【作者】 任佐华；

【导师】 肖启明； 李迪强； 张于光；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2009， 博士

【摘要】 土壤微生物是生态系统的重要成员,是土壤中最活跃的部分,它们在土壤中的数量与分布一定程度上反映出土壤肥力状况与植物营养的密切关系,同时也反映土壤、植被和气候等综合因素对土壤微生物的影响。因此土壤微生物多样性的保护和研究对系统生态保护具有重要意义。三江源自然保护区位于青海省南部,属于青藏高原腹地,海拔垂直差异明显,具有独特的自然地理环境,是我国面积最大、海拔最高、生物多样性最丰富和生态最敏感的国家级自然保护区,为开展全球变化相关研究提供了理想的实验场所。青藏高原高寒草原生态系统具有十分重要的生态地位,因其所处环境严酷、生态脆弱,在全球变化,特别是在人为干扰等因素的综合影响下,已呈现出严重的退化态势。研究三江源自然保护区高寒草原土壤微生物群落结构与功能,揭示其生态分布和区域特异性,为评价三江源自然保护区的生态环境、了解全球变化对三江源自然保护区土壤微生物的影响以及三江源自然保护区保护策略的制定提供科学依据。本研究采用传统分离培养方法对三江源自然保护区高寒草原生物多样性固定监测样地的土壤中主要微生物类群的数量进行研究,探讨其与土壤酶、微生物量碳氮和生态因子的相关性;采用末端限制性酶切片段长度多态性分析(T—RFLP)分子生物学技术对硝化基因(amoA)、反硝化基因(nirK和nosZ)、土壤细菌群落进行研究。同时探讨了不同海拔高度土壤微生物群落结构与功能的多样性及其主要影响因子。其主要研究结果如下:1.为了揭示青藏高原高寒草地土壤微生物活性组成和微生物量碳氮情况,同时探讨气候变化对土壤微生物的影响,以青藏高原腹地三江源自然保护区高寒草原土壤为研究对象,选择土壤质地、植被类型基本一致,海拔高度不同(3400-4200m)的4个样地,分析测定了土壤微生物(细菌、真菌、放线菌和部分生理功能微生物群)数量、土壤微生物量(碳、氮)、土壤酶(纤维素酶、蛋白酶、脲酶用、蔗糖酶)活性。结果表明:研究区域均含有较丰富的土壤有机碳和养分,微生物数量多少为细菌＞放线菌＞真菌,主要功能微生物菌群数量为氨化细菌＞好气性固氮菌＞硝化细菌＞亚硝化细菌,样地间的微生物生物量碳氮含量差异显著。相关性分析表明,除与亚硝酸细菌具有弱正相关性外,海拔高度与其它因子均具有负相关性,其中与细菌和氨化细菌具有极显著负相关性,与好气性固氮菌和硝酸细菌具有显著负相关性。因此,温度的升高可能明显的影响了三江源地区高寒草原的土壤微生物活性。2.研究证明应用Ultra clean soil DNA Isolation Kit直接从土壤中提取DNA的方法是一种快速、高效、稳定的方法,该方法无需特殊仪器,整个过程可在较短的时间内完成;为检测土壤微生物的群落结构、微生物的多样性和功能微生物的代谢途径研究提供了一种可行的方法。3.采用T—RFLP方法对三江源高寒草地海拔垂直海拔梯度分布下的5个样地土壤中硝化(amoA)微生物多样性及群落分布进行了研究,结果表明,硝化菌群在相临样地间相似率较高,推测地理距离可能是影响硝化菌群多样性分布的主要因素。硝化菌群多样性在样地ZD-1最高,样地GH最低:而且对硝化菌T—RFs多样性指数H′与土壤生物地理化学因子进行相关性分析,结果也发现amoA基因多样性指数H′与海拔高度表现出极其显著的正相关;由此可推断温度升高对硝化细菌多样性有一定的影响。4.本研究首次应用nirK和nosZ基因,通过T—RFLP技术探讨三江源自然保护区内高寒草地海拔垂直地理生态分布带中5个样地的反硝化微生物多样性和群落结构。结果表明,反硝化菌群在相临样地间相似率较高,样地间nirK基因相似率普遍低于nosZ基因相似率。nirK基因距离较近的样地间出现较高的相似率,地理距离可能是影响nirK基因反硝化菌群多样性分布的主要因素。而nosZ基因在样地间相似率都差异不很大,nosZ基因的分布跟空间距离相关性不大。nirK基因nosZ基因基本符合按地海拔梯度进行聚类。反硝化菌群多样性nirK基因在样地CD最高,在样地MD最低;其均匀度在样地GH最高,在样地MD最低;nosZ基因的多样性在样地GML最高,在样地MD最低;即反硝化细菌多样性与海拔高度变化相关性不明显,而对硝化菌T—RFs多样性指数H′与土壤生物地理化学因子进行相关性分析,结果也发现nirK和nosZ基因多样性指数H′与土壤含水量表现出极其显著的正相关;由此可推断土壤湿度的变化对反硝化细菌多样性有一定的影响。5.利用T—RFLP技术,分析了三江源高寒草甸沿海拔梯度分布的4个样地土壤中细菌群落结构和多样性状况,研究结果表明,海拔为4813m的样地土壤细菌多样性普遍高于其3个样地,地理距离的差异是影响此地区土壤细菌生态分布的因素。温度的上升对土壤细菌多样性的影响势必也不容忽视。此外,其它生态因子均与土壤细菌群落结构多样性及生态分布有一定相关性,因此,其它生态因子对土壤细菌多样性的影响也不能忽视。更多还原

【Abstract】 Soil microbes are important parts of the ecosystem and very active in soil . Thier quantity and distributions in soil can reflect the relationship between plant nutritions and soil fertility ,and the impacts of integrated factors such as soil, vegetation and climate, etc. Therefore, the protection and research of soil microorganism diversity of ecosystem are very important for ecological protection. Sanjiangyuan Nature Reserve located in the south of Qinghai Province, belonged to the Qinghai-Tibet Plateau, the altitude vary obviously vertically ,and has an unique geographic environment. Sanjiangyuan Nature Reserve is the largest and highest Nature Reserve in China.In addition,its biological diversity is very rich and being a very sensitive ecological National Nature Reserve , which provides an ideal laboratory for global ecological change research. Qinghai-Tibet Plateau alpine grassland ecosystem has an important ecological status, because of the tough environment, fragile ecosystem and face serious degradation under the climate changes