Knowledge Resource Center for Ecological Environment in Arid Area
| 青藏高原土壤固氮菌的分布格局以及对人类活动和环境变化的响应 | |
| 其他题名 | The distribution of soil diazotrophs on the Tibetan Plateau and their responses to human activities and environmental changes |
| 车荣晓 | |
| 出版年 | 2017 |
| 学位类型 | 博士 |
| 导师 | 崔骁勇 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 氮素是陆地生态系统初级生产力最重要的限制因子之一，生物固氮则是全球生态系统有效氮最主要的来源。因此作为生物固氮的执行者，固氮菌也一直是微生物生态学领域最受关注的研究对象之一。青藏高原是世界上平均海拔最高的高原，其独特的地理环境造就了该区域独有的动、植物群落特征，而这也预示着该区域土壤中很可能分布着独特的固氮菌类群。此外，作为典型的高寒陆地生态系统，青藏高原脆弱的生态环境正经受着全球变暖、过度放牧、草地退化及氮沉降速率逐年递增等一系列环境问题的威胁，而这些环境问题在高寒草甸地区表现得尤为突出。尽管青藏高原地区生态系统初级生产力深受氮素限制，且生物固氮也很可能是该区域最主要的氮素来源，但我们对该区域土壤固氮菌的分布格局及其驱动机制的了解仍近乎空白，有关高寒草甸土壤固氮菌对增温、放牧、退化及氮沉降等环境变化或人类活动的响应也知之甚少。基于此，我们通过采集遍布于青藏高原地区的54个地点的表层土壤样品（0-5 cm），结合样点信息调查（植被、气候等）、土壤理化性质分析以及基于nifH基因的固氮菌多度和群落结构分析，揭示了青藏高原草地土壤固氮菌的分布格局及其主要驱动机制。此外，我们还依托位于青藏高原东北部的中科院海北站增温、放牧实验平台和模拟氮沉降实验平台，结合三江源地区斑块状退化高寒草甸土壤样品的配对采集以及凋落物和磷添加室内土壤培养实验，初步探究了高寒草甸土壤固氮菌对增温、放牧、退化、氮沉降、凋落物和磷添加的响应。固氮菌多度及nifH基因的转录表达通过实时荧光定量PCR来测定，固氮菌群落结构则通过Miseq高通量测序或末端限制性酶切片段多态性（T-RFLP）来揭示。本论文的主要研究结果如下：（1）青藏高原地区土壤固氮菌多样性较高，在94%的分类水平上得到了1023个OTU，但主要为稀有种，仅有24个OTU占所有序列的比例超过1%。该区域土壤固氮菌多度及多样性均呈现出随降水量增加而增加的趋势，且高寒草甸土壤固氮菌多度及多样性均要显著高于高寒草原和荒漠。土壤固氮菌群落主要由蓝细菌（46.37%）及变形菌（44.75%）主导，且呈现出按降水量梯度分布的特征。固氮菌多度、多样性及群落结构与年均降水量，植物生物量，植物群落结构，土壤碳、氮、磷含量及土壤pH值、含水量等指标均呈现出显著的相关性。虽然地理距离与固氮菌群落结构的相关性也达到了显著水平，但仅能解释0.88%的固氮菌群落结构变异。（2）日动态分析发现高寒草甸土壤固氮菌nifH基因拷贝数、相对多度、nifH mRNA拷贝数、nifH基因平均表达量均未表现出显著的昼夜节律性变化。（3）增温处理显著降低了固氮菌的多度及相对多度，并显著改变了固氮菌的群落结构，但并未对固氮基因总转录表达量产生显著影响。在非增温处理下，放牧未对固氮菌产生显著影响。但在增温处理下，放牧显著降低了nifH基因的平均表达量，同时表现出降低nifH基因总转录表达量的趋势。（4）两倍及三倍于当地氮沉降量的施氮处理未对固氮菌产生显著的负面影响，甚至有增加固氮菌数量和相对多度的趋势，施氮量增加到五倍于当地氮沉降量时，固氮基因的转录表达受到了显著的抑制，这种抑制很可能是高氮处理下共生固氮菌比例的下降引起的。（5）高寒草甸退化秃斑的形成显著降低了包括氮素在内的大部分土壤养分的含量。但在退化程度较重的高寒草甸，退化秃斑内的土壤硝态氮含量却有显著增高的趋势，而amoA基因拷贝数及转录表达与硝态氮含量及其它土壤氮素动态具有极好的一致性，因而很可能在退化秃斑内土壤氮素的转化中起着十分重要的作用。固氮和反硝化功能基因多度及转录表达对退化的响应较弱，且趋势性不强，但固氮及反硝化功能群的群落结构对退化秃斑的形成十分敏感，并与多种土壤理化性质都表现出了较高的相关性。（6）凋落物添加在显著增加土壤铵态氮含量的同时，还显著地降低了土壤硝态氮和无机氮含量。尽管添加凋落物的同时施用磷素对土壤固氮菌nifH基因拷贝数影响不大，但在磷用量高时显著上调了固氮基因的转录表达。凋落物添加还显著地降低了土壤氨氧化功能群的多度，并表现出增加反硝化功能基因转录表达的趋势。上述研究不仅在一定程度上填补了世界范围内草地土壤固氮菌生物地理及固氮基因表达相关研究的空缺，也为了解青藏高原草地氮收支动态、调控生物固氮及完善“宏基因表达调控”研究框架等奠定了基础 |
| 英文摘要 | Nitrogen availability predominantly limits the ecosystem primary productivity globally, while biological nitrogen fixation (BNF) is the largest source of available nitrogen for our planet. Thus, as the performer of BNF, diazotrophs are always highly concerned in the field of microbial ecology. Tibetan Plateau, the highest plateau in the world. Its special geographical conditions have created unique communities of plants, animals and probably soil diazotrophs. Additionally, the vulnerable ecosystems there are suffering from a series of severe environmental problems, such as global warming, overgrazing, increasing nitrogen deposition, and grasland degradation. This is particularly serious in the alpine meadow regions. Although the primary productivity of the Tibetan Plateau is highly limited by the nitrogen availability, and BNF is speculated as the main source of available nitrogen there, we still know little about Tibetan diazotrophs. Even the particularly basic