BIOFERTILIZERS

Introduction
Chemical fertilizers are expensive. The ingredients used in chemical fertilizers are toxic to the
skin and respiratory system. The excessive use of fertilizers damages the plants and reduces
soil fertility. The leaching of chemical fertilizer causes it to reach the rivers leading to
eutrophication (algal bloom). The long-term use reduces the microbial activity and disturbs the
pH of the soil. Thus, an alternative to chemical fertilizer could be Biofertilizers.

History of biofertilizers
Biofertilizer concept goes back as early as 300 BC when our ancestors realized the importance
of legume crops bearing nodules. The commercial history of biofertilizers began in 1895 when
Nobbe and Hiltner introduced "Nitragin", a laboratory culture of Rhizobia. This was followed
by the discovery of Azotobacter, blue-green algae, and other microorganisms. In 1956, the first
commercial production of legume Rhizobium symbiosis began. In the 1960s, India began
commercial production of biofertilizers by V N Joshi. Many organisms capable of nitrogen
fixation, P solubilization, P mobilization, potassium solubilization and micronutrient
transformation in the soil were used as biofertilizers.

What are biofertilizers?

These are formulations comprised of living microbial cells, either a single strain or multiple
strains (mixed or consortium), that promote plant growth by increasing nutrient availability and
acquisition. The multifarious advantages of biofertilizers leads to its wide applicability in
sustainable agriculture.
Types of biofertilizers:

Importance of biofertilizers
• Enrichment of soil: They help to get high yield of crops by making the soil rich with
nutrients - Increase crop yield by 20-30%.
• Better suited than chemical fertilizers: Chemical fertilizers disturb the natural nutrient
content of the soil, but biofertilizers maintain that like before - Replace chemical
nitrogen and phosphorus by 25%.
• Not harmful for plants: Plant growth can be increased if biofertilizers are used, because
they contain natural components which do not harm the plants.
• Beneficial for soil microbes: Useful microorganisms necessary for the growth of the
plants can remain in the soil.
• Protects natural fertility of the soil: If the soil will be free of chemicals, it will retain its
natural fertility which will be beneficial for the plants as well as the environment.
• Plants are better adapted: Biofertilizers destroy those harmful components from the
soil which cause diseases in the plants. Plants can also be protected against drought and
other strict conditions by using biofertilizers.
• Economic: Biofertilizers are way more economic and can be readily prepared or
collected by the farmers can make use of them.
• Eco-friendly: They are environment friendly and protect the environment against
pollutants
Nitrogen Cycle:
Nitrogen (N) is an essential element for life and exists in a number of oxidation states. There
are four major microbial N transformations thus far: nitrification, denitrification, anammox,
and nitrogen fixation.
Nitrogen Fixation
Nitrogen gas (N2) is the most stable form of N and is a major reservoir for N on Earth. However,
only a relatively small number of prokaryotes are able to use N2 as a cellular N source by
nitrogen fixation. The N recycled on Earth is mostly already “fixed N”; that is, N in
combination with other elements, such as in ammonia (NH3) or nitrate (NO3-). In many
environments, however, the short supply of fixed N puts a premium on biological nitrogen
fixation, and in these habitats, nitrogen-fixing bacteria flourish.
The reduction of atmospheric gaseous nitrogen to ammonia is called nitrogen fixation. It is
carried out by free-living (require no host in order to carry out the process) or symbiotic (fix
nitrogen only in association with certain plants) diazotropic bacteria. The nitrogen fixing
bacteria includes free-living bacteria (e.g., Azotobacter, Azospirillum, Klebsiella, Clostridium,
and Methanococcus), bacteria living in symbiotic association with plants such as legumes
(Rhizobium, Bradyrhizobium, Frankia, Anabaena), and cyanobacteria (Nostoc and Anabaena).
The reduction of nitrogen to ammonia is catalysed by the enzyme nitrogenase. The reduction
of molecular nitrogen to ammonia is exergonic & has a high activation energy. Thus, the
nitrogen reduction is expensive and requires a large ATP expenditure. At least 8 electrons and
16 ATP molecules, 4 ATPs per pair of electrons, are required

