Rebecca Huot OSU undergraduate USFS biological technician Dr. Richard Cronn HHMI Mentor USFS molecular geneticist Diazotrophs in Shore Pine

Nitrogen’s Role in Biology Havlin, J. L. et al., Soil Fertility and Fertilizers, 6 th ed. (1999), Prentice Hall, Upper Saddle River, NJ, pg. 87 ~ 80% of Earth’s atmosphere is N 2 Often the most limiting nutrient in an ecosystem Crucial component of nucleic acids, amino acids, chlorophyll

N 2 + 8e - + 8H ATP  2NH 3 + H ADP + 16P i Nif genes present in free-living andNif genes present in free-living and symbiotic nitrogen fixers symbiotic nitrogen fixers NifH encodes the nitrogenase FeNifH encodes the nitrogenase Fe protein subunit protein subunit Nitrogenase & NifH Nitrogenase FeMoCo active site (N 2 binding site unknown; mechanism of subsequent reduction is not understood)

Free-LivingFree-Living SymbioticSymbiotic –Root Nodulating Legumes (Rhizobium)Legumes (Rhizobium) Alder, etc. (Frankia)Alder, etc. (Frankia) Cycads, Podocarps (Cyanobacteria)Cycads, Podocarps (Cyanobacteria) –Non-nodulating Recently being discovered widespreadRecently being discovered widespread N 2 -Fixing Microbe & Plant Interactions

1 of 4 varieties 1 of 4 varieties Dominate in nutrient-poor Dominate in nutrient-poor and disturbance-prone and disturbance-prone ecosystems ecosystems Shore Pine (Pinus contorta var. contorta) Successional species Successional species Roots of herbaceous and small woody plants Roots of herbaceous and small woody plants in close association with Shore Pine roots in close association with Shore Pine roots Prior work shows a potential association Prior work shows a potential association between Shore Pine and N 2 -fixer(s) between Shore Pine and N 2 -fixer(s) The Plant Why Shore Pine?

Previous Work Current Alder Dunes, Florence, OR Dr. C.Y. Li (USFS)Dr. C.Y. Li (USFS) –Cultured microbe from root tissue –Acetylene reduction assays from culture –In situ visualization –Putative identification = Yersinia sp. Michele Romanelli (Italy - M.S. student)Michele Romanelli (Italy - M.S. student) –Repeated microbial isolation and acetylene reduction assays; verified in situ localization –Acetylene reduction assay from fresh root tissue 7750 x SEM photo by Electron Microscopist of the Center for Biological Research of the Northwest Dr. Vladimir Lebsky (Mexico)

Hypothesis Fact: Symbiotic plant and microbe N 2 -fixing associations are found throughout the angiosperms N 2 -fixing associations are found throughout the angiosperms H I : Plant and microbe N 2 -fixing associations should be commonplace in gymnosperms that colonize N-limiting environments H II : Shore Pine should harbor one or more root-associated N 2 -fixing microbe

Study Objectives Verify N 2 -fixingVerify N 2 -fixing capacity in situ capacity in situ Determine microbe(s) distribution within Shore Determine microbe(s) distribution within Shore Pine Pine Ascertain geographic distribution Ascertain geographic distribution Identify microbe(s) Identify microbe(s)

Study Sites 3 Sites3 Sites –Nehalem Bay –Alder Dunes –Pistol River 11 trees per site11 trees per site –1 needle –1 branch –4 root (2 of 2) 10 acetylene reduction assays per site10 acetylene reduction assays per site –5 roots (two locations on one root) –2, 4 and 24 hr readings Nehalem Bay (Seaside)Alder Dunes (Florence)Pistol River (Gold Beach)

Shore Pine Roots 4 samples per tree (2 each 4 samples per tree (2 each from 2 roots) from 2 roots) Diameter range = Diameter range = 2.5 mm to 20.0 mm 2.5 mm to 20.0 mm Wash 1 x in 0.5% Tween detergent; Wash 1 x in 0.5% Tween detergent; 3 x in dH2O 3 x in dH2O

