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1. SEMINAR ON
BIOLOGICAL NITROGEN FIXATION WITH
SPECIAL REFERENCE TO RHIZOBIAL ROOT
NODULE SYMBIOSIS & ACTINORHIZAL
SYMBIOSIS
PAPER -304
SUBMITTED TO:
PROF. BIMAN KUMAR DUTTA
DEPT. OF ECOLOGY &
ENVTL. SCIENCE
Submitted by:
SUBRATA PAUL
Roll No. 31
M.Sc. 3 rd sem

2. INTRODUCTION
Nitrogen is required by all living organisms for
the synthesis of proteins, nucleic acids and other
nitrogen-containing compounds. The earth’s
atmosphere contains almost 80% nitrogen gas. It
cannot be used in this form by most living organisms
until it has been fixed, that is reduced (combined with
hydrogen), to ammonia. Green plants, the main
producers of organic matter, use this supply of fixed
nitrogen to make proteins that enter and pass through
the food chain. Microorganisms (the decomposers)
break down the proteins in excretions and dead
organisms, releasing ammonium ions. These two
processes form part of the nitrogen cycle.

3. REACTION MICRO-ORGANISM CONDITION
PROCESS
Nitrogen fixation Nitrogen-fixing bacteria eg Rhizobium
aerobic/anaerobic. The first step in the synthesis of virtually all
nitrogenous compounds. Nitrogen gas is fixed into forms other
organisms can use.
Ammonification (decay)
Ammonifying bacteria (decomposers).
The decomposers, certain soil bacteria and fungi, break
down proteins in dead organisms and animal wastes
releasing ammonium ions which can be converted to other
nitrogen compounds. Nitrification Nitrifying bacteria ,eg
Nitrosomonas &Nitrobacter
Aerobic Nitrification is a two-step process. Ammonia or
ammonium ions are oxidized first to nitrites and then to
nitrates, which is the form most usable by plants.
Denitrification Denitrifying bacteria anaerobic Nitrates are
reduced to nitrogen gas, returning nitrogen to the air and
completing the cycle.

4. BIOLOGICAL FIXATION
The reduction of nitrogen gas to ammonia is
energy intensive. It requires 16 molecules of ATP
and a complex set of enzymes to break the
nitrogen bonds so that it can combine with
hydrogen. Its reduction can be written as:
energy
N2 + 3H2 2NH2
Fixed nitrogen is made available to plants
by the death and lysis of free living nitrogen-fixing
bacteria or from the symbiotic association of some
nitrogen-fixing bacteria with plants.

5. RHIZOBIUM
Rhizobium is the most well known species of a group
of bacteria that acts as the primary symbiotic fixer of
nitrogen. These bacteria can infect the roots of
leguminous plants, leading to the formation of lumps or
nodules where the nitrogen fixation takes place. The
bacterium’s enzyme system supplies a constant source of
reduced nitrogen to the host plant and the plant furnishes
nutrients and energy for the activities of the bacterium.
About 90% of legumes can become nodulated.
In the soil the bacteria are free living and motile, feeding
on the remains of dead organisms. Free living rhizobia
cannot fix nitrogen and they have a different shape from
the bacteria found in root nodules. They are regular in
structure, appearing as straight rods; in root nodules the
nitrogen-fixing form exists as irregular cells called
bacteroids which are often club and Y-shaped.

6. ROOT NODULE FORMATION
Sets of genes in the bacteria control different
aspects of the nodulation process. One Rhizobium
strain can infect certain species of legumes but not
others e.g. the pea is the host plant to Rhizobium
leguminosarum biovar viciae, whereas clover acts as
host to R. leguminosarum biovar trifolii. Specificity
genes determine which Rhizobium strain infects
which legume. Even if a strain is able to infect a
legume, the nodules formed may not be able to fix
nitrogen.
Such rhizobia are termed ineffective. Effective
strains induce nitrogen-fixing nodules. Effectiveness
is governed by a different set of genes in the bacteria
from the specificity genes. Nod genes direct the
various stages of nodulation.

