AG. MICRO 6.

2
BIOPESTICIDES AND BIOFERTILIZERS
(2+1)

Department of Agricultural Microbiology

B A College of Agriculture
Anand Agricultural University
Anand-388110

Prepared By
Dr. H. K. Patel
B A College of Agriculture
Anand
Dr. Raghunandan B. L. Mr. M. M. Saiyad
AICRP- Biological Control College of Agriculture,
Anand Vaso

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 PART I:
BIOPESTICIDES

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 1. HISTORY AND CONCEPT OF BIOPESTICIDES
What is Biopesticide?
Bio means involving life or living organisms. Pesticide includes substance or mixture of
substances intended for preventing, destroying or controlling any pest. Biopesticide refers
introduction of any living organism such as microorganism, parasitoids, predators, botanicals,
biochemical etc that controls pests by biological non-toxic means e.g. Bacillus thuringiensis,
Beauveria, NPV, EPN, Trichograma, Hormones etc. All the living organisms or it‟s by products,
which are used and exploited for the control of pest/insect, are called biopesticides.
How biopesticides work?
Biologicals are used to control pests, pathogens and weeds by a variety of means.
Microbial biocontrols may include a pathogen or parasite that infects the target. Alternatively,
they might act as competitors or inducers of plant host resistance. Biochemical biocontrols can
also act through a variety of mechanisms. Some act by inhibiting the growth, feeding,
development or reproduction of a pest or pathogen. Still other biocontrols may be used to form a
barrier on the host, so as to act as a feeding or infection inhibitor.
History of Biopesticides
Plant extracts were likely the earliest agricultural biocontrols, as history records that
nicotine was used to control plum beetles as early as the 17th century. Experiments involving
biological controls for insect pests in agriculture date back as far as 1835, when Agostine Bassi
demonstrated that white muscadine fungus (Beauveria bassiana) could be used to cause an
infectious disease in silkworm. Experiments with mineral oils as plant protectants were also
reported in the 19th century. During the rapid institutional expansion of agricultural research
during the early 20th century, an ever-growing number of studies and proposal for biocontrols
were developed.
The first, and still most, widely used biocontrols included spores of the bacteria Bacillus
thuringiensis (Bt). In 1901, Bt was isolated from a diseased silkworm by Japanese biologist
Shigetane Ishiwata. Ernst Berliner in Thuringen, Germany, then rediscovered it ten years later in
a diseased caterpillar of flour moth. The Bt pathogen was classified in 1911 as type species
Bacillus thuringiensis and remains the most widely used biocontrols to this day.
In the early 1920s, the French began to use Bt as a biological insecticide. The first
commercially available Bt product, Sporeine, appeared in France in 1938. In the US in the
1950s, widespread use of biocontrols began to take hold as a host of research on Bt efficacy was
published.
In the latter half of the 20th century, research and development continued at a low level
because of the widespread adoption of cheaper but more toxic synthetic chemical insecticides.
During this time, new products were developed and applied; especially in niche markets where
petroleum based chemicals were not registered, not effective, or not economical. For example, in
1956, the Pacific Yeast Product Company developed an industrial process known as submerged
fermentation, which allowed production of Bt on a large scale. In 1973, Heliothis NPV was
granted exemption from tolerance and the first viral insecticide, Elcar received a label in 1975.

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 In 1977, Bacillus thuringiensis var. israelensis (toxic to flies) was discovered, and in
1983 the strain tenebrionis (toxic to beetles) was found. In 1979, the U.S. EPA registered the
first insect pheromone for use in mass trapping of Japanese beetles. In the 1990s, researchers
began testing kaolin clay as an insect repellent in organic fruit orchards. It was made
commercially available, particularly for use in organic systems, in 1999.
Biological development for the control of plant diseases has undergone a similar
transformation. During the early 20th century, studies of soil microbiology and ecology had led to
the identification of many different microorganisms that act as antagonists or hyperparasites of
pathogens and insect pests. A number of these were shown to be useful in field scale
inoculations, but few were developed commercially because of the rapid adoption of chemical
pesticides during that time period. Commercial success stories from the 1980s and 1990s include
products containing Agrobacterium radiobacter for the prevention of crown gall on woody crops
and Pseudomonas fluorescens for the prevention of fireblight in orchards where the streptomycin
had been overused and resistant pathogen populations were abundant. In the greenhouse and
potting mix industry, products containing a variety of microbes that suppressed soilborne
pathogens were introduced into the market.
As the costs of overusing such synthetic chemicals became apparent, there was
resurgence in academic and industrial research related to biocontrols development. And with the
rapid expansion of organic agriculture during the past decade, adoption rates have rapidly
increased. Because of this, development of new and useful biocontrols has continued to increase
rapidly since the mid 1990s. In fact, more than 100 biocontrols active ingredients have been
registered with the U.S. EPA Biologicals division since 1995. Many of these have been
introduced Biologicals division since 1995. Many of these have been introduced commercially in
a variety of products. Many of the active ingredients currently approved for use in the U.S.A. can
be found in publicly available databases.
Some examples of biocontrols developed in more recent years
Agrobacterium radiobacter Strain K84
Agrobacterium radiobacter Strain K84 is a naturally occurring bacterium found in many soils
and in plant root zones. This biocontrols is used in the greenhouse and nursery environment to
control crown gall, an important plant disease.
Bacillus spp.
Bacillus licheniformis, B. pumilus, and B. subtilis are naturally occurring soil bacteria with
fungicidal properties that together have become one of the fastest growing biocontrols in today‟s
market. Successes include uses as seed treatments or dressings, foliar application and soil-
applied control of diseases in a variety of crops.
Paecilomyces lilacinus
Paecilomyces lilacinus is used to control nematodes that attack plant roots in field crops
including many vegetables, fruit, turf, and ornamental crops.

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Trichoderma spp.
Trichoderma spp. is another biocontrols technology developed in the 1990 s that has been widely
commercialized in recent years. Trichodermais a genus of fungi that helps to control plant
disease by stimulating plant host defenses and growth, and, under certain conditions, parasitizing
harmful fungi within the plant root zone.
Azadirachtin
Azadirachtin is an insect growth regulator derived from neem tree seeds. Known to affect some
200 species of insects, azadirachtin disrupts insect feeding and inhibits its ability to molt as it
changes from the pupa to adult stage.
Beauveria bassiana
Beauveria bassiana is a naturally occurring soil fungus that grows as white mold. This insect
pathogen can be used to control a wide range of target pests, which become infected and develop
white muscadine disease, killing the pest within a matter of days.
Cydia pomonella granulo virus (CpGV)
CpGV is a natural pathogen of the codling moth, a major pest of tree fruits such as apples and
pears. Developed through research begun in the 1980‟s, commercial use of CpGV in both
organic and conventional systems has gained in popularity over the last ten years as codling
moth has displayed resistance to many traditional insecticides.
Dysphania ambrosioides
An extract of the plant Dysphania ambrosioides (syn. Chenopodium ambrosioides) is used to
control a number of sucking insect pests such as aphids, leafhoppers, whiteflies, and mites in
citrus, grapes, tree nuts, and vegetables. This product breaks down the pest‟s exoskeleton,
adversely affects its respiratory system, and interrupts its ability to navigate (find food).

