Introduction Nitrogen is one of the most essential nutrients for plant growth and development. However, plants are unable to use nitrogen in its natural atmospheric form. Many members of the legume family are able to develop symbiotic relationships with Rhizobia, which convert nitrogen to a usable form for plants in exchange for carbohydrates. The bacteria usually fix nitrogen for plants in structures called nodules, which develop on roots. Lupinus chamissonis (dune lupine) is a known nodulating species of the legume plant family. However, the identification of its microbial community remains unknown. As such, we seek to identify the bacterial associators of dune lupine and characterize their effect(s) on lupine development. Dune lupine is found natively along the California coast. In the Ballona wetlands and El Segundo sand dunes near Loyola Marymount University, there is an ongoing effort to understand the effects of urban runoff on sand dunes, where dune lupine grows. This study investigates how urban stresses, such as heavy metal contamination, can impact the partners in the Rhizobia-legume symbiosis. Materials and methods Nodules from L. chamissonis roots were surface sterilized, crushed, bacteria isolated, and pure cultures made of individual isolates. L. chamissonis and L. perennis seeds were grown axenically and inoculated with bacterial isolates. The 16S rDNA gene of selected strains were amplified by polymerase chain reaction (PCR) for sequencing analysis. Sequences were identified using BLAST to compare to public databases and a phylogenetic tree was generated. L. chamissonis and selected bacterial strains are being grown using media with various concentrations of zinc sulfate. Acknowledgments Funding was provided by the Loyola Marymount University Department of Biology. Special thanks to Elisabeth Ferris for her assistance throughout the project. Results Several of the bacterial isolates were chosen for further characterization and identified by 16S rDNA as Bradyrhizobium, Rhizobium, or Variovorax. The isolates did not nodulate L. perennis. Conclusions Nodulators of L.chamissonis have not yet been confirmed, though it likely one of the isolated bacteria from the nodules. High concentrations of zinc appears to stress L. chamissonis growth, causing longer shoots and shorter roots. Zinc also appears to stress the growth of the microbial community associated with L. chamissonis. Future work will characterize how the microbial community of L. chamissonis impacts plant growth and how zinc impacts this association. Decreasing numbers of dune lupine along its native California coast suggest that the species does not adapt well to the effects of urbanization. An increase of heavy metals due to urban runoff is likely to affect the growth of these plants, which provide ecosystem services to the near- extinct El Segundo Blue Butterfly, as well as other local wildlife. Continuing research on L. chamissonis and its nodulating symbiont is vital to ongoing restoration efforts of the Ballona wetlands. Michael Piña, Jessica Duong, Michelle Lum Department of Biology, Loyola Marymount University, Los Angeles, California Literature cited Y. Benhizia, H. Benhizia, A. Benguedouar, et al. Gamma proteobacteria can nodulate legumes of the genus Hedysarum. System. Appl. Microbiol. 27: A. Jarabo-Lorenzo, R. Perez-Galdona, J. Donate- Correa et al Genetic diversity of Bradyrhizobial populations from diverse geographic origins that nodulate Lupinus spp. and Ornithopus spp. Systematic and Appl. Microbiol. 26: For further information Please contact or More information on this and related projects can be obtained at A PDF-version of the poster is also available. Preliminary data shows that high concentrations of zinc stresses L. chamissonis. In high concentrations of zinc (100µM ZnSO ₄ ), L. chamissonis had longer shoots and shorter roots than those plants treated with lower concentrations of ZnSO ₄. Overall, plants grown in lower concentrations of ZnSO ₄ appeared healthier, with more robust leaves and a larger root system. Strain LCN3-4 showed the greatest resistance to increased zinc concentrations whereas strains LCN1-3, LCN3-1, and LCN3-2 were the most sensitive to zinc. Figure 4. Growth of nodule isolates on increasing concentration of zinc. Bacterial isolates from L. chamissonis nodules show decreased growth as the concentration of zinc sulfate increases, with the exception of strain LCN3-4. Effects of zinc on dune lupine and its microbial community No zinc5µM zinc 25µM zinc100µM zinc Figure 1. View of Ballona Wetlands in Los Angeles, CA Figure 2. Phylogenetic tree of L. chamissonis nodule isolates. Selected isolates were identified using BLAST. Ladder LCN3-1LCN1-3LCN2-2 + control LCN3-2LCN3-3 - control Ladder LCN5-3 Ladder LCN2-1LCN3-2 LCN3-6 + control - control A B C Figure 3. PCR products of 16S rDNA visualized by gel electrophoresis. (A,C) The positive control used was Burkholderia tuberum gDNA. The negative control used was water. (A,B,C) Red arrows denote band length of interest. Figure 5. Lupinus chamissonis grown in media containing zinc sulfate.