7. ROOT NODULE FORMATION STEPS
Root system with
root nodules

8. CONT……….
Root Hair

9. CONT……….
Root hair covered by
free-living Rhizobia

10. CONT…………..
root hair starts to
formation of an
infection curl

11. CONT……..
Formation of an
infection
thread through which
rhizobia enter root
cells

12. CONT………
infection thread
spreads
into adjacent cells

13. bacteroids are
released from the
infection thread

14. CONT……….
Bacteroids

15. BASIC TYPES OF ROOT NODULES
Determinate:
Short, predestined
lifespan (days-weeks)
New nodules form
& old nodules
sloughed off
(older branches)
as root grows
Soybean nodules
Indeterminate:
Longer lifespan
(many months)
New root cells
become infected
by old cells
Plants with apical
meristem
(alfalfa)

16. NITROGENASE
An enzyme called nitrogenase catalyses the
conversion of nitrogen gas to ammonia in
nitrogen-fixing organisms. In legumes it only
occurs within the bacteroids. The reaction
requires hydrogen as well as energy from ATP.
The nitrogenase complex is sensitive to oxygen,
becoming inactivated when exposed to it. This is
not a problem with free living, anaerobic
nitrogen-fixing bacteria such as Clostridium.
Free living aerobic bacteria have a variety of
different mechanisms for protecting the
nitrogenase complex, including high rates of
metabolism and physical barriers.

17. CONT.
Azotobacter overcomes this problem by
having the highest rate of respiration of any
organism, thus maintaining a low level of oxygen
in its cells. Rhizobium controls oxygen levels in
the nodule with leghaemoglobin. This red, iron-containing
protein has a similar function to that
of haemoglobin; binding to oxygen. This
provides sufficient oxygen for the metabolic
functions of the bacteroids but prevents the
accumulation of free oxygen that would destroy
the activity of nitrogenase. It is believed that
leghaemoglobin is formed through the
interaction of the plant and the rhizobia as
neither can produce it alone.

18. ACTINORHIZAL SYMBIOSIS: A HISTORICAL
PERSPECTIVE
The term “actinorhizal” was proposed at
the first international meeting on “Symbiotic Nitrogen
Fixation in Actinomycete nodulated Plants”, held at
Harvard Forest, to provide a convenient and more
positive designation for the field than the term “non-legume”
(Torrey and Tjepkema, 1979). Following the
first reproducible isolation of the microorganism by
the John Torrey’s group (Callaham et al., 1978),
which was a watershed event in the development of
Frankia-actinorhizal research, there was a rapid
increase internationally in the number of scientists
interested in Frankia and an exponential increase in
the number of isolates in culture..

19. TAXONOMY AND EVOLUTION OF THE HOST PLANT
AND NEW NODULATING GENERA
There are eight Angiosperm families known to be
nodulated by Frankia. Until 1979, only seven families
were commonly known to be actinorhizal hosts, when
Chaudhary (1979) reported that Datisca
(Datiscaceae) also forms Frankia symbioses.
Interestingly, Datisca was first described as a
nodulated plant by Severini (1922), but this report
had gone relatively unnoticed until Chaudhary’s
rediscovery. Three new genera of the Casuarinaceae
were defined by dividing the former genus Casuarina
into Casuarina, Allocasuarina, Gymnostoma and
Ceuthostoma (Johnson, 1980; 1982; 1988).
Nodulation of species in the first three of these
genera has been observed regularly

20. CONT………
This focused attention on the taxonomy
of Frankia and necessitated the
establishment of a classification system. At
the international conference on the “Biology
of Frankia”, held in Wisconsin in 1982, it was
agreed that criteria were lacking for a system
based on species names, and so a system
for numbering Frankia strains was proposed
in the first “Catalog of Frankia trains”
(Lechevalier, 1983; 1986), which is still in
use.