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 2. IMPORTANCE, SCOPE AND POTENTIAL OF BIOPESTICIDE
During recent years the concept of integrated pest management is arise but still farmers
have continued to rely heavily on chemical insecticides for pest control. The intensive and
indiscriminate use of insecticides for crop protection has created problems such as resistance of
insects to insecticides, resurgence and toxic residue in / on the crop plants and agro-ecosystem
pollution etc and also have adverse effects on the non target organisms such as pollinators,
parasitoids, predators and wild animals.
In order to overcome these problems, biological control is one of the safe approaches for
pest management. Insect control through an eco-friendly manner is no longer a dream because
many biological, botanical and natural pesticides are promoted successfully in the world market
by now. Insect pathogens are effectively demonstrated for pest control on different crops, since
the Second World War.
The chemical insecticides are valuable for control of insects, but due to their continuous
and over usage during last few decades have posed several serious problems.
 Development of resistance in pests.
 Resurgence of minor pests.
 Toxicity in the environment, Accumulation of pesticides residues.
 Biological magnification of pesticide residues through the food chain.
 General environmental pollution

In view of the several disadvantages associated with the unscientific use of pesticides in
agriculture, there is an urgent need for minimizing the use of chemical pesticides in the
management of insect pests through eco-friendly pest management tactics such as biological
control or biopesticide. Biopesticide aims at suppressing the pest in environmentally safe manner
without affecting other non target organisms. It provides the most effective, environmentally
sound and socially acceptable methods of managing diseases, pests and weeds".
Biopesticides:
In nature every ecosystem exists in a balance. Growth and multiplication of each
organism depends on the food chain, its predators, parasites, etc. In biological control system,
these interrelations are exploited. The natural enemy of a pest, disease or weed is selected; its
biology is studied for mass multiplication and utilizes the same to check the target pest. They are
also specific in their action and perish once their feed (i.e. the pest) is exhausted. Thus they are
based on natural principles; do not leave any residue, safe and economical. Among the
alternatives, biological control of pests is one of the important means for checking pest problems
in almost all agro-ecological situations.
Bio pesticides are living organisms, which can intervene the life cycle of insect pests in
such a way that the crop damage is minimized. The agents employed as biopesticides, include
parasites, predators and disease causing fungi, bacteria and viruses, which are the natural
enemies of pests.

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 Utilization of naturally occurring parasites, predators and pathogens for pest control is a
classical biological control. On the other hand, these bio agents can be conserved, preserved and
multiplied under Laboratory condition for field release. Once these bio-agents are introduced in
the field to build their population considerably, they are capable of bringing down the targeted
pest' population below economic threshold level (ETL). However, the crux lies in their mass
production and application at the appropriate time.

Advantages of biopesticides over chemical pesticides:

Biopesticides are preferred over chemical pesticides for the following reasons:
 No harmful residues.
 Target specific and safe to beneficial organisms like pollinators, predators, parasites etc.
 Growth of natural enemies of pests is not affected, thus reducing the pesticide application.
 Environmental friendly.
 Cost effective for long term use.
st nd
 Important component of IPM as 1 line and 2 line of defense, chemicals being the last
resort.

Difference between chemical pesticide and biopesticide

Factors Synthetic Pesticides Bio-pesticides
Cost effectiveness Cheap but increased Costlier but reduced
spraying cost number of applications
Persistence and residual effect High Low
Knockdown effect Immediate Delayed
Handling and Bulkiness Easy but danger and Bulky : Carrier based
Hazardous Easy : Liquid formulation
Pest resurgence More Less
Effect on Beneficial flora More harmful Not harmful
Target specificity Mostly broad spectrum Mostly host specific
Nature of control Curative Preventive
Shelf life More Comparatively Less
The market share of bio-pesticide is only 2% as compared to synthetic pesticide

Last three decade has witnessed a tremendous breakthrough in this aspect, especially on
standardization of production techniques of Trichoderma, Gliocladium, Paecilomyces,
Pseudomonas, NPV and Bacillus spp. to use them against many insect pests and diseases. The
popularity of biopesticides has increased in recent years, as extensive and systematic research
has greatly enhanced their effectiveness. Also, techniques for the mass production, storage,
transport and application of biopesticides have been improved in recent years.

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Commercial production of biopesticides:
Though there are more than about 300 biopesticide production units existing in the
country, as on today, they are able to meet the demand of only less than 10% of cropped area.
There exists a wide gap, which can only be bridged by setting up of more and more units for
production of biopesticides. This requires large-scale investment and private participation. Some
of the local small-scale industries have already started production and marketing of Beauveria
bassiana, Metarhizium anisopliae, Trichoderma spp., Paecilomyces lilacinus, and NPV. There is
a scope to enhance production and use of biological control agents in the days to come as the
demand is on the increase every year.
Few microbial pesticides marketed in India:
Sr. No. Name of Agent/s Common Product Names
1 Bacillus thuringiensis Delfin, Halt, Dipel, Biolap etc.
2 Nuclear polyhedrosis virus (NPV) HaNPV, SlNPV
3 Muscardine fungi, Beauveria bassiana Basina, Biosoft, Metarhizium, Beauveria
and Metarhizium anisopliae spores
4 Trichoderma viride Tricho-XP, Mnitor WP, Monitor S,
T. harzianum Trichomycil, T-2 Trichoderma spore
powder and granules, etc.
5 Pseudomonas fluorescence P. fluorescence wattable powder
6 Paecilomyces lilacinus Yorker, Tcicho X-P (Combi product)
7 Entomopathogenic nematodes Green commando, Soil commando,
Steinernema sp.
(More than 50 products are now available in India out of which majorities are produced by small-scale
regional producers and few products are also from State Agricultural Universities, but majority of them
are not registered products OR having provisional registrations)

Characteristics for development of a biopesticide:

 Efficacy: A highly effective biocontrol strain or other material must be obtained or produced.
Such strains must not only have appropriate mechanisms for biocontrol, but it should also (a)
be able to compete and persist in the environment in which it must operate, and (b) ideally,
be able to colonize and proliferate after application. But better to use existing natural strains,
which should be sufficiently effective.
 Cost: Inexpensive or less expensive production and formulation of the biocontrol agent or
other material must be developed. The production process must result in biomass with
excellent shelf life even under adverse storage conditions.
 Application: Delivery and application methods that permit the full expression of the
biocontrol agent. Delivery systems must ensure that biocontrol agents will grow well and
achieve their purpose. Delivery and application processes must be developed on a crop by
crop and application by application basis. No general solutions exist, and so biocontrol
systems must be developed for each crop. An effective method of use is to use the biocontrol

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 agent in conjunction with chemical pesticides. The chemicals provide good short-term
protection and the biocontrol provides long-term protection. As a consequence, yields
frequently are increased over use of the chemical alone.
 Market Potential: Considering the negative effects of indiscriminate case of pesticides,
importance for organic farming and promotion of sustainable farming practices it is estimated
that there will be further scope for new units, particularly in the states of Maharashtra,
Gujarat, Rajasthan, Madya Pradesh, Tamil Nadu, AP, UP, West Bengal and Karnataka,
where crops such as sugarcane, pulses, cereals and vegetable crops are grown in large scale.
At present, in some states, state government is purchasing the product from the private
parties and selling it to the individual farmers at a subsidized rate.