The nitrogenase enzymes is a complex system consisting of dinitrogenase and dinitrogenase

reductase. Dinitrogenase is a Molybdenum-iron containing protein (MoFe protein) with a
molecular weight of 2,20,000. Dinitrogenase reductase is joined with one or two Fe proteins
(MW 64,000). The MoFe protein - contains 2 atoms of molybdenum & 28 to 32 atoms of iron;
the Fe protein has 4 iron atoms. Nitrogenase is quite sensitive to O2 and must be protected
from O2 inactivation within the cell. This is done by rapid removal of O2 by respiration, O2
retarding slime layers or production of heterocyst. The symbiotic nitrogen-fixing bacteria can
consume almost 20% of the ATP produced by the host plant. Nitrogenase can also reduce a
variety of molecules containing triple bonds (e.g., acetylene, cyanide, and azide). The rate of
reduction of acetylene to ethylene is even used to estimate nitrogenase activity. The molecular
nitrogen has been reduced to ammonia, the ammonia can be incorporated into organic
compounds or can diffuse out of the bacterial cell and be assimilated in the surrounding legume
Nitrification
Nitrification, the oxidation of NH3 to NO3-, is a major process in well-drained oxic soils at
neutral pH, and is carried out by the nitrifying bacteria. Denitrification consumes NO3-,
whereas nitrification produces NO3- . If materials high in NH3, such as manure or sewage, are
added to soils, the rate of nitrification increases. Nitrification is a two-step aerobic process in
which some species oxidize NH3 to NO2- and then other species oxidize NO2- to NO3-. Many
species of Bacteria and at least one species of Archaea are nitrifiers. Although NO3- is readily
assimilated by plants, it is very soluble, and therefore rapidly leached or denitrified from
waterlogged soils. Consequently, nitrification is not beneficial for plant agriculture.
Ammonium, on the other hand, is positively charged and strongly adsorbed to negatively
charged soils. Anhydrous NH3 is therefore used extensively as an agricultural fertilizer, but to
prevent its conversion to NO3-, chemicals are added to the NH3 to inhibit nitrification.
One common inhibitor is a pyridine compound called nitrapyrin. Nitrapyrin specifically
inhibits the first step in nitrification, the oxidation of NH3 to NO2-. However, this effectively
inhibits both steps in nitrification because the second step, NO2- to NO3- conversion, depends
on the first.

Denitrification
Under most conditions, the end product of NO3- reduction is N2 and N2O. The reduction of
NO3- to these gaseous nitrogen compounds, called denitrification. On the one hand,
denitrification is a detrimental process. For example, if agricultural fields fertilized with nitrate
fertilizer become waterlogged following heavy rains, anoxic conditions can develop and
denitrification can be extensive; this removes fixed nitrogen from the soil. On the other hand,
denitrification can aid in wastewater treatment. By removing NO3- as volatile forms of N,
denitrification minimizes fixed N and thus algal growth when the treated sewage is discharged
into lakes and streams. The production of N2O and NO by denitrification can have other
environmental consequences. N2O can be photochemically oxidized to NO in the atmosphere.
NO reacts with ozone (O3) in the upper atmosphere to form nitrite (NO2-), and this returns to
Earth as nitric acid (HNO2). Thus, denitrification contributes both to O3 destruction, which
increases passage of ultraviolet radiation to the surface of Earth, and to acid rain, which
increases acidity of soils. Increases in soil acidity can change microbial community structure
and function and, ultimately, soil fertility, impacting both plant diversity and agricultural yields
of crop plants. Eg: Pseudomonas denitrificans

Each step is catalysed by nitrate reductase, nitrite reductase, nitric oxide reductase & nitrous
oxide reductase respectively.

Ammonification and Ammonia Fluxes

The ammonia that is released during the decomposition of organic nitrogen compounds –
ammonification. Another process contributing to the generation of NH3 is the dissimilative
reduction of nitrate to ammonia (DRNA) - respiratory reduction of NO3- to NH3. At neutral
pH, NH3 exists as ammonium (NH4+). Much of the NH4+ released by aerobic decomposition in
soils is rapidly recycled and converted to amino acids in plants and microorganisms. However,
because NH3 is volatile, some of it can be lost from alkaline soils by vaporization, and there
are major losses of NH3 to the atmosphere in areas with dense animal populations (for example,
cattle feedlots). On a global basis, however, NH3 constitutes only about 15% of the N released
to the atmosphere, the rest being primarily N2 or N2O from denitrification.

