Exploring the Symbiosis of Frankia Bacteria with the Invasive Shrub, Autumn Olive Andrew DeVries, Dr David Dornbos, Dr John Wertz, Calvin College Introduction Elaeagnus umbellata, Autumn Olive, is a deciduous, actinorhizal shrub and invasive in and non-native to Michigan. Autumn Olive, a Japan native, was introduced specifically to re-vegetate the low pH coal mine spoils in southern Illinois and Indiana and to reduce the erosion along roadsides after construction. It now thrives in soils particularly low in nitrogen: forest understories, prairies, and abandoned farm fields. Its advantage in nitrogen-poor soil is likely derived from nitrogen reduced by the symbiotic relationship with a bacteria species, Frankia. In 1978, a team of researchers at Harvard discovered the slow growing bacteria, Frankia, was responsible for the nitrogen fixing properties of sweet fern (another actinorhizal plant). Frankia is a nitrogen-fixing bacteria (has a nifH gene that allows for coding the nitrogenase enzyme) that takes nitrogen in its atmospheric form (N2) to ammonium (NH4+), which provides the plant with sufficient nitrogen, a growth limiting factor. This allows for Autumn Olive and similar actinorhizal plants to have a significant advantage, providing at least partial explanation for their ability to thrive in a nutrient-poor ecosystems. Objectives Our initial goal was to compare the Frankia in the Autumn Olive nodules among different geographic locations and among different environments due to morphological differences among plants in these areas. However, Frankia was not successfully cultured this summer, so the goal changed. The new goal was to compare the bacterial communities among different ecosystems (forest and meadow) and to identify secondary bacterial symbionts capable of reducing nitrogen by culturing bacteria and cloning vectors. Given that plant genetic composition is the same for meadow and forest Autumn Olive, I hypothesize that the bacterial communities found within the root nodules will also be the same. Because biological systems are incredibly complex, I hypothesize that secondary symbionts are possible, if not likely, to be at work in this symbiosis. Results Methods Noncultivated Bacteria Isolates Collection of Root Nodules: The root nodules were collected at Pierce Cedar Creek Institute (PCCI) and Wittenbach/Wege Environmental Center. The plants were dug out, placed into pots and taken directly to Calvin. Preparation of Root Nodules: The nodules were removed from the roots, washed with sterile water, sterilized in 70% ethanol (killing any surface soil bacteria), chopped with a sterile knife, and then homogenized. The homogenizer was rinsed with sterile water and then ethanol between each nodule. PCR Amplification: 16S and nifH: The extracted DNA was placed into a 96 well plate. General Bacteria and Frankia 16S rRNA-specific and Frankia-specific nifH primers were added to the extracted DNA along with GoTaq polymerase and dNTP’s. The 16S rRNA and/or nifH genes were amplified in PCR reactions. DNA Sequencing: In preparation for DNA sequencing, the PCR products were cleaned with ExoSAP-it, and the appropriate forward primers used in the PCR reactions were added. The cleaned products were then sent to Michigan State University for sequencing. Plating and Isolating the Bacteria: Plating the bacteria was done via spread method with dilutions ranging from 10-1 to 10-3. Later in the summer, the 10^-3 dilution was cut out to not waste plates (no bacteria would grow on this dilution). Bacteria were plated either with Novobiocin, an antibiotic, or without to prevent from contamination. DNA Extraction: One loop of isolated bacteria was placed in 500 microliters of sterile nanopure water, heated in a dry bath at 90 degrees Celcius for fifteen to thirty minutes, then frozen at -20 degrees Celcius. Non-Cultivation Based Microbial Analysis of Root Nodules: The nodules were removed from the roots, and homogenized as described in “Preparation of Root Nodules.” The total DNA from the root nodules was extracted and PCR amplified with the same primers as described above. PCR products were Topo-TA cloned into Top10 E.coli. After bluewhite screening, the E. coli were cultured in 96-well deep plates. Each clone was PCR amplified with the same primers as above, the PCR products cleaned, and sent to Michigan State University for sequencing. Results Types of Bacteria Found in AO Nodules Located in the Forest Types of Bacteria Found in Autum n Olive Nodules Located in the Meadow Gamma Proteobacteria Gamma Proteobacteria Bacilli Bacilli Beta Proteobacteria Beta Proteobacteria Alpha Proteobacteria Alpha Proteobacteria Actinomycetales Actinomycetales Acknowledgements David Dornbos, John Wertz Lori Keen Calvin College HHMI Michigan State University Piece Cedar Creek Institute Flat Iron Lake Preserve Wittenbach/Wege Environmental Center Figure 1. Cultivation-Based Analyses of Bacterial Communities: These pie charts show the differences in the bacterial classes of Autumn Olive root nodules between the forest ecosystem and the meadow ecosystem. The forest has a heavy amount of alpha proteobacteria, with very few bacilli species. The meadow system is majorly Gammaproteobacteria, with very few Actinomycetales. A Chi Squared Test was used to compare the two data sets, and it showed a significant difference in the communities (n = 94, p =0.0005) Figure 2. Detection of Putative Secondary Symbionts: Throughout the summer several bacteria were found that are closely related to organisms found to be important to the nitrogen economy of plants. Among the cultured bacteria, several nitrogen fixing bacteria were isolated including: Mesorhizobium, Bradyrhizobium, Sinorhizobium, and Herbaspirrillus seropedicae. Non-cultivation based bacterial identification, Sphingobium sp. and Variovorax paradoxus were found (along with Frankia). These species were also cultured, and have been shown to reduce nitrogen compounds. Frankia Variovorax Paradoxus Shingobium sp Ralstonia syzygii Figure 3. NifH-Based Detection of Putative Nitrogen Fixing Bacteria in Autumn Olive Root Nodules. Noncultivation based approaches showed high amounts of Frankia-related nifH as well as nifH from several other bacterial species. Conclusions Cultivation-based methods yielded no Frankia species from Autumn Olive root nodules. However, when non-cultivation based methods were used, Frankia-specific nifH genes were detected. A lack of cultivation success could be due to the fact that Frankia is a slow grower and it was either outcompeted by fast-growing bacteria or killed on plates that contained novobiocin (not all species of Frankia are resistant to this antibiotic). The cultivated bacterial communities of nodules in the forest and nodules in the meadow were statistically different. These differences in the Autumn Olive nodules depending on the ecosystem leads to some new, interesting questions: Are the nodules dependent on the bacterial soil compositions in a given ecosystem? What could the difference in bacterial communities mean to the growth of the Autumn Olive? This research raises the possibility that there may be secondary symbionts that assist in the Autumn Olive/Frankia symbiosis. Some Sphingobium are found to reduce nitrite to nitrate, an important step in the nitrogen cycle for plants. Variovorax paradoxus is known for promoting plant growth, and has been shown to reduce nitrite to ammonium. While this research gives evidence that they may be secondary symbionts, only through future experimentation can we confidently state that these bacteria are. References "Sphingobium Scionense Sp. Nov., an Aromatic Hydrocarbon-degrading Bacterium Isolated from Contaminated Sawmill Soil." International Journal of Systematic and Evolutionary Microbiology. N.p., n.d. Web. 30 Sept. 2013. Nishino, Shirely and Jim Spain. “Biodegradation of 3-Nitrotyrosine by Burkholderia sp. Strain JS165 and Variovorax paradocus JS171.” Applied and Environmental Microbiology. Web. 30 Sept 2013. Del Tredici, Peter. "A Nitrogen Fixation: The Story of the Frankia Symbiosis." (n.d.): n. pag. Web. 30 Sept. 2013. Benson, David R. 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