knowledge like their distribution pattern is still absent. Furthermore, there is also a paucity of knowledge on the responses of soil diazotrophs to warming, grazing, land degradation and nitrogen deposition. Therefore, this study aimed to elucidate the distribution pattern of soil diazotrophs on the Tibetan Plateau, and to reveal the responses of soil diazotrophs to human activities and environmental changes in the Tibetan alpine meadows.As for the distribution pattern of soil diazotrophs on the Tibetan Plateau, we collected surface soil samples (0-5 cm) from 54 sites which were evenly distributed on the Tibetan Plateau. To reveal the responses of soil diazotrophs to warming, grazing, and nitrogen deposition, soils were sampled from two field control experiments (one for warming and grazing; another for the simulation of nitrogen deposition) which were located at the Haibei alpine meadow research station. The effect of alpine meadow degradation on soil diazotrophs were determined using soil samples collected from three degraded alpine meadows in the “three-river sources region”. The effects of litter and phosphorus amendments on soil nitrogen cycling groups were revealed through a microcosm experiments using the soils collected from the Haibei alpine meadow research station. Copies of nifH gene and mRNA were measured using real-time PCR; the diazotroph community compositions were determined through terminal-restriction fragment length polymorphism (T-RFLP) or Miseq sequencing. The main findings are as follows:(1) The Tibetan soil diazotrophs were highly diverse, 1023 operational taxonomic units (OTUs) were obtained with an identity cut-off of 0.94. However, most OTUs were recognized as rare species, and only 24 OTUs occupied more than 1% of the total sequences. In this region, the soil diazotroph diversity and abundance showed significantly positive correlations with mean annual precipitation (MAP). The soil diazotroph diversity and abundance in the alpine meadow region were significantly higher than in the alpine steppe and desert regions. As for the community composition, the diazotroph communities were dominated by Cyanobacteria (46.37%) and Proteobacteria (44.75%), and also showed clear distribution tendency following the MAP gradient. In addition, the Tibetan diazotroph abundance, diversity, and community composition also showed significant correlations with plant composition and aboveground biomass; soil moisture, carbon, nitrogen and phosphorous contents; and soil pH values. Geographical distance was also significantly correlated with soil diazotroph community composition, but could only explain 0.88% of its variation. (2) In the alpine meadow, none of soil diazotroph abundance, relative abundance, and the transcription of nifH gene showed rhythmically diurnal dynamics.(3) Warming significantly reduced the abundance and relative abundance of soil diazotrophs, and also significantly altered the soil diazotroph community composition, but exerted no significant effects the transcription of nifH gene. Under the warming treatments, grazing significantly decreased the average transcription of nifH gene, and also tended to decrease the nifH mRNA copies.