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

这个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的生物地球化学过程有关的细菌和真菌。该研究的重点是执行氮循环过程的微生物的功能多样性，因为N是该生态系统中对树木生长最具限制性的营养物质。浓度主要是生产和消费的过程NO3因为他们的关键作用,为植物生长提供N和因为环境的重要性的N损失地下和地表水NO3或大气中N2 o .特定酶的功能多样性,生产和消费在土壤NO3位于特定的网站在安德鲁斯方面N-cycling已被证明的变化对人的影响(木材收获,燃烧)或自然演替事件（放线根到针叶树物种的过渡）将被检查。(1)研究原始针叶林采伐后次生演替过程中关键氮循环微生物的功能多样性变化；(2)确定草地草甸和针叶林植被对关键氮循环微生物功能多样性的影响；(3)确定放线根固氮共生的自然高N输入对关键N循环微生物功能多样性的影响；(4)研究外生菌根席的存在如何影响关键氮循环微生物的功能多样性。利用基于聚合酶链反应（PCR）的方法确定了关键N循环微生物的多样性。PCR引物将被选择为特定于编码选定N循环酶的基因的引物，集中于产生或消耗NO3 -的过程，如NH3单加氧酶，同化NO3 -还原酶和异化NO2 -还原酶。用末端限制性片段长度多态性（TRFLP-PCR）或不使用限制性内切酶（长度异质性或LH-PCR）酶切的凝胶电泳，根据大小对PCR产物进行分离，从而确定PCR产物的多样性。这些功能多样性的测量将与它们各自的过程速率的测量相关，以检查功能酶的类型及其在生态系统中的功能之间是否存在联系。如果发现过程速率和执行这些功能的酶系统的多样性之间有意义的关系，将尝试对这些基因进行测序，分离代表这些功能群的微生物，并通过16S rDNA测序对其进行表征。获得的任何分离株将在当地保存，并应要求提供给其他人。本研究产生的序列将存入GenBank。从历史上看，在安德鲁斯进行的创造性前沿研究和教育活动在增加对温带针叶林生态系统功能的理解方面发挥了重要作用。此外，这些活动已被用来推动更复杂的土地使用政策的发展和接受，以管理这一生产性生态系统。这个项目将引入分子生态学到这个生态系统，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。

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.