21. NEW NODULATED GENERA
New nodulated genera in the Rhamnaceae
(Colletia, Trevoa, Talguenea and Retanilla), which are
native to South America, have also been discovered
and Frankia strains from these shrubs characterised
(Caru, 1993), whereas nodulation of Rubus has now
been discounted (Stowers, 1985).
Nodulation of genera in new families, e.g. Atriplex of
the Chenopodiaceae (Caucas and Abril, 1996),
always requires careful, independent confirmation.
“Nodules”, often called tubercles in older literature,
which are produced by mycorrhizal or other forms of
microbial infection, may easily be confused with
Frankia nodulation. Arbuscular mycorrhizal nodules,
which like actinorhizas are modified lateral roots,
have been reported recently for Gymnostoma
(Duhoux et al., 200

22. INFECTION AND NODULE DEVELOPMENT
The application of electron microscopy facilitated further detailed
observations of infection pathways and nodule development (Berry
and Sunell, 1990). One of the most important developments was the
recognition of two different infection pathways used by Frankia
hyphae.
The “traditional” pathway, which occurs in genera such as Alnus,
Myrica and Casuarina, involved penetration of deformed root hairs,
followed by intracellular penetration of cells of the root cortex.
The“alternative” pathway, which was first recognised in
Elaeagnus (Miller and Baker,1985), involved epidermal
penetration, followed by intercellular colonisation of the cortex,
before intracellular penetration of mature cortical cells and
ultimately the host cells of the developing nodule.
Furthermore, whereas hyphae in intracellular infections are
encapsulated in a host-derived matrix of polysaccharides,
cellulose,

23. CONT…..
Hemicellulose and pectin (Berg, 1990), during
intercellular colonisation, the hyphae are not
encapsulated until they penetrate the host cells. The
molecular signals that initiate infection remain unknown.
No convincing evidence of either nod genes or Nod-factor
homologs has been demonstrated in Frankia (Cérémonie
et al., 1998a; 1998b), although a root hair-deforming
factor is produced constitutively by some Frankia strains
(van Ghelue et al.,1997; Cérémonie et al., 1998b).
Because of their role as signal molecules in
legume symbioses, flavonoids excreted by the host plant
have been examined, but clear evidence for their
involvement in nodulation has not been obtained (Benoit
and Berry, 1997; Hughes et al., 1999)

24. LIFE WITH OXYGEN
Nitrogen fixation by Frankia in both the
free-living and symbiotic state is supported
by aerobic metabolism and is maximal at
about atmospheric O2 partial pressures, so
special mechanisms must be in place to
protect nitrogenase from inactivation by O2.
Early identification of vesicles as the
probable site of nitrogen fixation in cultured
Frankia was confirmed by immunogold
labelling of nitrogenase (Tjepkema et al.,
1981; Meesters et al., 1987)

25. ECOLOGY AND APPLICATIONS
Traditional techniques of ecological
physiology were employed in the Himalayas to
determine the contributions of Alnus nepalensis
to nutrient cycling and primary production in
agroforestry, both in different aged plantations
and in naturally regenerated landslip sites
(Sharma et al., 1998). These studies showed
clearly how uptake of recycled mineral nitrogen
replaced the high-energy processes of both
nitrogen fixation and nodule production as soil-nitrogen
concentration increased with stand age
(Sharma and Ambasht, 1988).

26. CONCLUTION
Nitrogen is essential to living things,
because atmospheric nitrogen plant can not
use directly from the atmosphere. Rhizobium
bacteria is also very important for the growth
of certain plant like pea, bean,soyabean etc.
Actinorhizal symbioses are exceptional
because of their present and potential uses
in forestry, agroforestry, revegetation, &
improvement of soils.

27. REFERENCES
 Symbiotic Nitrogen Fixation Technology, edited by
Gerald H. Elkan
 http://helios.bto.ed.ac.uk/bto/microbes/nitrogen.htm
 www.aob.oxfordjournals.org
 https://www.boundless.com/microbiology/textbooks/boun
dless-microbiology-textbook/microbial-ecology-
16/microbial-symbioses-196/the-legume-root-… 1/10
 http://mmbr.asm.org/content/63/4/968.full
 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC98982/
 http://en.wikipedia.org/wiki/Rhizobia
 http://www.sciencedaily.com/releases/2008/03/08030407
5746.htm
 The Plant Cell, Vol. 7, 869-885, July 1995 O 1995
American Society of Plant Physiologists
 http://dx.doi.org/10.5772/56991