Future prospects for biopesticide research, production and use in India:

Due to chemical pesticide problems in India, there is an urgent need to promote
environmental friendly biopesticides in the country. Moreover recent Government policies also
favor production and use of biopesticide.
 More potential industrial fermentation technique for production of biopesticides needs to
be developed to fulfill the ever increasing demand in the market.
 Large-scale field demonstrations at farmer‟s fields are required to increase awareness and
adoption of biopesticides for pest management.
 Use of effective biopesticides should be accelerated in integrated management and
economics needs to be worked out.
 Interest to be focused on developing new methods of biological control. In particular,
identification of proteins and the genes encoding them from various microbial agents and
newer microbes.

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 3. DEFINITIONS AND CLASSIFICATION OF BIOPESTICIDES
The term biopesticides defines compounds that are used to manage agricultural pests by
means of specific biological effects rather than as broader chemical pesticides. It refers to
products containing biocontrol agents – i.e., natural organisms or substances derived from
natural materials (such as animals, plants, bacteria, or certain minerals), including their genes or
metabolites, for controlling pests. According to the FAO definition, biopesticides include those
biocontrol agents that are passive agents, in contrast to biocontrol agents that actively seek out
the pest, such as parasitoids, predators, and many species of entomopathogenic nematodes. Thus
biopesticides cover a wide spectrum of potential products that can be classified in major four
groups as:
1. Microbial pesticides
2. Botanical pesticides
3. Biochemical/Semiochemical pesticides
4. Plant incorporated Protectants (PIPS).

Classification of biopesticides:
1. Microbial pesticides and other entomopathogens:
Pesticides that contain microorganisms, like bacteria, fungi, or virus, which attack
specific pest species, or entomopathogenic nematodes as active ingredients. Although most of
these agents attack insect species (called entomopathogens; products referred to as
bioinsecticides), there are also microorganisms (i.e., fungi) that control weeds (bioherbicides).
They suppress pest by producing a toxin specific to the pest, causing a disease or preventing
establishment of other microorganisms through competition or other modes of action.
Microbial insecticides are a new form of pesticide that works by infecting selected insect
populations. Though this sounds potentially dangerous, but it is actually safe, since the
application of microbial insecticides is specific to the species one is trying to kill. Microbial
insecticides have no effect on animal populations, unless diminishing a certain bug in the area
interrupts the food chain. Each type usually works against only one type of insect.
Types of microbial pesticides:
There are five main categories of microbial pesticides based on the active ingredient used
as bellow.
1) Bacteria based: Bacterial biopesticides are probably the most widely used and cheaper than
the other methods of pest bioregulation. Insects can be infected with many species of bacteria but
those belonging to the genus Bacillus are most widely used as pesticides.
Bacillus thuringiensisI (Bt):
 Discovered in Japan in early 20th century and first become a commercial product in France in
1938.
 Control lepidopterous pests like American bollworm in cotton and stem borers in rice.
 Bt has developed many molecular mechanisms to produce pesticidal toxins i.e. Cry and VIP;
most of toxins are coded for by several cry genes.

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 Since its discovery in 1901 as a microbial insecticide, Bt has been widely used to control
insect pests important in agriculture, forestry and medicine.
 When ingested by pest larvae, Bt releases toxins which damage the mid gut of the pest,
eventually killing it.
 Main sources for the production of Bt preparations are the strains of the subspecies kurstaki,
galeriae, dendrolimus for agriculture use.
Bacillus popilliae:
 A naturally occurring bacterium that is used for control of white grub.
 Cause „milky spore disease‟ in the larvae of the grub or beetle and establish a resident
population capable of causing mortality over several seasons if soil conditions are appropriate.
 Bacillus popilliae was the first insect pathogen to be registered in the U.S. as microbial
control agent.

2) Fungi based: Fungi often act as important natural control agents that limit insect populations.
Most of the species that cause insect diseases spread by means of asexual spores called conidia.
Where viable conidia reach a susceptible host, free water or very high humidity is usually
required for germination. Unlike bacterial spores or virus particles, fungal conidia can germinate
on the insect cuticle and produce specialized structures that allow the fungus to penetrate the
cuticle and enter the insect's body. In most instances, as fungal infections progress, infected
insects are killed by fungal toxins, not by the chronic effects of parasitism. Many important
fungal pathogens attack eggs, immatures, and adults of a variety of insect species.
Beauveria bassiana:
 This common soil fungus has a broad host range that includes many beetles and fire ants. It
infects both larvae and adults of many species.
 Habitat: Foliage and soil
 Insect Host: White flies, beetles & caterpillars (including Helicoverpa sp.)
Metarhizium anisopliae:
 This common soil fungus has a broad host range that includes many beetles and other soft
body insects.
 Habitat: Foliage and soil
 Insect host: Frog hoppers, beetles
Vericillium lecanii:
 This fungus has been used in greenhouses to control aphids and whiteflies.
 Habitat: Glasshouse foliage
 Insect host: Aphids, whiteflies & scales.
Nomuraea rileyi:
 Naturally occurring epidemic infections of Nomuraea rileyi cause dramatic reductions in
populations of soybean foliage-feeding caterpillars.
Hirsutella thompsonii:
 It is a pathogen of the citrus rust mite.
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3) Virus based:
 The larvae of many insect species are vulnerable to devastating epidemics of viral diseases.
 The viruses that cause these outbreaks are very specific, usually acting against only a single
insect genus or even a single species.
 Specificity of the virus-host relationship makes viruses an ideal candidate for use in the
control of specific pest population.
 The most important group of viruses used for biocontrol belongs to the highly host-specific
family of Baculoviridae, which are pathogenic for invertebrates.
 Baculoviruses have been found in over 700 species of invertebrates, mainly Lepidoptera and
are considered an effective and selective means for biological insect control.
 Based on the morphology of their occlusion bodies (OBs), they can be distinguished between
nucleopolyhedroviruses (NPVs) and granuloviruses (GVs).
 The OBs are composed of a crystalline protein matrix, mainly consisting of a single protein,
the so-called „polyhedrin‟ in NPVs and „granulin‟ in GVs.
A. Polyhedrosis virus
 Polyhedrosis virus where many virus particles are embedded in each protein crystal.
 When they occur in the host cell nucleus, they are called Nuclear Polyhedrosis Virus
(NPV).
 When they occur in the host cytoplasm they are called Cytoplasmic Polyhedrosis Virus
(CPV).
B. Granuloisis virus
 Granulosis virus where only one virus is contained in each protein crystal.
 They either develop in the nucleus or cytoplasm of the host cells (NGV or CGV).
 The viral DNA replicates in the nuclei of the host cells and then spreads throughout the body
of the larvae, turning it into a “virus factory.”
 The infected insect stops feeding within a few days, dies and disintegrate.
 HaNPV (Helicoverpa NPV): It is highly effective on H. armigera, pest of cotton, gram, pea,
pigeon pea, tomato, cabbage, ground nut, millets and oilseeds.
 SlNPV (Spodoptera NPV): It is highly effective against S. litura caterpillar, pest of cotton,
gram, pigeon pea, cabbage, tomato, chillies & oilseeds.
4) Nematode based:
 Entomopathogenic nematodes (EPNs) are insect-specific parasites in the genera Steinernema
(Steinernematidae) and Heterorhabditis (Heterorhabditidae) that are obligatory associated
with symbiotic bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) which are
responsible for rapidly killing host insects.
 After entering a host insect, the infective juvenile stage (IJ) of EPNs releases its symbiotic
bacteria. In addition to killing the host, the bacteria digest host tissues, and produce antibiotics
to protect the host cadaver from saprophytes and scavengers.