Anammox
Ammonia can be oxidized under anoxic conditions, which is referred as anoxic ammonia
oxidation (Annamox). The organisms responsible for it includes Brocadia anammoxidans,
Kuenenia, Anammoxoglobus, Jettenia, and Scalindua. NH3 is oxidized anaerobically with NO2-
acting as the electron acceptor, forming N2 as the final product. In the anammox reaction, NO2-
is first reduced to nitric oxide (NO) by nitrite reductase, and then NO reacts with ammonium
(NH4+) to yield N2H4 by activity of the enzyme hydrazine hydrolase. N2H4 is then oxidized to
N2 plus electrons by the enzyme hydrazine dehydrogenase.
Phosphate Solubilization

Phosphorus is the most important key element in the nutrition of plants, next to nitrogen (N).
It plays an important role in virtually all major metabolic processes in plant including
photosynthesis, energy transfer, signal transduction, macromolecular biosynthesis and
respiration and nitrogen fixation in legumes. Although P is abundant in soils in both inorganic
and organic forms, it is a major limiting factor for plant growth as it is in an unavailable form
for root uptake. Inorganic P occurs in soil, mostly in insoluble mineral complexes, some of
them appearing after frequent application of chemical fertilizers. These insoluble, precipitated
forms cannot be absorbed by plants. Organic matter is also an important reservoir of
immobilized P that accounts for 20–80% of P in soils. Only 0.1% of the total P exists in a
soluble form available for plant uptake because of its fixation into an unavailable form due to
P fixation.

The term P fixation is used to describe reactions that remove available phosphate from the soil
solution into the soil solid phase. There are two types of reactions (a) phosphate sorption on
the surface of soil minerals and (b) phosphate precipitation by free Al3+ and Fe3+ in the soil
solution. It is for this reason that soil P becomes fixed and available P levels have to be
supplemented on most agricultural soils by adding chemical P fertilizers, which not only
represent a major cost of agricultural production but also impose adverse environmental
impacts on overall soil health and degradation of terrestrial, freshwater and marine resources.
Thus, increased P levels have been identified as a main factor for eutrophication of surface
waters that may lead to algal blooms. The repeated and injudicious applications of chemical P
fertilizers, leads to the loss of soil fertility by disturbing microbial diversity, and consequently
reducing yield of crops.

Moreover the efficiency of applied P fertilizers in chemical form rarely exceeds 30% due to its
fixation, either in the form of iron/aluminium phosphate in acidic soils or in the form of calcium
phosphate in neutral to alkaline soils. The realization of all these potential problems associated
with chemical P fertilizers together with the enormous cost involved in their manufacture, has
led to the search for environmental compatible and economically feasible alternative strategies
for improving crop production in low or P-deficient soils. The use of microbial inoculants
(biofertilisers) possesing P-solubilizing activities in agricultural soils is considered as an
environmental-friendly alternative to further applications of chemical based P fertilizers.

Occurrence and isolation of PSM

Solubilization of insoluble P by microorganisms was reported by Pikovskaya (1948). Typically
such microorganisms have been isolated using cultural procedures with species
of Pseudomonas and Bacillus bacteria and Aspergillus and Penicillium fungi being
predominant. These organisms are ubiquitous but vary in density and mineral phosphate
solubilizing (mps) ability from soil to soil or from one production system to another. Both
bacterial and fungal strains exhibiting P solubilizing activity are detected by the formation of
clear halo (a sign of solubilization) around their colonies. In addition
to Pseudomonas and Bacillus, other bacteria reported as P-solubilizers
include Rhodococcus, Arthrobacter, Serratia, Chryseobacterium, Gordonia, Phyllobacterium,
Delftia sp., Azotobacter , XanthomonasEnterobacter, Pantoea, and Klebsiella. Furthermore,
symbiotic nitrogenous rhizobia, which fix atmospheric nitrogen into ammonia and export the
fixed nitrogen to the host plants, have also shown PS activity e.g, Rhizobium leguminosarum
bv. Trifolii. In addition to bacteria, fungi and actinomycetes, algae such as cyanobacteria and
mycorrhiza have also been reported to show P solubilization activity.

Mechanism of P-solubilization by PSM

The main P solubilization mechanisms employed by soil microorganisms include: (1) release
of complexing or mineral dissolving compounds e.g. organic acid anions, siderophores,
protons, hydroxyl ions, CO2, (2) liberation of extracellular enzymes (biochemical P
mineralization) and (3) the release of P during substrate degradation (biological P
mineralization).

