Source: UNIVERSITY OF WASHINGTON submitted to
NITROGEN FIXATION WITHIN POPLAR BY ENDOPHYTIC BACTERIA
 
PROJECT DIRECTOR: Doty, S.
 
PERFORMING ORGANIZATION
ECOSYSTEM SCIENCES
UNIVERSITY OF WASHINGTON
SEATTLE,WA 98195
 
NON TECHNICAL SUMMARY: Professor Doty of the University of Washington's College of Forest Resources has discovered a bacterial endophyte within the stems of poplar plants (cottonwood trees; Populus), which are fast-growing deciduous, non-leguminous trees. Sequencing of ribosomal RNA genes of the poplar endophyte revealed that the bacteria is Rhizobium tropici. This diazotrophic (nitrogen-fixing) bacterium is known for its ability to nodulate an exceptionally wide range of legumes. However, there were previously no reports of this bacterium within non-legumes. If this rhizobium could fix atmospheric dinitrogen within the stems of poplar, then the plants would be able to grow in nitrogen-free medium. Indeed, cuttings of poplar grow well in hydroponic solution lacking nitrogen salts, unlike control plants of tobacco and unnodulated clover. If the endophytic rhizobium is truly providing fixed nitrogen within the stems of poplar trees, it would represent a new nitrogen-fixing symbiosis. If these four objectives can be met, the further study of the interaction between poplar and endophytic diatrophic bacteria would be a significant contribution to our understanding of how poplar, and possibly other nonleguminous plants, can obtain fixed nitrogen for growth.
 
OBJECTIVES: Under the hypothesis that poplar plants can grow under nitrogen limited conditions due to the presence of endophytic nitrogen-fixing bacteria, the following points need to be demonstrated: Poplar, both wild and laboratory hybrids, can grow under nitrogen limitation due to nitrogen fixation. Poplar contain diazotrophic endophytic bacteria. Internally-sterilized poplar cannot grow under nitrogen limitation. Sterilized poplar with the addition of poplar extract containing the bacteria can grow under nitrogen limitation.
 
APPROACH: To lessen our dependency on fossil fuels and to provide a more natural method of increasing plant growth, we require a greater understanding of nitrogen-fixing symbioses. The implication of the discovery of rhizobium within forest trees is profound, since nitrogen-fixation within a non-legume without the requirement for nodulation could provide an avenue for bringing nitrogen fixation to non-leguminous agricultural crops.
 
CRIS NUMBER: 0202663 SUBFILE: CRIS
PROJECT NUMBER: WNZ-1120 SPONSOR AGENCY: OCI
PROJECT TYPE: STATE PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Jul 1, 2004 TERMINATION DATE: Jun 30, 2005

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
203249911002.2100%

CLASSIFICATION HEADINGS
KA203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants
S2499 - Plant research, general
F1100 - Bacteriology
G2.2 - Increase Efficiency of Production and Marketing Systems


RESEARCH EFFORT CATEGORIES
BASIC (N/A)%
APPLIED 100%
DEVELOPMENTAL (N/A)%

KEYWORDS: nitrogen fixation; populus; endophytes; nitrogen fixing bacteria; stress tolerance; bacteriology; nitrogen; nutrient availability; hybrids; tree growth; extracts; rna; ribosomes; genes; bacterial genetics; gene expression; rhizobium; rna sequences; nodulation; symbiosis; symbionts

PROGRESS: Jul 1, 2004 TO Jun 30, 2005
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.

IMPACT: 2004-07-01 TO 2005-06-30 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.

PUBLICATION INFORMATION: 2004-07-01 TO 2005-06-30
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.

PROJECT CONTACT INFORMATION
NAME: Doty, S.
PHONE: 206-543-5774
FAX: (N/A)