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 After two to three reproductive cycles, when host nutrients are depleted, infective juveniles
are produced and begin leaving the host insect. This stage is capable of immediately infecting
a new host or may persist for months in the absence of a host.
 The entomogenous nematodes Steinernema feltiae, S. riobravis, S. carpocapsae and
Heterorhabditis heliothidis are the species most commonly used in insecticidal preparations.
 Within each of these species, different strains exhibit differences in their abilities to infect and
kill specific insects.
 In general, however, these nematodes infect a wide range of insects.
 On a worldwide basis, laboratory or field applications have been effective against over 400
pest species, including numerous beetles, fly larvae, and caterpillars.
5) Protozoa based:
 Protozoa are single-celled eukaryotic organisms that exist in both water and soil.
 The protozan Nosema locustae is known to be a natural biocontrol agent of many grasshopper
species.
 Nosema infects at least 90 species of grasshoppers.
 It is non-toxic to humans and other mammals.
 It infects and weakens young grasshoppers and adversely affects female grasshoppers‟ ability
to reproduce.

2. Botanical pesticides:
 These are naturally occurring plant material that may be crude preparation of the plant parts
ground to produce a dust or powder or liquid that can be used in full strength or dilute form in
a carrier such as clay, talc or diatomaceous earth.
 Several plant based insecticides as nicotinoids, natural pyrethrins, rotenoids, neem products
etc are used.
 They are generally acting in one of two ways 1) Contact poison and 2) Stomach poison.
 There are about 25,000 plant species evaluated among which, 2500 found useful for pest
management and around 1005 exhibited insecticidal activity including 384 anti-feedants, 297
repellents, 27 attractants and 31 possess growth inhibiting properties.
 Neem tops the list of 2,500 plant species that are reported to have pesticidal properties and is
regarded as the most reliable source of eco-friendly biopesticidal property.
 “Azadirachtin” affects the reproductive and digestive process of pest.
 Neem products (Neem Seed Kernel Extract: NSKE) are effective against more than 350
species of arthropods, 12 species of nematodes, 15 species of fungi, three viruses, two species
of snails and one crustacean species.
 Majority of formulations contain 300 to 1,500 ppm azadirechtin.
 Recent formulations contain 10,000 to 50,000 ppm.
 Chrysanthemum (Tanacetum), Nicotiana, Derris are other members.

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 Examples:
Name Plant part used Active ingredients
Neem (Azadirachta indica) Oil and leaves Azadirachtin
Castor (Ricinus communis) Leaves and oil Ricin
Onion (Allium cepa) Bulb Oleic acid, α asarone, β
asarone
Custard apple (Annona squamosa) Leaves and bark Annonin, squamocin

 Some of the plant product registered as biopesticides

Plant product used as Target Pests
biopesticides
Limonene and Linalool Fleas, aphids and mites, also kill fire ants, several types of flies,
paper wasps and house crickets
Neem A variety of sucking and chewing insect
Pyrethrin Ants, aphids, roaches, fleas, flies, and ticks
Rotenone Leaf-feeding insects, such as aphids, certain beetles (asparagus
beetle, bean leaf beetle, Colorado potato beetle, cucumber beetle,
flea beetle, strawberry leaf beetle, and others) and caterpillars, as
well as fleas and lice on animals
Ryania Caterpillars (European corn borer, corn earworm, and others) and
thrips
Sabadilla Squash bugs, harlequin bugs, thrips, caterpillars, leaf hoppers, and
stink bugs

3. Biochemical/Semiochemical pesticides:
The chemical messages that trigger various behavioral responses are collectively referred
as semiochemicals. Pesticides based on these naturally occurring substances that control pests by
non-toxic mechanisms, in contrast to chemical pesticides that contain synthetic molecules that
directly kill the pest. Biochemical pesticides include substances that interfere with growth or
mating, such as insect growth regulators, or substances that repel or attract pests, such as
pheromones.
Pheromones:
Biochemical pesticides include substances, such as insect sex pheromones, which
interfere with mating, as well as various scented plant extracts that attract insect pests to traps.
Man-made pheromones are used to disrupt insect mating by creating confusion during the search
for mates or can be used to attract male insects to traps. Pheromones are often used to detect or
monitor insect populations or in some cases, to control them.
Pheromones are chemicals emitted by living organisms used to send messages to
individuals usually of the opposite sex of the same species. Pheromones of hundreds of insect
species have been chemically elucidated, including the sex pheromone. When used in

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combination with traps, sex pheromones can be used to determine what insect pests are present
in a crop and what plant protection measures or further actions might be necessary to assure
minimal crop damage. If the synthetic attractant is exceptionally effective and the population
level is very low, some control can be achieved with pheromone traps or with the "attract and
kill" technique.
Insect Growth Regulators:
An insect growth regulator (IGR) is a substance (chemical) that inhibits the life cycle of
an insect. Two main groups of insect growth regulators (IGRs) being used in a commercial scale
are the juvenile hormone analogues (JHAs) and the chitin synthesis inhibitors (CSIs).
JHA:
 Five different formulations of methoprene are available commercially for the control of
mosquitoes, flies, beetle ants and fleas.
 Another, JHA, fenoxycarb, used for control of several fruit pests.
 Pyriproxyfen effective against sucking pests.
 Diofenolan for control of scale insects and lepidopteran pests.
CSI:
 Acyl urea group used for control of foliage feeders and tissue borers.
 Diflubenzuron used against several pests of cotton.

