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

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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.