A) Inorganic P solubilization: by P-solubilizing microorganisms occurs mainly by

organic acid production, either by: (i) lowering the pH, or (ii) by enhancing chelation
of the cations bound to P (iii) by competing with P for adsorption sites on the soil (iv)
by forming soluble complexes with metal ions associated with insoluble P (Ca, Al, Fe)
and thus P is released. The lowering in pH of the medium suggests the release of
organic acids by the P-solubilizing microorganisms via the direct oxidation pathway
that occurs on the outer face of the cytoplasmic membrane. These acids are the product
of the microbial metabolism, mostly by oxidative respiration or by fermentation of
organic carbon sources (e.g., glucose) or such organic acids can either directly dissolve
the mineral P as a result of anion exchange of phosphate by acid anion or can chelate
Fe, Al and Ca ions associated with P. Thus, the synthesis and discharge of organic acid
by the PSM strains into the surrounding environment acidify the cells and their
surrounding environment that ultimately lead to the release of P ions from the P mineral
by H+ substitution for the cation bound to phosphate. The prominent acids released by
PSM in the solubilization of insoluble P are gluconic acid, oxalic acid, citric acid, lactic
acid, tartaric acid, aspartic acid. The other mechanism is the production of H2S, which
react with ferric phosphate to yield ferrous sulphate with concomitant release of
 phosphate. The solubilization of insoluble P by inorganic acid (e.g. HCl) has also been
reported, although HCl was able to solubilize less P from hydroxyapatite than citric
acid or oxalic acid. H+excretion originating from NH4+assimilation- most probable
reason for solubilization without acid production is the release of protons
accompanying respiration or NH4+ assimilation

B) Organic P solubilization is also called mineralization of organic phosphorus. Organic

P may constitute 4–90% of the total soil P. Such P can be released from organic
compounds in soil by enzymes:
(i) Non-specific acid phosphatases (NSAPs), which dephosphorylate phospho-
ester or phosphoanhydride bonds of organic matter. Typically, acid
phosphatases predominate in acid soils, whereas alkaline phosphatases are more
abundant in neutral and alkaline soils.
(ii) phytases, which specifically cause release of P from phytate degradation. In its
basic form, phytate is the primary source of inositol and the major stored form
of P in plant seeds and pollen, and is a major component of organic P in soil.
(iii) phosphonatases and C–P lyases, that cleave the C–P bond of
organophosphonates.

Characteristics of different types of Biofertilizer:

Rhizobium – A Symbiotic Nitrogen Fixer
Several microbial genera are able to form nitrogen-fixing nodules with legumes. These include
Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium, and
Rhizobium. The genus Rhizobium is a prominent member of the rhizosphere community. This
bacterium also can establish a symbiotic association with legumes and fix nitrogen for use by
the plant. Rhizobium infects and nodulates specific legume hosts. The bacterium contains a
large plasmid that encodes information that is not used when it grows as a free-living organism
in the soil, but is vital for infection and nodulation of the susceptible host. Ability of legumes
(soybeans, clover, alfalfa, beans, and peas) to grow without nitrogen fertilizer saves farmers
millions of dollars in fertilizer costs yearly. Rhizobia (symbionts) are species of Alpha- or
Betaproteobacteria that can grow freely in soil or can infect leguminous plants and establish a
symbiotic relationship.

