Progress 07/01/04 to 06/30/05

Outputs
Objective One: Poplar can grow under nitrogen limitation due to nitrogen fixation. With the RRF award, I was able to purchase 15N2 labeled gas for repeated exposures of the poplar in nitrogen-free medium. After 30 days, I collected the new growth for 15N analysis. The dosed plant had twice the level of 15N incorporated into the new tissue than had undosed plant tissue, demonstrating that nitrogen fixation did occur. However, the plants grew poorly in these small vials, so I purchased an instrument that can quantify nitrogen fixation without enclosing the plants. The Qubit apparatus measures the hydrogen gas by-product of nitrogen fixation. The owner of the company designed a stem chamber specifically for my experiments. I collected cuttings of cottonwood both from greenhouses and from the Snoqualmie River site and grew the cuttings in nitrogen-free solution until they had extensive roots. We are in the process of testing the cuttings for nitrogen fixation now. Objective Two: Poplar contain endophytic bacteria. With this award, I was able to finish enough experiments to complete a descriptive paper on the first endophyte I found within poplar. This paper is now in press in Symbiosis. We also isolated many more endophytes from poplar in its native habitat at the Snoqualmie River. We did PCR amplification of the nitrogenase gene, and found two isolates with an especially clear signal. We verified the identity of the gene by sequencing, and then sequenced the 16S gene of these isolates. The endophytes are a close match to Rahnella aquatilis that was also isolated recently as an endophyte from Norway Spruce. These isolates from wild poplar grew well on medium without ammonium or nitrate (Ashbys Nitrogen-Free Medium). The bacteria grew in the presence of oxygen so perhaps the extreme levels of exopolysaccharide produced by these strains help to protect nitrogenase from inhibitory oxygen. These two isolates will be the focus of further studies (see Objective Four). Objective Three: Sterile poplar cannot grow under limitation We grew many sterile tips of poplar for this experiment, and demonstrated that sterile poplar plantlets cannot grow in the nitrogen limited medium. There is little to no growth, and the leaves become pale. Objective Four: Poplar containing endophytes can grow under nitrogen-limitation In an effort to satisfy Kochs postulates, we grew sterile cuttings of cottonwood and aspen, inoculated them either with the endophyte or with the type strain of Rhizobium tropici, or mock-inoculated them, and transferred them to nitrogen-free medium. In some cases, the plants inoculated with the endophyte stayed green longer than the other test plants, but overall, none of the plants were healthy. Therefore, either the inoculation technique was insufficient to allow effective colonization of the plant or the isolate under study is not the primary provider of fixed nitrogen. Since non-sterile cuttings of cottonwood grow vigorously in nitrogen-free medium, I will repeat these experiments with the other isolates described above under Objective Two.

Impacts
The impact of this award was substantial. As a junior faculty member, I had no grants as Principal Investigator (I have two as co-P.I. but on another topic) and I had no grants or publications on the subject of this proposal. It had been a side project for several years while I was a post-doc. But when I received this award, I was able to immediately hire personnel as well as purchase the supplies I needed, produce enough data for a manuscript, and have enough preliminary data for the writing of several federal grant proposals.

Publications

• Identification of an endophytic Rhizobium in stems of Populus. 2005. Sharon L. Doty, Megan R. Dosher, Glenda L. Singleton, Allison L. Moore, Benoit Van Aken, Reinhard F. Stettler, Stuart E. Strand, and Milton P. Gordon. Symbiosis 39(1):27-36.