Nitrogen Fixation
101
Jude Maul, USDA-ARS, Sustainable Agricultural Systems Lab
Beltsville, MD, 20770
jude.maul@ars.usda.gov
And
Julie Grossman, North Carolina State University
Department of Soil Science
julie_grossman@ncsu.edu
Nitrogen Fixation
101
Jude Maul, USDA-ARS, Sustainable Agricultural Systems Lab
Beltsville, MD, 20770
jude.maul@ars.usda.gov
And
Julie Grossman, North Carolina State University
Department of Soil Science
julie_grossman@ncsu.edu
Nitrogen Fixation
Today we will discuss:

Who fixes N? Types of bacterial N-fixers

Focus on legumes – nodulation and species specific relationships

How do we measure N-fixation?

Ways we are working on improving N-fixation in cropping systems.

Quick example of our work with hairy vetch
Nitrogen Fixation

More than 99% of N on Earth is unavailable!

N2 must be “fixed” by prokaryotes into ammonia to be used for
metabolic processes.

N is a primary constituent of proteins and nucleic acids, therefore
essential for life.
Legume based cropping system
N2
Crop residue
Biological
Nitrogen
Fixation
Mineral Nitrogen
Decomposition
Who can fix nitrogen?
Associative
N2
Phototrophic
Symbiotic
• Use C from rhizodeposits or
decaying wood
• Genera represented:
Azospirillum, Herbaspirillum,
Burkholderia
• Many tropical grasses have
them
Who can fix nitrogen?
Associative
• Use C from rhizodeposits or
decaying wood
• Genera represented:
Azospirillum, Herbaspirillum,
Burkholderia
• Many tropical grasses have them
Phototrophic
• Cyanobacteria in rice
paddies
• i.e. Azolla water fern and
Anabaena azollae symbiont
• High N-fixation ability - > 100
kg N ha-1 yr
• Biological soil crusts have
phototrophic N-fixers too
N2
Symbiotic
Who can fix nitrogen?
Associative
• Use C from rhizodeposits or
decaying wood
• Genera represented:
Azospirillum, Herbaspirillum,
Burkholderia
• Many tropical grasses have them
Phototrophic
• Cyanobacteria in rice
paddies
• i.e. Azolla water fern and
Anabaena azollae symbiont
• High N-fixation ability - > 100
kg N ha-1 yr
• Biological soil crusts have
phototrophic N-fixers too
N2
Symbiotic
• Legumes and rhizobia
• Actinorhizal plants and
Frankia
Who can fix nitrogen?
• Use C from rhizodeposits or
decaying wood
• Genera represented:
Azospirillum, Herbaspirillum,
Burkholderia
• Many tropical grasses have
them
Associative
N2
Phototrophic
Symbiotic
• Cyanobacteria in rice
paddies
• i.e. Azolla water fern and
Anabaena azollae symbiont
• High N-fixation ability - > 100
kg N ha-1 yr
• Biological soil crusts have
phototrophic N-fixers too
• Legumes and rhizobia
• Actinorhizal plants and
Frankia
Associative Fixers with tropical grasses:
Colonization of the corn root surface by A. brasilense at
the root elongation zone
Photo courtesy of Y.Okon (2002)
Azospirillum brasilense
(Patriquin, 1982)
Who can fix nitrogen?
• Use C from rhizodeposits or
decaying wood
• Genera represented:
Azospirillum, Herbaspirillum,
Burkholderia
• Many tropical grasses have
them
Associative
N2
Phototrophic
Diazotroph (def): Bacteria that
use N2 as their sole source of N.
Symbiotic
• Cyanobacteria in rice
paddies
• i.e. Azolla water fern and
Anabaena azollae symbiont
• High N-fixation ability - > 100
kg N ha-1 yr
• Biological soil crusts have
phototrophic N-fixers too
• Legumes and rhizobia
• Actinorhizal plants and
Frankia
Photosynthetic diazatrophs:
Cyanobacteria of different forms
•Common in aquatic
areas
•Photosynthesize
•Protect O2 through
membranes or
heterocysts
filamentous
Cyanobacteria in association with plants