4. Plant-Incorporated Protectants (PIPs):

These include pesticidal substances that are produced in genetically modified
plants/organisms (GMO) (i.e., through the genetic material that has been incorporated into the
plant). One approach, to reduce destruction of crops by phytophagous pests, is to genetically
modify plants to express genes encoding insecticidal toxins. The adoption of genetically
modified (GM) crops has increased dramatically in the last years. Genetically modified (GM)
plants possess a gene or genes that have been transferred from a different species.
The production of transgenic plants that express insecticidal δ-endotoxins derived from
the soil bacterium Bacillus thuringiensis (Bt plants) were first commercialized in the US in 1996.
The expression of these toxins confers protection against insect. The lethality of Bt endotoxins is
highly dependent upon the alkaline environment of the insect gut, a feature that assures these
toxins are not active in vertebrates, especially in humans. These proteins have been
commercially produced, targeting the major pests of cotton, tobacco, tomato, potato, corn, maize
and rice, notably allowing greater coverage by reaching locations on plants which are
inaccessible to foliar sprays.
There are numerous strains of Bt, each with different Cry proteins and more than 375 Cry
proteins have been identified. Most Bt maize hybrids express the Cry1Ab protein, and a few
express the Cry1Ac or the Cry9C protein, all of which are targeted against the European corn
borer (Ostrinia nubilalis Hubner) (Lepidoptera), a major pest of maize in North America and
Europe. Some recent maize hybrids express the Cry3Bb1 protein, which is targeted against the
corn rootworm complex (Diabrotica spp.).

15
 Cotton expressing the Cry1Ac protein is targeted against the cotton bollworm
(Helicoverpa), which is a major pest of cotton; potato expressing the Cry3A or Cry3C is targeted
against the Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera), which is a
major pest of potato; and Cry4 proteins are targeted against some Diptera, such as certain flies
(e.g., Lycoriella castanescens Lengersdorf) and mosquitoes (e.g., Culex pipiens L.).

16
 4. VIRULENCE, PATHOGENICITY AND SYMPTOMS OF
ENTOMOPATHOGENIC PATHOGENS AND NEMATODES
1. Entomopathogenic bacteria
1) Bacillus thuringiensis
The most commonly used microbial agents to control noxious insects world over is
Bacillus thuringiensis. The efficacy of Bt is quite impressive than other biopesticide and easily
available in market. Amongst all insect pathogenic microbes, Bt has received prime attention and
the product based on this bacterium is widely demonstrated among all the biological insecticides.
Bt is a gram positive, aerobic, sporulating bacterium that synthesizes crystalline proteins
during later stage of growth as secondary metabolites which is highly toxic to agriculturally
important pest especially caterpillars belonging to lepidopterans at very low concentration. The
outstanding feature of Bt is the production of protenaceous delta (δ) endotoxin (crystal) that
accounts up to 30 % of total protein content of the bacterium.
The crystal contains insecticidal proteins that may vary in type, quantity as well as
toxicity against different groups of insects depending on the bacterial strain and place of
isolation. Bt δ endotoxin (cry proteins) has acquired acceptability as eco-friendly biopesticides
because of its specificity towards insects having alkaline gut and by enlarge safer to higher
animals. Bt is under extensive use for control of insects in agriculture, horticulture, forestry and
also for mosquito control over past three decades.
Mode of action:
The crystal protein of Bt is effective to insects having specific gut pH (alkaline) and /or
the specific gut membrane structures required for binding of the toxin. The protein toxin binds to
specific receptors present in the midgut epithelial membrane and disrupts membrane‟s ion
exchange potential and osmotic equilibrium causing cell lyses and finally this damage leads to
gut paralysis and ultimately cause death of the susceptible host. Infected insect stop feeding and
die due to the combined effect like starvation and tissue damage. Bt spores have also capacity to
cause disease (septicemia) in the insect by invading in heamocoel system.
The mode of action of Bacillus thuringiensis is summarized in the following sequential
stages:
1. Ingestion of sporulated Bacillus thuringiensis by an insect larva.
2. Solubilization of the crystalline ICP in the midgut at high pH (alkaline, >9.0).
3. Activation of the ICP by gut proteases enzymes.
4. Binding of the activated ICP to specific receptors in the midgut cell membrane.
5. Insertion of the toxin in the cell membrane and formation of pores and channels in the gut
cell membrane, followed by destruction of the epithelial cells.
6. Subsequent Bacillus thuringiensis spore germination. Septicemia may enhance mortality.
Symptoms:
Larvae infected by Bt become inactive, stop feeding and have watery excretion. The head
capsule may appear to be larger compared to body size. The larva becomes flaccid and dies,
usually within days. A typical putrefied smell comes out from dead cadavers. The body content

17
turns brownish-black as they decompose, while in case of other bacteria it may turn red or
yellow.

Figure: Mode of Action of B. thuringiensis on caterpillars.

Bt was first found to affect a range of lepidopteran insects, the major agricultural pests.
Subsequently, discovery of newer strains expanded its host range. The host range of Bt is
constantly widening with the large amount of commercial interest and at present, extensive
screening programmes are in progress world over. Although most of the Bt strains are toxic to
lepidoptera, few strains are known to be toxic against Coleopterans (root–grubs), Dipterans
larvae especially mosquitoes, lice, mites and phytonematodes too (Table).
Table: Host range of B. thuringiensis
Order Susceptible families
Insecta: Most of the lepidopteran families susceptible
Lepidopterans Sphingidae - Hawk moths
Pieridae - Cabbage worms
Lymantridae - Tussock moths
Tortricidae - Leaf roller moth
Noctuidae - Cutworms
Dipterans Culcidae - Mosquitoes
Simuliidae - Black flies
Anisopodidae - Gnats
Chironomidae - Nudges
Psychodiae - Moth flies
Tipulidae - Crane flies
18
Coleopteran Cyrysomelidae - Leaf beetles
Phthiraptera Philopteridae - Bird lice
Treichodectidae - Mammalian lice
Arachnida: Dermanyssidae - Animal mites
Acari Tetranichidae - Plant mites
Nematoda: Trichostrongylidae - Animal parasitic
Strongylida Tylenchidae - Plant parasitic

2) Bacillus popilliae
Bacillus popilliae is a highly fastidious bacterium that is the primary etiological agent of
the so called milky diseases of scarab larvae. These insects are the immature stages of beetles,
such as the Japanese beetle Popillia japonica, that are important grass and plant pests belonging
to the coleopteran family Scarabaeidae.
The term “milky disease” is derived from the opaque white color that characterizes
diseased larvae and results from the accumulation of sporulating bacteria in larval hemolymph
(blood). The disease is initiated when grubs feeding on the roots of grasses or other plants ingest
the bacterial spores. The spores germinate in the midgut and vegetative cells invade the midgut
epithelium, where they grow and reproduce, changing in form as they progress toward invasion
of the hemocoel (body cavity). After passing through the basement membrane of the midgut, the
bacteria colonize the blood over a period of several weeks and sporulate, reaching populations of
108cells/ml. For larvae that ingest a sufficient number of spores early in development, the disease
is fatal. Dead larvae in essence become bag of spores that serve as a source of infection for up to
30 years.
Despite decades of research, suitable media for the growth and mass production of P.
popilliae in vitro have not been developed. Thus, the technical material (i.e., spores) used in
commercial formulations is produced in living, field-collected scarab larvae.
2. Entomopathogenic fungi
Agostino Bassi in 1835, first time formulated the germ theory by the use of white
muscardine fungus on the silkworm that was then named in his honor as Beauveria bassiana and
gave the idea of using insect infecting fungi for the control of insect pest management.
Mode of action:
The development of fungal infections in terrestrial insect is largely influenced by
environmental conditions. High humidity is vital for germination of spores and transmission of
the pathogen from one insect to another. Unlike bacteria and virus, which must be consumed,
toxicity from entomopathogenic fungi most often occurs from contact of the fungal conidia with
the host cuticle. With most entomopathogenic fungi, disease development involves following
steps which are popularly known as „Mycosis‟:
1. Attachment of the infective units like spore or conidia or zoospores to the insect
epicuticle.
2. Germination of the infection unit on the cuticle.

