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Diazotrophs in Shore Pine

Diazotrophs in Shore Pine. Rebecca Huot OSU undergraduate USFS biological technician Dr. Richard Cronn HHMI Mentor USFS molecular geneticist. Nitrogen’s Role in Biology. ~ 80% of Earth’s atmosphere is N 2 Often the most limiting nutrient in an ecosystem

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Diazotrophs in Shore Pine

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

  2. Nitrogen’s Role in Biology ~ 80% of Earth’s atmosphere is N2 Often the most limiting nutrient in an ecosystem Crucial component of nucleic acids, amino acids, chlorophyll Havlin, J. L. et al., Soil Fertility and Fertilizers, 6th ed. (1999), Prentice Hall, Upper Saddle River, NJ, pg. 87

  3. Nitrogenase & NifH • N2 + 8e- + 8H+ + 16ATP  2NH3 + H2 + 16ADP + 16Pi • Nif genes present in free-living and • symbiotic nitrogen fixers • NifH encodes the nitrogenase Fe • protein subunit www.jic.bbsrc.ac.uk/staff/simon-george/ Nitrogenase FeMoCo active site (N2 binding site unknown; mechanism of subsequent reduction is not understood)

  4. N2-Fixing Microbe & Plant Interactions • Free-Living • Symbiotic • Root Nodulating • Legumes (Rhizobium) • Alder, etc. (Frankia) • Cycads, Podocarps (Cyanobacteria) • Non-nodulating • Recently being discovered widespread www.emc.maricopa.edu/faculty/ farabee/BIOBK/nodule_2.gif

  5. The Plant • 1 of 4 varieties • Dominate in nutrient-poor • and disturbance-prone • ecosystems Shore Pine (Pinus contorta var. contorta) Why Shore Pine? • Successional species • Roots of herbaceous and small woody plants • in close association with Shore Pine roots • Prior work shows a potential association • between Shore Pine and N2-fixer(s)

  6. Previous Work2000 - CurrentAlder Dunes, Florence, OR • 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) • 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 NorthwestDr. Vladimir Lebsky (Mexico)

  7. Hypothesis Fact: Symbiotic plant and microbe N2-fixing associations are found throughout the angiosperms HI: Plant and microbe N2-fixing associations should be commonplace in gymnosperms that colonize N-limiting environments HII: Shore Pine should harbor one or more root-associated N2-fixing microbe

  8. Study Objectives • Verify N2-fixing • capacity in situ • Determine microbe(s) distribution within Shore • Pine • Ascertain geographic distribution • Identify microbe(s)

  9. Nehalem Bay (Seaside) Alder Dunes (Florence) Pistol River (Gold Beach) Study Sites • 3 Sites • Nehalem Bay • Alder Dunes • Pistol River • 11 trees per site • 1 needle • 1 branch • 4 root (2 of 2) • 10 acetylene reduction assays per site • 5 roots (two locations on one root) • 2, 4 and 24 hr readings http://www.oregon101.com

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

  11. Analytical Methods • Enzymatic Assay • Acetylene reduction • Genetic Assay • Fall 2002 thru Spring 2003 • Use degenerate primers to amplify 16S rDNA, NifH gene from Li’s isolate • Cloned & sequenced to design microbe-specific NifH primers • Summer 2003 • Collect tissue • Extract DNA • Screen all tissues and trees for presence/absence of N2-fixing bacteria

  12. Enzymatic Assay:Acetylene Reduction Chemical similarity • Gaseous Nitrogen NN (converted to NH3) • Acetylene HC CH (converted to CH2=CH2)

  13. Acetylene Reduction Results Why aren’t Shore Pine acetylene reduction results reproducible between studies? Insignificant ethylene detected in all samples * Nitrogenase activity could not be confirmed in Shore Pine root samples • Sporadic spatial distribution of N2-fixers • Seasonal variation in microbe population sizes • Different climatic conditions at time of sampling

  14. Genetic Assay • DNA Extraction • ~ 75-100 mg of tissue; • FastDNA Kit (Q-Biogene) • Screen Tissues for NifH gene sequence • Restrictive PCR (isolate-specific NifH) • Relaxed PCR (all NifH – in progress) • Verify sequences (in progress) • Screen tissues for 16S rDNA diversity (in progress) • “universal” marker for all prokaryotes • identify how many microbe 16S present • sequence each unique microbe 16S rDNA found

  15. NifH PCR Screening M M M + + + + + + + + + M = Marker = Positive Control = NifH identical to original isolate = Other Potential N2-fixers M + + + + + + Roots 7 putative “NifH” size classes identified Needles Branches

  16. NifH Results • 33 trees sampled (3 sites) • 14 trees (42%) tested positive for NifH identical to original isolate • 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) • Root colonization by NifH isolate is geographically widespread

  17. NifH Results • 63% of NifH positives found in 5.1 mm – 10.0 mm root diameter class • 12% of all root samples tested positive for NifH gene • While widespread, N2-fixers are not uniformly distributed in roots NUMBER OF SAMPLES WITH NifH GENE PRESENT 1-2 3-4 5-6 7-8 9-10 11-12 13-24

  18. Summary • 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 • elucidate a possible new plant-microbe symbiotic relationship • provide an additional native specie for use in reclamation and restoration projects • be a comparison between method sensitivity: Acetylene Reduction assay vs. NifH PCR amplification • show that further investigation should be pursued Performed biochemical and DNA-based surveys for N2-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 N2-fixing bacteria isolate (~ 5 mm) NifH-PCR surveys appear more sensitive than Acetylene Reduction (measures presence of gene, not true nitrogenase activity)

  19. Acknowledgements Support Supervisors 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 HHMI URISC

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