Root nodule formation

The Rhizobium infection process is controlled by the bacA gene that is required to establish
the nodule. The establishment of the nodules involves a newly discovered plant gene called
nin. This complex infection process, appears to involve a series of molecules produced by the
host plant that lead to the exchange of recognition signals. Flavonoid inducers produced by the
plant play a major role in this process by stimulating the Rhizobium to synthesize specific Nod
factors that activate the host symbiotic processes necessary for root hair infection and nodule
development. After bacterial attachment, the root hairs curl and the bacteria induce the plant to
form an infection thread that grows down the root hair. The Rhizobium then spreads within the
infection thread into the underlying root cells as noted in figure below. At no time does the
Rhizobium actually enter the plant cytoplasm while it is in the infection thread! When the
bacteria are released from the infection thread into the host cell the Rhizobium is enclosed by
a plant-derived membrane, called the peribacteroid membrane, to form a bacteroid. Further
growth and differentiation lead to the development of a nitrogen-fixing form, a structure called
a symbiosome. At this point, specific nodule components such as leghemoglobin, which protect
the nitrogen fixation enzymes from oxygen, are produced to complete the nodulation process.
Leghemoglobin functions as an “oxygen buffer,” cycling between the oxidized (Fe3+) and
reduced (Fe2+) forms of iron to keep unbound O2 within the nodule low.
The symbiosomes within mature root nodules are the site of nitrogen fixation. Within these
nodules, the differentiated bacteroids reduce atmospheric N2 and form ammonia (the primary
product) and alanine; these compounds are released into the host plant cell, assimilated into
various other nitrogen-containing organic compounds, and distributed throughout the plant.
Because reduced nitrogen is the nutrient most commonly limiting plant growth, biological
nitrogen fixation, as exemplified by the Rhizobium-legume symbiosis, is of major importance
to agricultural productivity and the biogeochemical nitrogen cycle needed to sustain life on
Earth.
After the Rhizobium cells have developed into the final differentiated bacteria, they no longer
can revert back to bacteria capable of reproduction and have been lost to the “gene pool.” The
fittest members of the bacterial community, which have achieved nodulation, thus have
sacrificed their own reproduction! A major goal of biotechnology is to introduce nitrogen
fixation genes into plants that do not normally form such associations. Recently it has been
possible to produce modified lateral roots on nonlegumes such as rice, wheat, and oilseed rape;
the roots are invaded by nitrogen-fixing bacteria. It appears that the infection begins with
bacterial attachment to the root tips. Although these modified root structures have not yet been
found to fix useful amounts of nitrogen, they do enhance rice production.
Azospirillum – An associative N2 fixer
In 1925, Beijerinck described N2 fixing bacteria – Spirillum lipoferum. These are
microaerophilic, non-symbiotic, diazotrophic bacteria. They associative N2-fixers – colonizes
the root and infects cortex also. They are Gram-negative and do not form spores. Azospirillum
are aerobic, but can also function as microaerobic diazotrophs. Promote growth of plants and
increases growth of root hairs and the number of lateral roots on cotton plants resulting in a
better uptake of nutrients by the host plant. It colonize roots of rice, maize, wheat, sorghum
(and other cereals), sugarcane, cotton, grasses, and vegetables such as tomato, pepper, beets,
sweet potato, and eggplant. When inoculated onto cereal roots, Azospirillum strains multiply
and form small aggregates, mainly in zones of root elongation and root hair. Reside in the
intercellular spaces of the plant’s vascular system, take dissolved N2 from the sap flow, and
convert it into ammonia, nitrogen and amines for use by the plants. Examples includes:
Azospirillum lipoferum, A. amazonense and A. Brasilense

Azotobacter – A free-living N2 fixer

Beijerinck isolated Azotobacter chroococcum. Azotobacter is an aerobic, free-living,
nonsymbiotic N2-fixer residing in soil and rhizosphere. They are found in slightly acidic to
neutral soil. They are Gram-negative, rod shaped, aerobic bacteria, produces an insoluble
black-brown pigment containing melanin due to oxidation of tyrosinase. The optimum
environmental conditions for Azotobacter are: temperature (25-30), high humidity, aeration,
pH (7.2-7.6), high salt concentration etc. Examples include: A. vinelandii , A. chroococcum, A.
beijerincki and A. insignis. Each cell produce a resting structure called cyst (double thick cell
wall). Cysts are dormant cells that are resistant to unfavorable conditions (desiccation &
exposure to UV light ), which germinate under favorable condition. They have been found to
produce indole acetic acid, gibberellic acids, vitamins etc. It is recommended as biofertilizer
for rice, wheat, millets, cotton etc.

Pseudomonas fluorescens
Pseudomonas is a plant-based saprophyte that lives on or inside the plants. It is a saprophyte
creates a greenish fluorescence pigment that has the potential to protect plants from fungal
growth. Pseudomonas fluorescens that lives in the plant tissues stops the invasion of pathogens
within the cells, promoting the generation of plant growth-stimulating hormones while
increasing disease resistance. It improves the soil properties by improving soil permeability
and oxygen availability to the roots. Proper oxygen supply will prevent root diseases, fungal
formations and root rots. Improves the uptake of nutrients by roots. Pseudomonas fluorescens
living within the plant tissues promote the generation of growth promotion hormones. It
increases resistance to diseases, acting as a biocontrol agent.