19
 3. Penetration of the cuticle, either directly by germ tube or by infection pegs from
appresoria.
4. Multiplication of the hyphal body in the haemocoel.
5. Death of the host: Growth of the mycelia phase with invasion of virtually all host organs
and penetration of the hyphae from the interior through the cuticle to the exterior of the
insect.
6. Production of infective units on the exterior of the insect.

Salient features of mycosis:

I. Spore germination: the adhesion of spores into the insect‟s integument may be a passive
mechanism and involve mucilaginous material. Spore germination largely depends on the
environmental humidity and temperature. Optimum temperature for development,
pathogenicity and survival of the fungi merely falls between 20 to 30˚C. Very high
humidity (~ 80 %) is required for spore germination. the germination spore form a germ
tube that serves as a penetration hyphae and an appressorium may also be produced.
II. Penetration: the mode of penetration is determined by the characteristics of the
integument, its thickness, sclerotization and the presence of antifungal and nutritional
substances. The larvae are more susceptible immediately after moulting before the cuticle
is fully hardened. the process of penetration through the cuticle by hyphae germinating
from spores involves chemical and physical forces. The enzymes detected on germ tubes
are proteases, aminopeptidases, lipase, esterase and chitinase. The germ tube produces
appressorium, when the integument is hard. Fungi can infect insect through spiracles and
other body openings.
III. Replication: The fungi produce hyphal bodies and may avoid the immune system of the
insect by (i) Developing protoplasts that are not recognized by the haemocysts, (ii) forming
hyphal bodies that multiply and disperse rapidly and (iii) by producing mycotoxins. After
death of the insect, the fungus grows saprophytically in the haemocoel to form a mycelia
mass that forms a hard or firm sclerotium. The reproductive spores are produced within the
sclerotium or sporengiophore or conidiophores.
Mycotoxins: Symptoms such as partial or general paralysis, sluggishness, and decreased
irritability in mycosed insects are consistent with the action of neuromuscular toxins. B.

20
 bassiana and M. anisopliae produced significant amounts of toxic compounds within their
hosts. For example, the toxins Beauvericin, Bassianolide, Isarolides and Beauverolides
have been isolated from B. bassiana infected hosts, toxins Destruxins (DTXs) and
Cytochalasins have been isolated from M. anisopliae infected hosts.
IV. Spore dispersal: The spores of many fungi are forcibly discharged and carried by wind.
Certain fungi from conidia that are enclosed in mucilaginous slime. Such conidia may
attach to a passing insect or other invertebrates for dispersal.
Symptoms of mycosis:
1. Loss of appetite, irritability and paralyses.
2. Discoloured patches on integuments and increased acidity in blood.
3. The body hardens and covered by dense white mycelial mat or growth.
4. Mummified larvae/insect adheres to leaves, stem and fruiting body with upright position
on its prolegs at the time of death.
5. Death occurs within 4-7 days depending on host insects and environmental conditions.
Host range:
1. Beauveria bassiana: They are used particularly to control sucking pests and caterpillars
infesting crop plants. These fungi are used to control the caterpillars of yellow stem borer and
leaf folder of rice, white grub of groundnut, sugarcane pyrilla, coconut rhinoceros beetle,
caterpillars of pulses, tomato and cotton, diamond back moth, leaf eating caterpillars of
tobacco and sunflower etc.
2. Metarrhizium anisopliae: This fungus is used to control mainly coconut rhinoceros beetle,
groundnut cut worm, rice brown plant hopper, diamond back moth and early shoot borer, top
shoot borer and internode borer of sugarcane.
3. Lecanicelium lecanii: This fungus is mainly used to control whiteflies, aphids, thrips, brown
plant hopper, scale insects, mealy bugs and other sucking insect pests of crop plants.
4. Nomuraea rileyi: It is used to control pod borers, cut worms, cabbage borers etc.
5. Hirsutella thompsonii: These fungi are used to control different hoppers and bug pests,
whiteflies, red mites etc.
3. Entomopathogenic virus
Viruses in the family Baculoviridae, a diverse family of more than 700 viruses, are the
best known of all the insect viruses. This is because they are very common in some of our most
important insect pests, disease symptoms are easily recognized and they have the most potential
for development as microbial insecticides. The major reason for interest in baculoviruses is their
environmental safety. They are often, genus or species specific and safe to non target organisms.
Mode of action / Life cycle of baculoviruses
The baculovirus life cycle involves two distinct forms of virus. Occlusion derived virus
(ODV) is present in a protein matrix (polyhedrin or granulin) and is responsible for the primary
infection of the host while the budded virus (BV) is released from the infected host cells later
during the secondary infection.

21
 Typically, the initial infection occurs when a susceptible host insect feeds on plants that
are contaminated with the occluded form of the virus. The protein matrix dissolves in the
alkaline environment of the host midgut (stomach), releasing ODV that then fuse to the columnar
epithelial cell membrane of the host intestine and are taken into the cell in endosomes.
Nucleocapsids escape from the endosomes and are transported to nucleus. Viral transcription and
replication occur in the cell nucleus and new BV particles are budded out from the lateral side to
spread the infection systemically. During budding, BV acquires a loosely fitting host cell
membrane with expressed and displayed viral glycoproteins.

Baculovirus infection can be divided to three distinct phases: Early (0–6 h post-infection),
Late (6–24 h p.i.) and Very late phase (18–24 to 72 h p.i.).
While BV is produced in the late phase, the ODV form is produced in the very late phase
acquiring the envelope from host cell nucleus and embedded in the matrix of occlusion body
protein. These occlusion bodies are released when cells lyse to further spread baculovirus
infection to next host. The extensive lysis of cells frequently causes the host insect to literally
disintegrate, thus the reason for the historic name "wilting disease." The complete ODV-
polyhedrin particles are resistant to heat and light inactivation, whereas the naked BV virion is
more sensitive to environment.
When infecting a caterpillar, the advanced stages of infection cause the host to feed
without resting, exhibit negative geotropism and then to climb to the higher parts of trees,
including exposed places they would normally avoid due to the risk of predators. This is an
advantage for the virus if (when the host dissolves) it can drip down onto leaves which will be
consumed by new hosts.

22
Symptoms:
 Discoloration (brown and yellow)
 Stress
 Decomposition (liquefication)
 Lethargy (slow movement to no movement at all; refusal to eat)
 Hanging body on to the top branches of the plant.