Analytical Methods Enzymatic AssayEnzymatic Assay –Acetylene reduction Genetic AssayGenetic Assay –Fall 2002 thru Spring 2003 Use degenerate primers to amplify 16S rDNA, NifH gene from Li’s isolateUse degenerate primers to amplify 16S rDNA, NifH gene from Li’s isolate Cloned & sequenced to design microbe-specific NifH primersCloned & sequenced to design microbe-specific NifH primers –Summer 2003 Collect tissueCollect tissue Extract DNAExtract DNA Screen all tissues and trees for presence/absence of N 2 -fixing bacteriaScreen all tissues and trees for presence/absence of N 2 -fixing bacteria

Chemical similarity –Gaseous Nitrogen N  N (converted to NH 3 ) (converted to NH 3 ) –Acetylene HC  CH (converted to CH 2 CH 2 ) (converted to CH 2 =CH 2 ) Enzymatic Assay: Acetylene Reduction

Why aren’t Shore Pine acetylene reduction results reproducible between studies? Sporadic spatial distribution of N 2 -fixersSporadic spatial distribution of N 2 -fixers Seasonal variation in microbe population sizesSeasonal variation in microbe population sizes Different climatic conditions at time of samplingDifferent climatic conditions at time of sampling Acetylene Reduction Results Insignificant ethylene detected in all samples * Nitrogenase activity could not be confirmed in Shore Pine root samples

Genetic Assay DNA Extraction ~ mg of tissue;~ mg of tissue; FastDNA Kit (Q-Biogene) FastDNA Kit (Q-Biogene) Screen Tissues for NifH gene sequence Restrictive PCR (isolate-specific NifH)Restrictive PCR (isolate-specific NifH) Relaxed PCR (all NifH – in progress)Relaxed PCR (all NifH – in progress) Verify sequences (in progress)Verify sequences (in progress) Screen tissues for 16S rDNA diversity (in progress) “universal” marker for all prokaryotes“universal” marker for all prokaryotes identify how many microbe 16S presentidentify how many microbe 16S present sequence each unique microbe 16S rDNA foundsequence each unique microbe 16S rDNA found

NifH PCR Screening Needles 7 putative “NifH” size classes identified Roots + M MM = Marker = Positive Control = NifH identical to original isolate = Other Potential N 2 -fixers + M M Branches

33 trees sampled (3 sites)33 trees sampled (3 sites) 14 trees (42%) tested positive for NifH identical to original isolate14 trees (42%) tested positive for NifH identical to original isolate Found primarily in root tissue (16 of 19 cases)Found primarily in root tissue (16 of 19 cases) NifH gene found in 2 branches and 1 needle sample (roots from these trees tested positive for NifH)NifH gene found in 2 branches and 1 needle sample (roots from these trees tested positive for NifH) NifH Results Root colonization by NifH isolate is geographically widespread Root colonization by NifH isolate is geographically widespread

63% of NifH positives found in 5.1 mm –63% of NifH positives found in 5.1 mm – 10.0 mm root diameter class 10.0 mm root diameter class 12% of all root samples tested positive for NifH gene12% of all root samples tested positive for NifH gene While widespread, N 2 -fixers are not uniformly distributed in rootsWhile widespread, N 2 -fixers are not uniformly distributed in roots NifH Results NUMBER OF SAMPLES WITH NifH GENE PRESENT

Summary Performed biochemical and DNA-based surveys for N 2 -fixation in coastal Shore Pine NifH verified in roots of plants at all three locations Enzymatic and genetic assays indicate sporadic microbe distribution within plant and ecosystem Confirmed “ideal” root diameter for Li’s N 2 -fixing bacteria isolate (~ 5 mm) NifH-PCR surveys appear more sensitive than Acetylene Reduction (measures presence of gene, not true nitrogenase activity) has potential to be enhanced work to be continued: identify other putative n-fixers 16S work I hope this study serves to increase our understanding of chemical processes involved in succession 2.elucidate a possible new plant-microbe symbiotic relationship 3.provide an additional native specie for use in reclamation and restoration projects 4.be a comparison between method sensitivity: Acetylene Reduction assay vs. NifH PCR amplification 5.show that further investigation should be pursued

Acknowledgements SupportSupervisors Dr. Richard Cronn Dr. C.Y. Li Dr. Dave Myrold Dr. Bernard Bormann Lab Assistance Sue Huber Sarah Shaffar Field Crew Shaun Sims John Schenk Funding from USDA USFS HHMIURISC