Bacillus amyloliquefaciens
It a root-colonizing biocontrol bacterium. It can improve soil nutrient availability, including
improving nitrogen supply, solubilizing phosphate and potassium, and producing siderophores.
It can change the soil microbial community and improve the availability of minerals and plant
growth conditions. Bacillus amyloliquefaciens can secrete hormones and volatile organic
compounds (VOCs) associated with plant cell growth and root development and further
improve nutrient uptake by plants. It can enhance plant resistance against biotic stresses from
soil pathogens through competition of niches and nutrients, producing substances such as cyclic
lipopeptides, polyketides, and VOCs to antagonize pathogens directly, and induction of system
resistance of the plants

Cyanobacteria
It is also referred as Blue-green algae (Eg: Anabaena, Nostoc, Plectonema, Tolypothrix,
Aulosira, Cylindrospermum). In water-logging conditions the cyanobacteria multiply, fix
atmospheric N2 and release it into the surrounding in the form of amino acids & other growth
promoting substances. Algalization refers to the application of blue green algae to be used as
biofertilizer. The main objective of it is to develop low cost indigenous technology for mass
production of cyanobacteria, to isolate a better N2 fixing strains, to develop starter inoculum
and to study benefits on both economy & ecology.

Hapalosiphon
These are filamentous cyanobacteria. It has a thin sheath, true branching, and intercalary
heterocysts a cyanobacteria that can be used as a biofertilizer in paddy fields. It can be found
in tropical and temperate regions, often in stagnant waters, swamps, and the littoral zone of
lakes and ponds. It is beneficial for non-leguminous crops like paddy, jowar, mize, ragi,
turmeric, ginger, and cardamom.

Azolla
Azolla is an aquatic heterosporous fern which contains endophytic cyanobacterium, Anabaena
azollae in its leaf cavity. It became popular in Vietnam, China, Indonesia, Philippines, India &
Bangladesh. Eg: Azolla caroliniana, A. filiculoides, A. mexicana, A. microphylla, A.nilotica,
A.pinnata & A.rubra. It shows tolerance against heavy metals like As, Hg, Pb, Cu, Cd, Cr etc.
A. pinnata absorbs heavy metals into cell walls and vacuoles through specific metal resistant
enzymes.

Frankia
A member of Actinomycetes, Frankia (named after the discoverer, Frank 1880) is a symbiotic
structure with the roots of non-leguminous plants called actinorhiza. Eg: Alnus, Hippophae,
Allicasuarina, Casuarina, Gymnostoma, Coriaria etc. Frankia grows very slowly & has a very
long log phase. It makes it difficult to frow Frankia on mass scale. It is microaerophile &
requires pH range of 6.7-7.0. Hyphae are thin, slender, poorly branched and pigmented. Spores
are produced inside the sporangia in clusters intra-hyphally or terminally. In N2 free media, it
produces round or spherical vesicles – nitrogen fixing centre. Spores get attached on surface of
root hairs, resulting in curling. It produces growth substances like auxins, cytokinins,
gibberilins etc

Mycorrhiza
Mycorrhizae are fungus-root associations, first discovered by Albert Bernhard Frank in 1885.
The term “mycorrhizae” comes from the Greek words meaning fungus and roots. These
microorganisms contribute to plant functioning in natural environments, agriculture, and
reclamation. The roots of about 95% of all kinds of vascular plants are normally involved in
symbiotic associations with mycorrhizae. Five mycorrhizal associations have been described.
These include both non-septate and septate fungi. There are endophytic arbuscular mycorrhizae
(AM) that form arbuscules and sometimes vesicles, septate types associated with orchids, and
those that form endomycorrhizal relationships with ericoid plants such as blueberries. In the
endophytic mycorrhizae, the fungus penetrates the plant cells where it forms characteristic
structures, including arbuscules and coils. Vesicles are not consistently observed. In addition,
ectendomycorrhizae are formed by basidiomycetes. These have sheaths and intracellular coils.

The Mycorrhiza functions in delivers P from organic polymers, other micronutrients, influence
carbon cycling, improve soil aggregation, protect against stress such as drought, serve as
biocontrol agents for the plant, help interactions with neighboring plants, and range of possible
interactions with other soil microbial groups.
Ectomycorrhizae
It forms an extensive sheath around the outside of the root. They are found mainly on the roots
of forest trees, especially conifers, beeches, and oaks - Pinaceae, Betulaceae, Fagaceae, and
Diperocarpaceae in tropical, subtropical, and arid environments. The both long and short roots
show fungal colonization. Plants can form multiple mycorrhizal associations, that help equalize
resource availability. Eg includes: Cennococcum, Pisolithus and Amanita. The fungal hyphae
between the cortical cells are called as Hartig net. Through the intercellular space, minerals
and nutrient materials are exchanged between the fungus and the plant. The ectomycorrhizae
also exhibit synergistic interactions with other plant-beneficial organisms.