Host range: Larvae of major lepidopteran insect including codling moth, Spodoptera litura,
Helicoverpa armigera, H. zea, H. virescens, Autographa californica and many more.
4. Entomopathogenic nematodes
Entomopathogenic nematodes occur naturally in soil environments and locate their host
in response to carbon dioxide, vibration and other chemical cues. They infect many different
types of insects living in the soil like the larval forms of moths, butterflies, flies and beetles as
well as adult forms of beetles, grasshoppers and crickets. Species in two families,
Heterorhabditidae and Steinernematidae have been effectively used as biological insecticides in
pest management programs.
Mode of action / Life cycle of EPN
The infective juvenile stage (IJ) is the only free living stage of entomopathogenic
nematodes. The juvenile stage penetrates the host insect via the spiracles, mouth, anus, or in
some species through intersegmental membranes of the cuticle and then enters into the
hemocoel. Both Heterorhabditis and Steinernema are mutualistically associated with bacteria of

23
the genera Photorhabdus and Xenorhabdus, respectively. The juvenile stage release symbiotic
bacteria from their intestines into the hemocoel. The bacteria multiply in the insect hemolymph
and the infected host usually dies within 24 to 48 hours. After the death of the host, nematodes
continue to feed on the host tissue, mature and reproduce. The progeny nematodes develop
through four juvenile stages to the adult. Depending on the available resources one or more
generations may occur within the host cadaver and a large number of infective juveniles are
eventually released into environment to infect other hosts and continue their life cycle.
Symptoms

The insect cadaver becomes red if the insects are killed by heterorhabditids and brown or
tan if killed by steinernematids. The color of the host body is indicative of the pigments
produced by the monoculture of mutualistic bacteria growing in the hosts.
Searching Behavior
Entomopathogenic nematodes use two search strategies: ambushers or cruisers.
Ambushers such as St. carpocapsae have an energy conserving approach and lie-in-wait
to attack mobile insects (nictitating) in the upper soil. Cruisers like St. glaseri and H.
bacteriophora are highly active and generally subterranean, moving significant distances
using volatile signals and other methods to find their host underground. Therefore, they
are effective against less mobile pests such as white grubs. Some nematode species such
as St. feltiae and St. riobrave use an intermediate foraging strategy (combination of
ambush and cruiser type) to find their host.

Host range
The major pests that are killed using these nematodes include white grubs, black vine
weevil, turf grass pests, mole crickets, weevils and cutworms.

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 5. MASS PRODUCTION TECHNOLOGIES OF BIOPESTICIDES

5.1 Scope for Production of Biopesticides

Though there are about 140 bio-pesticide production units existing in the country as on
today, they are able to meet the demand of only less than 1% of cropped area. There exists a
wide gap, which can only be bridged by setting up of more and more units for production of Bio-
pesticides. There is a scope to enhance production and use of biological control agents in the
days to come as the demand is on the increase every year. This requires large scale investment
and private participation
To achieve optimum results, bio-pesticide facilities are to be set up in areas which have
appropriate climatic conditions. Because temperature control is less costly in locations where
there are no extreme conditions. Besides the climatic conditions, the proximity of the location to
the market is also important. However, care must be taken that the production facilities are set up
at least a quarter of a mile away from farming areas, so as to prevent the contamination of
production facilities by insecticides from the farming areas. Also, as air pollution can damage
bio-pesticides, the production should be located away from industrial and urban areas.
5.2 Methods in production of biopesticides
5.2.1 Solid state fermentation
Solid-State Fermentation (SSF) SSF utilizes solid substrates like bran, bagasse, and paper
pulp. The main advantage of using these substrates is that nutrient-rich waste materials can be
easily recycled as substrates. In this fermentation technique, the substrates are utilized very
slowly and steadily, so the same substrate can be used for long fermentation periods. Hence, this
technique supports controlled release of nutrients. SSF is best suited for fermentation techniques
involving fungi and microorganisms that require less moisture content. However, it cannot be
used in fermentation processes involving organisms that require high aw (water activity), such as
bacteria. The most used substrates are starchy and lignocellulosic materials. Some of the
common substrates used in solid state fermentation are wheat bran, rice and rice straw, hay, fruit
and vegetable waste, paper pulp, bagasse, coconut coir, and synthetic media. Spores produced
using SSF were more stable and resistant to stress than those produced using liquid culture or on
agar. Most of the microorganisms used in SSF are filamentous fungi due to their capability to
grow on solid surfaces with low free water, but also some bacteria with biocontrol ability have
been produced using SSF.
5.2.2 Liquid state fermentation (lf)/ submerged fermentation (smf)
SmF utilizes free flowing liquid substrates, such as molasses and broths. The bioactive
compounds are secreted into the fermentation broth. The substrates are utilized quite rapidly;
hence need to be constantly replaced/supplemented with nutrients. This fermentation technique is
best suited for microorganisms such as bacteria that require high moisture content. An additional
advantage of this technique is that purification of products is easier. Some common substrates
used in submerged fermentation are soluble sugars, molasses, liquid media, fruit and vegetable
juices, and sewage/waste water.

25
 SmF is primarily used in the extraction of secondary metabolites that need to be used in
liquid form. SmF is usually implemented in case of bacterial enzyme production, due to the
requirement of higher water potential. SSF is preferred when enzymes have to be extracted from
fungi, which require lesser water potential. More than 75% of the industrial enzymes are
produced using SmF, one of the major reasons being that SmF supports the utilization of
genetically modified organisms to a greater extent than SSF. This is highly critical due to the fact
that the metabolism exhibited by microorganisms is different in SSF and SmF, and the influx of
nutrients and efflux of waste materials needs to be carried out based on these metabolic
parameters. Any slight deviation from the specified parameters will result in an undesirable
product.
5.3 Mass production of Trichoderma
For mass multiplication of Trichoderma the following steps should be followed
sequentially as noted below:
1) Take about 200 gm of grains in autoclavable bags [71 (B) x 111 (H) and add equal amount of
tap water.
2) After filling the bags, keep a 1.51 inches PVC pipe at the top of the cover and tied it with a
rubber band.
3) Close PVC pipe mouth using cotton plug.
4) Boil the grains in a 10-20 liter pressure cooker with water inside it for a period of 40 minutes.
5) The grains are cooled at room temperature after sterilization.
6) Transfer the bags into inoculation chamber.
7) Inoculate with 1-2 bits of Trichoderma mother culture in each bag inside the chamber with
all the help of inoculation loop/spatula. Shake the bags properly for mixing the fungal culture
all over the grains.
8) Keep the inoculated bags at the room temperature (25-30 C)
9) Observe the inoculated bags if there is mycelial growth, do not disturb the inoculated bag. If
mycelial growth is not observed, shake the inoculated bag.
10) Once Trichoderma sporulation (green color) takes place shake the bags every alternate day
for about 5 to 7 days in order to spread and allow the Trichoderma growth and further
sporulation.
11) Transfer the grains with fully grown Trichoderma mycelia & sporulation into cleaned plastic
trays and cover it with blotter/newspaper. Keep these plastic trays for further sporulation and
drying for about 3-4 days at room temperature. Mix the transferred Trichoderma colonized
grains once in every day for up to 3-4 days with the help of spatula for enhancing sporulation
and drying.
12) The Trichoderma will be ready for use as soil application or the grounded fine powder for
seed treatment and or foliar application.
13) From 1 kg sorghum grains approximately 500 gm dried biomass of Trichoderma including
grains can be produced, which could be utilized directly for soil application for one hectare
after mixing in kg of well decomposed compost or Farm Yard Manure (FYM). The dry

