA 基因分布,而只有总碳影响氨氧化古菌amoA 基因分布。
英文摘要:       Global changes of environment and climate have been extensively highlighted. In marine systems, the growth of greenhouse gases emission will acidify the ocean and threaten the marine ecosystems, whereas in terrestrial systems, the global warming accelerates glacier retreat, leading to sea level rise. The following transgression endangers the ecosystems and human’s living conditions in coastal areas. The increasing frequency of climate change related disastrous weather seriously affects the production and living conditions of human beings. However, researchers primarily study on the responses of plants and soil carbon pools, but rarely focus on the response of below-ground microbial ecosystems to the global changes. There is an interrelationship between above-ground and below-ground ecosystems. It has great scientific significance in fully understanding the consequences of global changes of environment and climate and making adaptive and correspondent strategies to study the response of below-ground microbial ecosystems to the global changes. In this study, we used the ammonia oxidizers, which play an important role in global nitrogen cycle, as a biological indicator. The molecular methods, such as quantitative PCR, clone library and restriction fragment length polymorphism (RLFP),were used to determine the effects of simulated global climate changes on the abundances and community structures of ammonia-oxidizing bacteria and archaea. To our knowledge, this study first time to use the ammonia oxidizers to link three different processes of global changes. In addition, these simulated global climate change experiments have all been operated more than ten years. The results are listed as follows: 1) The free air CO2 enrichment (FACE) in Duke forest includes four treatments: ambient CO2 and unfertilized (AU), ambient CO2 and fertilized (AN), elevated CO2 and unfertilized (EU), elevated CO2 and fertilized (EN). The elevated CO2 experiment was conducted since 1996, and in 2005, one half of each plot was executed with N fertilization. N fertilization alone significantly increased the abundance of bacterial amoA genes in comparison with control plots. In the other hand, a negative correlation between the abundance of archaeal amoA genes and soil 13C indicated a positive effect of CO2 enrichment on the growth of ammonia-oxidizing archaea. However, those positive effects were mitigated by the N fertilization addition in EN treatment. Results of RFLP and phylogenetic tree analysis showed that for archaeal amoA genes, the ratio of soil cluster 5 to 2 in AN treatment was higher than other treatments. For bacterial amoA genes, 5 clusters (cluster_1,cluster_2,cluster_9,cluster_11 and cluster_3a) were obtained from EU and EN treatments, and the proportion of cluster_2 in EU treatment decreased but that of cluster_9 and cluster_11 increased. Moreover, there are 3 clusters (cluster_1,cluster_2 and cluster_9) and 4 clusters (cluster_1,cluster_2,cluster_4 and cluster_9 ) in AU and AN treatment, respectively, and the proportion of cluster_2 in both treatment was higher than 50%. In conclusion, elevated CO2 shifted the community composition of ammonia-oxidizing bacteria, but did not affect that of ammonia-oxidizing archaea. Finally, CCA results indicated that soil pH significantly affected the distribution of ammonia oxidizers. 2) The long-term soil warming experiment was conducted in Flakaliden of Sweden in cluded four treatments: irrigation - no warming (IC), irrigation– warming (Ih), irrigation + fertilization - no warming (ILC), and irrigation + fertilization - warming (ILh). The nutrient optimization experiment began in 1987 and that of soil warming began in 1995. Results of quantitative PCR showed that the abundance of bacterial amoA genes was 2-3 orders of magnitude larger than that of archaea. The ratio of population size between bacterial amoA genes and bacterial 16S rRNA genes indicated that fertilization rather than soil warming affected the abundance of ammonia-oxidizing bacteria, whereas soil warming rather than fertilization influenced the abundance of ammonia-oxidizing archaea. Only cluster_2 was detected in ILC and ILh treatments. In addition, the ratio of soil cluster 5/2 in ILC and ILh treatments were higher than that of IC and Ih. Finally, CCA results showed fertilization was the dominant factor to explain genotype-environment relationship of ammonia-oxidizing bacteria and archaea. 3) The long-term forest prescribed burning experiment was located in Peachester national forest, Queensland, Australia. The prescribed burning method includes three methods: control plots, 2-yr burned and 4-yr burned. The soil was sampled in both sections: 0-10cm and 10-20cm. The prescribed burning was begun in 1972. Soil (bio)chemical properties data showed that 2-yr burned significantly increased soil pH and decreased soil total carbon, total nitrogen, DOC, DON and microbial biomass carbon.However, the impact of 4-yr burned was insignificant on soil (bio)chemical prop-erties, except for the pH increased. In addition, the effect of fire on the subtop soil was insignificant. In topsoil, linear regression analysis showed that the abundance of bacterial amoA genes was positively correlated with soil pH and negatively correlated with DOC/DON ratio, whereas the abundance of archeal amoA genes was negatively correlated with soil pH, microbial biomass carbon (MBC) and nitrogen (MBN) but positive correlated with soil C/N ratio. In topsoil, bacterial amoA copies in 2-yr burned plots were significantly higher than that in control plots. Results of amoA gene RFLP and phylogenetic tree analysis indicated that fire significantly change the community composition of both ammonia oxidizers. For example, in the topsoil, the ratio of soil cluster 2 to 5 of archaeal amoA gene decreased from 249 in control plots to 3.7 and 6.9 in 2-yr and 4-yr burned plots, respectively, in the subtop soil, that of archaeal amoA gene decreased from 9.8 in control plots to 4.1 and 3.0 in 2-yr and 4-yr burned plots, respectively. The shift of ratio of cluster 2 to 11 indicated an effect of fire on the community structure of ammonia-oxidizing bacteria. In the topsoil, the ratio of cluster 2 to 11 decreased from 10.5 in 2-yr burned plots to 3.2 and 2.6 in control and 4-yr burned plots, respectively, in the subtop soil, the ratio of cluster 2 to 11 decreased from 5.2 in 2-yr burned plots to 2.4 and 3.9 in control and 4-yr burned plots. In addition, the molecular variance analysis results indicated a significant effect of fire on the community composition of ammonia-oxidizing bacteria. Finally, CCA results showed that factors which shaped the amoA gene distribution were different in both soil sections. In the topsoil, soil DOC significantly affected the distribution of bacterial amoA genes whereas nitrate-nitrogen, MBN and DOC significantly influenced the distribution of archeal amoA genes. In subtop soil, soil pH and total carbon determined the genotype-environmental relationship of bacterial amoA genes, whereas only total carbon shaped the distribution of archaeal amoA genes.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/35158
Appears in Collections:中澳联合土壤环境研究室_学位论文

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Recommended Citation:
龙锡恩. 三种森林土壤氨氧化微生物对全球变化因子的响应及其机制[D]. 北京. 中国科学院研究生院. 2012.
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