SUBMITTED TO:

PROF. BIMAN KUMAR DUTTA

DEPT. OF ECOLOGY &

ENVTL. SCIENCE

SEMINAR ON BIOLOGICAL NITROGEN FIXATION WITH

SPECIAL REFERENCE TO RHIZOBIAL ROOT NODULE SYMBIOSIS & ACTINORHIZAL

SYMBIOSIS

PAPER -304

Submitted by:

SUBRATA PAULRoll No. 31

M.Sc. 3 rd sem

INTRODUCTION

Nitrogen is required by all living organisms forthe synthesis of proteins, nucleic acids and othernitrogen-containing compounds. The earth’satmosphere contains almost 80% nitrogen gas. Itcannot be used in this form by most living organismsuntil it has been fixed, that is reduced (combined withhydrogen), to ammonia. Green plants, the mainproducers of organic matter, use this supply of fixednitrogen to make proteins that enter and pass throughthe food chain. Microorganisms (the decomposers)break down the proteins in excretions and deadorganisms, releasing ammonium ions. These twoprocesses form part of the nitrogen cycle.

REACTION MICRO-ORGANISM CONDITION

PROCESSNitrogen 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 ,egNitrosomonas &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.

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.

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 rhizobiacannot 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.

ROOT NODULE FORMATION

Sets of genes in the bacteria control differentaspects of the nodulation process. One Rhizobiumstrain can infect certain species of legumes but notothers e.g. the pea is the host plant to Rhizobiumleguminosarum biovar viciae, whereas clover acts ashost to R. leguminosarum biovar trifolii. Specificitygenes determine which Rhizobium strain infectswhich legume. Even if a strain is able to infect alegume, the nodules formed may not be able to fixnitrogen.

Such rhizobia are termed ineffective. Effectivestrains induce nitrogen-fixing nodules. Effectivenessis governed by a different set of genes in the bacteriafrom the specificity genes. Nod genes direct thevarious stages of nodulation.

ROOT NODULE FORMATION STEPS

Root system with

root nodules

CONT……….

Root Hair

CONT……….

Root hair covered by

free-living Rhizobia

CONT…………..

root hair starts to

formation of an

infection curl

CONT……..

Formation of an

infection

thread through which

rhizobia enter root

cells

CONT………

infection thread

spreads

into adjacent cells

bacteroids are

released from the

infection thread

CONT……….

Bacteroids

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)

NITROGENASE

An enzyme called nitrogenase catalyses theconversion of nitrogen gas to ammonia innitrogen-fixing organisms. In legumes it onlyoccurs within the bacteroids. The reactionrequires hydrogen as well as energy from ATP.The nitrogenase complex is sensitive to oxygen,becoming inactivated when exposed to it. This isnot a problem with free living, anaerobicnitrogen-fixing bacteria such as Clostridium.Free living aerobic bacteria have a variety ofdifferent mechanisms for protecting thenitrogenase complex, including high rates ofmetabolism and physical barriers.

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.

ACTINORHIZAL SYMBIOSIS: A HISTORICAL

PERSPECTIVE

The term “actinorhizal” was proposed atthe first international meeting on “Symbiotic NitrogenFixation in Actinomycete nodulated Plants”, held atHarvard Forest, to provide a convenient and morepositive designation for the field than the term “non-legume” (Torrey and Tjepkema, 1979). Following thefirst reproducible isolation of the microorganism bythe John Torrey’s group (Callaham et al., 1978),which was a watershed event in the development ofFrankia-actinorhizal research, there was a rapidincrease internationally in the number of scientistsinterested in Frankia and an exponential increase inthe number of isolates in culture..

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’srediscovery. Three new genera of the Casuarinaceaewere defined by dividing the former genus Casuarinainto Casuarina, Allocasuarina, Gymnostoma and Ceuthostoma (Johnson, 1980; 1982; 1988). Nodulation of species in the first three of these genera has been observed regularly

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.

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

INFECTION AND NODULE DEVELOPMENTThe 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,

CONT…..

Hemicellulose and pectin (Berg, 1990), duringintercellular colonisation, the hyphae are notencapsulated until they penetrate the host cells. Themolecular signals that initiate infection remain unknown.No convincing evidence of either nod genes or Nod-factorhomologs has been demonstrated in Frankia (Cérémonieet al., 1998a; 1998b), although a root hair-deformingfactor is produced constitutively by some Frankia strains(van Ghelue et al.,1997; Cérémonie et al., 1998b).

Because of their role as signal molecules inlegume symbioses, flavonoids excreted by the host planthave been examined, but clear evidence for theirinvolvement in nodulation has not been obtained (Benoitand Berry, 1997; Hughes et al., 1999)

LIFE WITH OXYGEN

Nitrogen fixation by Frankia in both thefree-living and symbiotic state is supportedby aerobic metabolism and is maximal atabout atmospheric O2 partial pressures, sospecial mechanisms must be in place toprotect nitrogenase from inactivation by O2.Early identification of vesicles as theprobable site of nitrogen fixation in culturedFrankia was confirmed by immunogoldlabelling of nitrogenase (Tjepkema et al.,1981; Meesters et al., 1987)

ECOLOGY AND APPLICATIONS

Traditional techniques of ecological physiology were employed in the Himalayas to determine the contributions of Alnus nepalensisto 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).

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.

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/boundless-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/2008/03/080304075746.htm

The Plant Cell, Vol. 7, 869-885, July 1995 O 1995 American Society of Plant Physiologists

http://dx.doi.org/10.5772/56991