Endomycorrhizae
The fungus becomes deeply embedded within the root tissue. It grows intracellularly and form
coils, swellings, or minute branches. Found in wheat, corn, beans, tomatoes, apples, oranges,
and many other commercial crops, as well as most grasses. Types include: Arbuscular
Mycorrhizae, Orchid endomycorrhizae and Ericoid endomycorrhizae. The other type
mycorrhizae includes Arbutoid ectendomycorrhizae, which form intracellular hyphae and an
extracellular sheath.

Arbuscular Mycorrhizae (AM fungi)

Eg: Glomeromycota
It is an obligate plant mutualist. AM colonize more than 85% of all terrestrial plants, including
most grassland species and many crop species. AM fungi produce plant growth substances that
induce morphological alterations in the roots, stimulating formation of the mycorrhizal state.
AM fungal spores germinate to form hyphopodia (or appressoria), followed by inter- and
intracellular penetration by hyphae forming a branched intracellular structures called
“arbuscules”. Arbuscules main site of exchange of C, P, water, and other nutrients. AM fungi
produce runner hyphae (extraradicular hyphae). These hyphae are finer than roots with high
surface to volume ratio. The phytohormones produced by the plant stimulate fungal
metabolism as well as hyphal branching.

Ericoid mycorrhiza
Ericoid mycorrhizas tend to be host-specific and colonize only the plants in the family
Ericaceae. The fungus develops inside epidermal cells, forming coils that give rise to
independent infection units. No sheath is formed. Eg: Ascomycetes. It plays an important role
in mobilizing the organic nutrients in the soil. The fungus is endophytic. The “arbutoid” type,
found in members of Ericaceae tribe Arbuteae and subfamilies Pyroloideae and
Monotropoideae, the association is ectendotrophic, i.e. the fungus grows within and also
ensheaths the root tissue.

Orchid mycorrhiza
Orchid mycorrhizas tend to be host-specific and colonize only the plants in the family
Orchidaceae. It involve partially or completely achlorophyllous plants (for some part of their
life) and fungi of the basidiomycete group. It is critically important during orchid germination.
The orchid seed has virtually no energy reserve and obtains its carbon from the fungal
symbiont. Coils produced by orchid mycorrhizae occur mostly in the inner layers of the root

Microbial inoculants
These are living microbes, which when applied to seed, plant, or soil promote growth by the
supply of essential nutrients such as N, P, and other mineral nutrients.

Inocula
The inocula can be solid or liquid; bacterial or fungal; pure culture or mixed culture inocula.
Even spores, hyphal fragments, and mycorrhizal root residues can be used. It should be mixed
with carrier such as peat, clay, or fly ash. Since, Mycorrhiza has an obligate requirement for
plant. Roots of plants used for propagation such as sorghum, maize, and onion can be used for
Mycorrhizal inocula. Substrates such as sand, soil, or other materials such as zeolite and perlite
are used to mass produce AM fungal inoculum in pots, bags, or beds.

Carriers
Carriers should provide a favourable microenvironment for the PGPM. They should ensure
their viability with adequate shelf life of the inoculant formulation. They should be easily
available, stable, economical, eco-friendly. They should be easy to apply and have good
moisture-holding capacity and pH-buffering capacity.

These carriers can be organic materials peat, coal, clay, saw dust, wheat bran, peat
supplemented with chitin-containing materials or inorganic materials such as vermiculite,
perlite, silicates, kaolin, and bentonite.
In the case of solid inocula, the size of the granules or beads used for immobilization of the
microbe may vary from 75 μm to 250 μm. Liquid inoculants can be broth cultures, suspensions
in solutions of humic acid, or suspensions in mineral or organic oils or oil-in-water suspensions.
Liquid or powder-type inoculants can be used to coat the seeds, for root dipping at the time of
transplantation of seedlings, or apply directly into the furrow (or seed beds) or as a foliar spray.
Humic acid has been a popular carrier for a number of microbial inoculant formulations for
producing a stable inoculant at ambient temperature. Peat has been commonly used as a carrier
for PGPR, particularly for rhizobial inoculants, due to its wide availability and a long history
of field trials. Organic polymers such as alginate (d-mannuronic acid and l-glucuronic acid
polymer), hydroxyethylcellulose, and carrageenan have also been suggested as inoculum
carriers. Different polymers entrap or encapsulate the microorganisms, which are slowly
released over a period of time. Alginate appears to be a popular carrier in this group. It is the
most common polymer used for encapsulation of microbes.

