NIMO Course of Study


Created by George Rice, Montana State University


Root culture close-ups above show cultivated diazotrophs from roots. The culture is inoculated by stabbing clean roots into growth media containing various carbon sources, but no combined nitrogen. Bacterial growth from root surfaces can be seen as off-white smears.



Methods:


Non-destructive measurements of plant primary productivity at all sampling stations were initiated in the first year of this project ( 2003—2004) to record key sediment physical and pore-water chemical parameters. (Baseline determined in order to evaluate edaphic and biotic parameters likely to influence the distribution of diazatrophs in this environment.)

  • N2 fixation (acetylene reduction) rates in rhizosphere sediments was measured.

  • Rhizosphere microbiota DNA was extracted from rhizosphere cores and washed plant roots in each of the vegetation zones under study.

  • The gene encoding a key component of the nitrogenase complex, nifH, was used in this study as a molecular marker for diazotrophs. (The nifH gene is found exclusively in diazotrophs, and evolves slowly in a manner consistent with other genes that are useful for phylogenetic analysis.)

  • nifH sequences were amplified from the DNA recovered from the rhizosphere microbiota.

  • Diazotrophs in each study zone were then profiled using denaturing gradient gel electrophoresis (DGGE) of the newly characterized nifH amplicons.

Two students on a sampling transect in the salt marsh.



Results:


The characterization of over 900 new nifH diazotrophic sequences in this project has led to the following important discoveries.

  • Examination of newly characterized nifH sequences has revealed distinct patterns showing that different types of diazotrophs exist in different salt marsh vegetation zones.

  • Numerous new nifH sequences characterized in this project reflect novel types of organisms that had not been previously described.

  • A pure culture collection is being developed and isolation efforts have produced many new bacterial taxa, which are currently being characterized.

  • Characterization of newly isolated nifH sequences has shown the capacity of many familiar bacterial groups to fix nitrogen. Many of these bacterial groups had not previously been known to be nitrogen fixers.

Perspective of the marsh in the project sampling area.



Copyright on all images and material by Charles R. Lovell 2005.



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