(4) Treatments with low and middle (equivalent to 2 and 3 times of local nitrogen deposition rate, respectively) nitrogen addition rates exerted no adverse effects on the soil diazotrophs, and even tended to increase the abundance and relative abundance of soil diazotrophs. However, the treatments with high nitrogen addition rate (equivalent to five times of local nitrogen deposition rate) significantly depressed the transcription of soil nifH genes, which might be caused by the decrease of the relative abundance of symbiosis diazotroph under the treatments with high nitrogen deposition rate. (5) The patch degradation of alpine meadows significantly lowered the contents of most soil nutrients (e.g. carbon, nitrogen and phosphorous contents), but also tended to increase soil nitrate nitrogen contents at the heavily degraded alpine meadows. The copies of amoA gene and archaeal amoA mRNA significantly increased under patch degradation, which were positively correlated with soil δ15N values and nitrate nitrogen contents, but were negatively correlated with most of other soil nutrient properties. These results suggested that the increased abundance of ammonia-oxidizing microbes might play a vital role in affecting the soil nitrogen dynamics under the patch degradation. The responses of nitrogen-fixing and denitrifying functional gene abundance and transcription to the patch degradation were generally weak and showed high inconsistency across the study sites. However, the community structures of diazotrophs and most denitrifiers were much more sensitive to patch degradation, and were significantly correlated with several different soil properties.(6) Plant litter addition significantly increased soil dissolved organic carbon and ammonium nitrogen contents, but also significantly decreased the soil inorganic and nitrate nitrogen contents. Although neither litter nor phosphorous amendments significantly affected the diazotroph abundance, litter with high amount phosphorous amendment significantly up-regulated the transcription of nifH gene. The litter amendments significantly decreased the abundance of soil nitrifiers, but also tended to increase the transcription of denitrifying functional genes. The aforementioned findings not only fill in the knowledge gap on the biogeography of diazotrophs and the regulation of nitrogen fixing gene expressions in soil, but also provide bases for the estimation of Tibetan grassland nitrogen budget, the regulation of soil BNF, and the improvement of the research system on “the regulation of metagene expression”. |
| 中文关键词 | 青藏高原 ; 高寒草甸 ; 固氮菌 ; 气候变化 ; 放牧 ; 生物地理 |
| 英文关键词 | Tibetan Plateau alpine meadow nitrogen-fixing microorganisms climate changes grazing biogeography |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 生态学 |
| 来源机构 | 中国科学院大学 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/287909 |
| 推荐引用方式 GB/T 7714 | 车荣晓. 青藏高原土壤固氮菌的分布格局以及对人类活动和环境变化的响应[D]. 中国科学院大学,2017. |
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