Application methods

Application methods for the delivery of PGPM to crops in the field are relatively limited.
Farmers are not keen on purchasing specialized equipment to be used for microbial-based
products. Formulated inocula should be readily applied using standard farming machinery to
make it appealing to farmers. Application of liquid inoculant is often more convenient for the
farmer because of less time required, ease of application, and the equipment routinely used on
a farm can be used. The development of inexpensive and efficient technologies for the efficient
delivery of inocula, probably by modification of sprayers and sprinklers normally used for plant
irrigation, could facilitate use of PGPM formulations. Soil inoculation can be done either with
solid or liquid formulations. Normally, the carrier is mixed with the inoculum in the factory,
but it could be mixed by the farmer prior to application, especially when liquid formulations
are used. Depending on the particular inoculant formulation, the inocula can be used for seed
coating, for dipping seedlings, direct application to the furrow, or as foliar application. The use
of fertilizers that were produced by mixing organic matrices and insoluble phosphates with the
addition of selected P-solubilizing microorganisms can also be considered for applying PGPM
to crops. Such fertilizers increase the availability of nutrients (particularly of P) to plants and
also improve the tolerance of the plant to pathogens. Using PGPM strains that form stable and
effective biofilms could be a strategy for producing commercially viable inoculant
formulations

Monoculture and Co-culture Inoculant Formulations

Ex: R. leguminosarum bv. Trifolii, Bradyrhizobiuim, P. fluorescens, Azospirillum,
A. brasilense and Sinorhizobium meliloti
Trichoderma spp. and Pseudomonas spp.
mycorrhiza and PGPR

Polymicrobial Inoculant Formulations

It is a formulation containing a consortium of beneficial microbes.
Ex: Klebsiella pneuminiae, P. fluorescens (or P. putida), and Citrobacter freundii
P. fluorescens was selected for its N2-fixing ability, Klebsiella pneumoniae for its P-
solubilizing ability, and C. freundii, for its ability to produce toxic substances that inhibited
nearly 50% of the rice rhizosphere population
P. fluorescens, Pseudomonas striata, Paenibacillus (Bacillus) polymyxa, B. subtilis,
Azospirillum, Rhizobium, Azotobacter, T. harzianum, Trichoderma viride, Saccharomyces
cerevisiae and Lactobacillus

Constrains in Biofertilizer Development

1. Soil temperature
Microbial activity is stronger when the soil temperature is between 15 and 30 degrees Celsius
Soil temperature fluctuates a lot and it varies seasonally, daily and even hourly depending on
solar radiation
majority of bio-inoculants used in the biofertilizer business operate better at ideal soil
temperatures of 28°C to 35°C
2. Soil pH
Changes in soil pH, on the other hand, have a significant impact on signalling molecule
synthesis, plant root attachment, nodule formation and soil longevity
Soil acidity has an impact on the nodulation process and nitrogen fixation, from rhizobia
survival and multiplication in the soil to infection and nodulation and finally nitrogen fixation
Rhizobium spp. are less acid tolerant than Bradyrhizobium spp
When soil pH is decreased below 5.5, Bradyrhizobium japonicum nodulation in soybean is
greatly influenced.
Agrochemical residues in soil
3. Native microbial population
When biofertilizer strains are introduced to the soil, they compete with the soil’s natural
microbes
4. Predatory organism
Protozoan grazing, especially that of naked amoeba, the most common bacterial grazer in soil,
Protozoans feed on specific bacterial strains in the soil.
Three nematode species (Caenorhabditis elegans, Acrobeloides thornei and Cruznema sp.)
dramatically reduced Pseudomonas species and Bacillus subtilis colonisation in the wheat
rhizosphere.
5. Issues related to microbial strain development
Isolated species/strains may perform better for a single crop and have a limited host range

The following are some of the essential characteristics of an efficient bio-inoculant strain:
• Ability to compete with native microbial population.
• It should not antagonistic to beneficial microorganism.
• Should have higher survival rate in soil.
• Must tolerant to drought, salinity and heavy metal stress.
• Must grow in soil with low organic matter content.
• Should establish positive relationship with the host plant.
• Must express its PGP characters at maximum rate.