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
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
Nitrogenase & NifH
• 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/
N2 + 8e- + 8H+ + 16ATP  2NH3 + H2 + 16ADP + 16Pi
Nitrogenase FeMoCo active site
(N2 binding site unknown; mechanism of
subsequent reduction is not understood)
N2-Fixing Microbe & Plant Interactions
• Free-Living
• Symbiotic
– Root Nodulating
www.emc.maricopa.edu/faculty/
farabee/BIOBK/nodule_2.gif
• Legumes (Rhizobium)
• Alder, etc. (Frankia)
• Cycads, Podocarps (Cyanobacteria)
– Non-nodulating
• Recently being discovered widespread
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)
Previous Work
2000 - Current
Alder Dunes, Florence, OR
• Dr. C.Y. Li (USFS)
– Cultured microbe from root tissue
7750 x
– Acetylene reduction assays from culture
– In situ visualization
– Putative identification = Yersinia sp.
SEM photo by Electron
Microscopist of the Center for
Biological Research of the
Northwest Dr. Vladimir
Lebsky (Mexico)
• Michele Romanelli (Italy - M.S. student)
– Repeated microbial isolation and acetylene reduction assays;
verified in situ localization
– Acetylene reduction assay from fresh root tissue
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
Study
Objectives
• Verify N2-fixing
capacity in situ
• Determine microbe(s) distribution within Shore
Pine
• Ascertain geographic distribution
• Identify microbe(s)
Study Sites
• 3 Sites
– Nehalem Bay
– Alder Dunes
– Pistol River
Nehalem Bay (Seaside)
• 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
Alder Dunes (Florence)
Pistol River (Gold Beach)
http://www.oregon101.com
Shore
Pine
Roots
• 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
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
Enzymatic Assay:
Acetylene Reduction
Chemical similarity
– Gaseous Nitrogen NN
(converted to NH3)
– Acetylene HC CH
(converted to CH2=CH2)
Acetylene Reduction Results
Insignificant ethylene detected in all samples
* Nitrogenase activity could not be confirmed in Shore
Pine root samples
Why aren’t Shore Pine acetylene reduction
results reproducible between studies?
• Sporadic spatial distribution of N2-fixers
• Seasonal variation in microbe population sizes
• Different climatic conditions at time of sampling
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
NifH PCR Screening
Roots
M
7 putative
“NifH” size
classes
identified
+
+
M
+
M
+
+
+
+
+
+
Needles
Branches
+
+
M
+
+
+
M = Marker
+ = Positive Control
= NifH identical to original isolate
= Other Potential N2-fixers
NifH Results
GEOGRAPHIC LOCATION OF ROOTS
WITH NifH BAND (N=16)
Alder Dunes
31%
Pistol River
19%
Nehalem Bay
50%
• 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
NifH Results
• 63% of NifH
positives found
in 5.1 mm –
10.0 mm root
diameter class
ROOT DIAMETERS THAT CONTAIN NifH
WITH
OF SAMPLES
NUMBER
NUMBER
OF SAMPLES
WITH
NifH
GENE
PRESENT
NifH GENE
PRESENT
6
5
• 12% of all root
samples tested
positive for
NifH gene
4
3
2
1
0
1-2
3-4
5-6
7-8
9-10
11-12
ROOT DIAMETER (mm)
Nehalem Bay
Alder Dunes
Pistol River
13-24
• While
widespread,
N2-fixers are
not uniformly
distributed in
roots
Summary
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)
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