around the world, especially when integrated factors,such as the human disturbance accelerate the degradation. Researchs on the structure and function of soil microbial communities of Sanjiangyuan alpine grassland Nature Reserves ,unfold the soil microbes ecological distribution and regional specificity can provide a scientific basis for evaluating ecological evironment of the Sanjiangyuan Nature Reserve, and help us to understand the impacts of global climate changes on soil microbes of Sanjiangyuan nature reserve, then we can regulating pretective strategies base on these datas to protect the source of the Sanjiang Nature Reserve well.We use traditional isolation methods of microbes in this study to research the number of major soil microbial groups of alpine grassland biodiversity in monitoring plots of the Three Rivers Nature Reserve, and to examine its relationship with soil enzymes, microbial biomass carbon and nitrogen and the relevance of ecological factors; We use terminal restriction fragment length polymorphism (T-RFLP) molecular biology technology to study the diversity of nitrification gene(amoA), denitrification genes (nirK and nosZ), and the soil bacterial community. We also discussed the soil microbil diversity of the structure and function at different altitudes and its major impact factor. The main result are as follows:1. Soil of Qinghai-Tibet Plateau alpine grassland in Sanjiangyuan Nature Reserve was chosen to research ,for revealing the composition of soil microbial activity and microbial biomass carbon and nitrogen, and exploring the potential impacts of climate change on the microorganism. Soil samples from 4 site of different altitude, with similar texture and vegetation type, were analyzed. We test microorganism number (bacteria, fungi, actinomycetes and some physiological functions of microorganism), microbial biomass (C, N) and enzyme activities (cellulase, protease, urease, and sucrase). The results showed that all soil samples were rich in organic carbon and nutrients, the number of microorganisms as bacteria> actinomycetes> fungi, the main function of the number of microbial flora were ammonifiers> aerobic azotobacteria> nitrate bacteria> nitrobacteria , and there were signicant difference in microbial biomass (C, N) among those samples. Correlation analysis showed that the altitude had negative correlation with all factors except nitrite bacteria, and the altitude have negative correlation significantly with bacteria and ammonifiers. The altitude have negative correlation significantly with aerobic azotobacteria and nitrate bacteria. Therefore, the increasing temperature might affecte the activity of microorganisms in soil of Qinghai-Tibet Plateau alpine grassland in Sanjiangyuan Nature Reserve significantly.2. We concluded that genomic DNA can be extracted from soil effectively in a short time with ’Ultra clean soil DNA Isolation Kit’ , and without special equipments. High quality DNA can be used in detecting community and diversity of soil microorganism, it is also helpful for studying metabolism pathway of function microbes.3. We study the amoA bacteria community structure and diversity of five soil