Azolla used in Asia for centuries
in association with rice
Who can fix nitrogen?
• Use C from rhizodeposits or
decaying wood
• Genera represented:
Azospirillum, Herbaspirillum,
Burkholderia
• Many tropical grasses have
them
Associative
N2
Phototrophic
Diazotroph (def): Bacteria that
use N2 as their sole source of N.
Symbiotic
• Cyanobacteria in rice
paddies
• i.e. Azolla water fern and
Anabaena azollae symbiont
• High N-fixation ability - > 100
kg N ha-1 yr
• Biological soil crusts have
phototrophic N-fixers too
• Legumes and rhizobia
• Actinorhizal plants and
Frankia
Legume/rhizobia
symbiosis
Most terrestrial N2-fixing symbioses
involve a N2-fixing prokaryote and a
photosynthetic host.
How the nodules form

Chronological sequence between
plant and bacteria

Highly specific! A specific
bacterial species with one, or a
limited number of, plant species
Early visible steps in infection and nodule
formation by Rhizobium
 Steps in root hair infection in
Photos courtesy of J.Gen.
Microbiol.
clover:
*Rhizobium attachment to root
hairs,
*Localized enzyme production
causing wall softening and
root-hair curling
*penetration by ~20 rhizobia,
with fresh cell wall material
deposited around them
*Infection thread formation and
movement of the rhizobia
down the root hair
Nodule morphology
depends on the plant,
not the bacteria
Soybean root nodules
Clover root nodules are tiny!
Pea nodules large and round
Infection thread
O2
C
N
Mechanisms to avoid oxygen

High respiratory rate – Azotobacter

Specialized cells – heterocysts

Avoidance – anaerobic metabolism

O2 regulating proteins - leghemoglobin
Effective
nodules
contain
leghemoglobin
Nodules vary
in their
morphology
Energetics of N2 Fixation
Marschner, Mineral Nutrition of Higher Plants
N fixation decreases as soil
N fertility increases
Fixed N
Soil N pool
N additions
Drinkwater, 2004
Factors affecting BNF in the field
Photo by P. Graham

Acid soil factors

Temperature

Water availability

Soil nutrients (P, Mo, Fe, S, Ca,
Co, B, Ni)

Competition and persistence

Contact of rhizobia with fertilizer
or fungicide

Presence of suitable rhizobia
(see Inoculation)
When is it desirable to inoculate?
When a species is new to a
region, yield increases following
inoculation can reach 30-40% .

When the legume hasn’t been
previously cultivated in the region, or
hasn’t been cultivated for quite some
time

When environmental conditions are
unfavorable and could limit rhizobial
survival in soil

When soil analysis shows N
deficiency

When previous sowings have shown
good nodulation but little evidence of
nitrogen fixation
Commercial inoculant often
uses peat as a carrier
Qualities of a good inoculant strain
 Nodulates and
fixes N with
all the varieties of legume for
which it is recommended.
 Competes well with native
rhizobia in the soil
 Persistent over time in the soil
 Tolerant of environmental
conditions in the soil
 Genetically stable
 Grows well in simple and
economic culture media
Resident rhizobia can represent a barrier
to inoculant nodule formation
 Rhizobia in soil are from either


Inoculant, or
indigenous (native) soil populations
 Native rhizobia can be beneficial, or
problematic if they compete with
inoculant strain and are less efficient in
fixing N (Obaton et al., 2002; Botha et. al, 2004)

Competitiveness of a rhizobia strain is its
capacity to form nodules on a legume in the
presence of other rhizobia of the same
specificity.
Research
 Great advancements!
 Function for many N-fix genes

identified
Entire genetic code unraveled for
some strains
 However…
 Inoculant quality and availability often


poor
Improved N-fixation rarely considered
a breeding objective
Impacts of management on
nodulation and nitrogen fixation
mostly unknown