Diversity of Nitrogen-Cycling Microorganisms at the H.J. Andrews LTER

H.J. Andrews LTER 氮循环微生物的多样性

• 批准号: 9977933
• 负责人: David Myrold
• 金额: $ 56.14万
• 依托单位: Oregon State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-10-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9977933&HistoricalAwards=false
• 关键词: Diversity; Nitrogen; Cycling; Microorganisms; H.J.

This Microbial Observatory is dedicated to the study of bacteria and fungi central to biogeochemical processes of coniferous forest ecosystems associated with the H.J. Andrews Experimental Forest, located in the Cascade Mountains of Central Oregon. The research focuses on the functional diversity of microorganisms that perform nitrogen cycle processes because N is the most limiting nutrient to tree growth in this ecosystem. Concentration is primarily on the processes that produce and consume NO3 - because of their pivotal role in supplying N for plant growth and because of the environmental importance of N losses to ground and surface waters as NO3 - or to the atmosphere as N2 O. The functional diversity of specific enzymes that produce and consume NO3 - in soils located at specific sites at the Andrews where aspects of N-cycling have been shown to change in response to the influence of man (timber harvest, burning) or to natural successional events (actinorhizal to coniferous species transitions) will be examined. The following objectives are being pursued: (1) To determine how the functional diversity of key N-cycling microorganisms changes during secondary succession following the clear cutting of old-growth coniferous forest; (2) To determine the influence of grassland meadow and coniferous vegetation on the functional diversity of key N- cycling microorganisms; (3) To determine how naturally high inputs of N from actinorhizal N2 -fixing symbioses affect the functional diversity of key N cycling microorganisms; and (4) To examine how the presence of ectomycorrhizal mats affects the functional diversity of key N cycling microorganisms. The diversity of key N cycling microorganisms is being determined using methods based on the polymerase chain reaction (PCR). PCR primers will be selected that are specific to genes that code for selected N cycling enzymes, concentrating on those processes that produce or consume NO3 -, such as NH3 monooxygenase, assimilatory NO3 - reductase, and dissimilatory NO2 - reductase. The diversity of the PCR products will be determined by separating them by size using gel electrophoresis either with terminal restriction fragment length polymorphism (TRFLP-PCR) or without (length heterogeneity or LH-PCR) digestion with restriction enzymes. These measures of functional diversity will be related to measurements of their respective process rates to examine whether there is a link between the types of functional enzymes and their functioning in theecosystem. If meaningful relationships among the process rates and the diversity of the enzyme systems that carry out these functions are found, attempts will be made to sequence these genes, isolate the microorganisms representative of these functional groups, and characterize them by 16S rDNA sequencing. Any isolates obtained will be maintained locally and made available to others by request. Sequences generated by this research will be deposited in GenBank. Historically, creative cutting-edge research and educational activities conducted at the Andrews have played a major role in increasing understanding of how temperate coniferous forest ecosystems function. Furthermore, these activities have been used to drive thedevelopment and acceptance of more sophisticated land-use policies for managing this productive ecosystem. This project will introduce molecular ecology to this ecosystem and use it to further an understanding of how disturbance and plant succession influence the microbial communities that drive the biogeochemical processes.

该微生物观测站致力于研究与位于俄勒冈州中部喀斯喀特山脉的 H.J. 安德鲁斯实验森林相关的针叶林生态系统生物地球化学过程的核心细菌和真菌。该研究重点关注执行氮循环过程的微生物的功能多样性，因为氮是该生态系统中树木生长最限制的营养物质。 主要关注产生和消耗 NO3 的过程，因为它们在为植物生长提供氮方面发挥着关键作用，也因为氮以 NO3 的形式流失到地下水和地表水，或以 N2 O 的形式流失到大气中对环境的重要性。产生和消耗 NO3 的特定酶的功能多样性位于安德鲁斯特定地点的土壤中，其中氮循环的各个方面已被证明会因人类的影响而发生变化。 （木材采伐、燃烧）或自然演替事件（放线根到针叶树种的过渡）将受到审查。 正在追求以下目标：（1）确定主要氮循环微生物的功能多样性在老针叶林砍伐后的次生演替过程中如何变化； （2）确定草原草甸和针叶植被对关键氮循环微生物功能多样性的影响； (3) 确定来自放线菌固氮共生体的自然高氮输入如何影响关键氮循环微生物的功能多样性； (4) 研究外生菌根垫的存在如何影响关键氮循环微生物的功能多样性。 关键氮循环微生物的多样性正在使用基于聚合酶链反应（PCR）的方法进行测定。 将选择对编码所选 N 循环酶的基因特异的 PCR 引物，集中于产生或消耗 NO3 - 的那些过程，例如 NH3 单加氧酶、同化 NO3 - 还原酶和异化 NO2 - 还原酶。 PCR 产物的多样性将通过使用凝胶电泳按大小分离来确定，无论是使用末端限制性片段长度多态性 (TRFLP-PCR) 或不使用限制性酶消化（长度异质性或 LH-PCR）。这些功能多样性的测量将与其各自的加工速率的测量相关，以检查功能酶的类型与其在生态系统中的功能之间是否存在联系。 如果发现处理速率和执行这些功能的酶系统多样性之间有意义的关系，将尝试对这些基因进行测序，分离代表这些功能组的微生物，并通过 16S rDNA 测序来表征它们。 获得的任何分离株将在本地保存，并根据要求提供给其他人。本研究产生的序列将存入GenBank。 从历史上看，安德鲁斯开展的创造性前沿研究和教育活动在增进人们对温带针叶林生态系统功能的了解方面发挥了重要作用。此外，这些活动还被用来推动更复杂的土地利用政策的制定和接受，以管理这个富有成效的生态系统。该项目将分子生态学引入该生态系统，并利用它来进一步了解干扰和植物演替如何影响驱动生物地球化学过程的微生物群落。