samples, which vertically distributed in Sanjiang cold grassland ranged from altitude, T-RFLP method was used for analysis. The results showed that there were high similarity between neighborhood samples, geography distance might the main factor upon diversity of amoA bacteria community. amoA bacteria diversity increase with altitude increasing, the highest diversity was observed in sample ZD-1 and the lowest diversity happened in sample GH. The correlativity analysis between the T-RFs diversity indexe H’ of amoA bacteria and soil geochemistry factors showed that the T-RFs diversity indexe H’ of amoA bacteria is significant correlative with altitude, therefore, the increasing temperature might have definite influence on amoA bacteria diversity.4 We applied nirK and nosZ genes for study firstly, with T-RFLP techniques to evaluate the five plots denitrification microbial diversity and community structure of above sea level of vertical distribution of geo-ecological zone of alpine grassland Sanjiangyuan Nature Reserve. The results showed that there were high similarity between denitrifying bacteria. nirK gene similar rates were lower than similar rates of nosZ genes generally. nirK gene from the nearer plot has a higher similaritie rates , geographical distance may be the main factor which affected the diversity and the distribution of denitnfication nirK gene of flora.The similar rates of the nosZ gene in samples has not much differences , and nosZ gene distance has little relationship relevance with distribution. nirK gene and nosZ gene are in line with an elevation gradient according to the cluster. the highest nirK denitrifying bacteria diversity was observed in sample CD-like gene, and the lowest diversity happened in sample MD; its highest uniformity is in sample GH plot, and the lowest is in the sample MD; the diversity of nosZ gene is the highest in GML sample, and the lowest is in MD sample; that is to say the diversity of denitrifying bacteria associated with the change of altitude was not obvious, while the analysis of T-RFs nitrifier diversity index H ’and of soil bio-geo-chemical of the relevant factors also found that nirK and nosZ gene diversity index H ’and soil water content showed an extremely significant positive correlation;thus it can be infered that the changes in soil moisture have a certain impact on the diversity of denitrifying bacteria5. By using of T-RFLP techniques ,we can evaluated the distribution of bacterial community structure and diversity from four soil samples of Sanjiangyuan alpine meadow along an altitudinal gradient.The results showed that the plot with altitude of 4813m got a higher bacterial diversity in soil than the other plot with different altitude, and the geographical distance was the factors which affected the ecologically distribution of soil bacteria in this region. Temperature rise on the impact of the diversity of soil bacteria can not be ignored. In addition, other ecological factors are related to the diversity of soil bacterial community structure and ecological distribution, therefore,the mpact of other ecological factors on the diversity of soil bacteria can also not be ignored.更多还原