26
 biomass powder along with 0.5% Carboxy Methyl Cellulose (CMC) can be utilized for seed
treatment @ 10 g/kg seed.
5.4 Mass production of Pseudomonas
5.4.1 Preparation of mother culture using the king’s B medium
Peptone : 20.0 g
K2HPO4 : 1.5 g
Mg SO4 : 1.5 g
Glycerol : 10 ml
Distilled water : 1000 ml
Dispense the above broth into conical flasks and autoclave at 15 lb pressure for 15
minutes. After cooling inoculate with a loop of Pseudomonas and incubate for 2 days.
5.4.2 Mass production:
For mass multiplication of Pseudomonas the following steps should be followed sequentially
as noted below:
1) Prepare Kings B Medium and transfer into conical flask or fermentor depending on the
requirement. Sterilize the medium at 15 lb pressure for 15 minutes.
2) The media should be cooled at room temperature after sterilization.
3) Inoculate the conical flask/ fermentor with Pseudomonas mother culture @ 5%
4) Keep the inoculated vessels at the room temperature (30- 35oC)
5) Once bacterial growth starts, shake the flasks at every 4-6 hours for about 3 to 4 days in order
to spread and allow the bacterial growth.
6) After 7-8 days Pseudomonas will be ready to use. Transfer the liquid media with bacterial
growth into cleaned plastic trays and add fine talc material @ 1:3 (bacterial media: talc).
7) Mix the material properly and allow them to dry at room temperature.
8) The mixed formulation will be ready for use as soil application or for seed treatment and or
foliar application
5.5 Mass production of Beauveria bassiana and Metarhizium anisopliae in carrot broth
1) Cut the carrot into small pieces (40 g), wash in potable water
2) Transfer the carrot pieces in to conical flask (250 ml) and add 150 ml of water.
3) Plug the conical flasks with cotton and autoclave for 15 min at 15 psi.
4) Allow the flasks to cool and take to laminar flow chamber for inoculation.
5) Transfer the loopful of B. bassiana or M. anisopliae aseptically.
6) Incubate the flasks at room temperature.
7) Harvest the spores in 12-15 days.
5.6 Mass production of Bacillus thuringensis
The fermentation of the different isolates of B.t., regardless of subspecies, have some
general characteristics in common. They all use sugar (usually glucose, molasses, or starch),
producing acid during the fermentation. In general, they have similar requirements for proteins
+
or protein hydrolysates, can use NH salts, and respond similarly to minerals. However, the
4

27
individual isolates are unique entities, and a particular medium that may support good growth or
toxin production by one isolate may be less satisfactory for another. Different isolates of B.t. may
produce toxins with different spectra of activities.
The "log-phase" of any bacterial fermentation is that period during which the organism is
vigorously growing and rapidly dividing. This first phase lasts 16-18 hours. Sporulation is
complete 20-24 hours after inoculation, although the cells have not yet lysed. Lysis is complete
by 35-40 hours.
5.7 Mass production of nucleopolyhedrosis virus (NPV)
Mass production of Nuclear Polyhedrosis Virus (NPV) on commercial scale is restricted
to in vivo procedures in host larvae which are obtained by
 Field collection from cotton, pigeon pea and chickpea – H. armigera
 Mass culturing in the laboratory in semi synthetic diet – H. armigera
Some small scale producers use field – collected larvae for mass production of NPV in
spite of the following constraints.
 Collection of a large number of larvae in optimum stage (late IV / early V instars) is
time-consuming and can be expensive in terms of labour and transportation costs.
 Wild populations of insects may carry disease causing organisms like microsporidians,
cytoplasmic polyhedrosis virus, stunt virus and fungal pathogens which will affect both
virus production and quality.
 Transportation of a large number of larvae with cannibalistic behaviour will be a difficult
task.
 Parasitized larvae collected from the field will die prematurely yielding little virus.
5.7.1 Production procedure
The NPV of H. armigera is propagated in early fifth instar larvae. The virus is multiplied
in a facility away from the host culture laboratory. The dose of the inoculum used is 5 x 105
polyhedral occlusion bodies (POB) in 10 ml suspension. The virus is applied on to the
semisynthetic diet (lacking formaldehyde) dispensed previously in 5 ml glass vials. A blunt end
polished glass rod (6 mm) is used to distribute the suspension containing the virus uniformly
over the diet surface. Early fifth instar stage of larvae are released singly into the glass vials after
inoculation and plugged with cotton and incubated at a constant temperature of 25 oC in a
laboratory incubator. When the larvae exhausted the feed, fresh untreated diet is provided. The
larvae are observed for the development of virosis and the cadavers collected carefully from
individual bottles starting from fifth day. Approximately, 200 cadavers are collected per sterile
cheese cup (300 ml) and the contents are frozen immediately. Depending upon need, cadavers
are removed from the refrigerator and thawed very rapidly by agitation in water.
5.7.2 Processing of NPV
The method of processing of NPV requires greater care to avoid losses during
processing. The cadavers are brought to normal room temperature by repeatedly thawing the
container with cadaver under running tap water. The cadavers are homogenized in sterile ice cold
distilled water at the ratio 1: 2.5 (w/v) in a blender or precooled all glass pestle and mortar. The

28
homogenate is filtered through double layered muslin and repeatedly washed with distilled
water. The ratio of water to be used for this purpose is 1: 7.5-12.5 (w/v) for the original weight of
the cadaver processed. The left over mat on the muslin is discarded and the filtrate can be semi-
purified by differential centrifugation. The filtrate is centrifuged for 30-60 sec. at 500 rpm to
remove debris. The supernatant is next centrifuged for 20 min at 5,000 rpm. Then the pellet
containing the polyhedral occlusion bodies (POB) is suspended in sterile distilled water and
washed three times by centrifuging the pellet in distilled water at low rpm followed by
centrifugation at high rpm. The pellet finally collected is suspended in distilled water and made
up to a known volume, which is necessary to calculate the strength of the POB in the purified
suspension.

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 6. METHODS OF APPLICATION OF BIOPESTICIDES
The suitable method of application of biopesticides is essential for effective and sustainable
pest and disease control. The following are the major methods by which biopesticides are
delivered in crop ecosystem for the management of insect pests and diseases.
1) Seed treatment
2) Seedling root dip
3) Soil application
4) Foliar spray
6.1 Method of application of Trichoderma and Pseudomonas
6.1.1 Seed treatment: Seed treatment is a term that describes both products and processes. The
usages of specific products and specific techniques can improve the growth environment for the
seed, seedlings and young plants. Seed treatment is an alternative approach to introduce
Trichoderma spp. and Pseudomonas spp. into the soil. This method requires smaller amounts of
biological material than soil treatment.
6.1.1.1 Seed dressing: This is the most common method of seed treatment. The seed is dressed
with either a dry formulation or wet treated with a slurry or liquid formulation. Dressings can be
applied at both farm and industries. Low cost earthen pots can be used for mixing biopesticides
with seed or seed can be spread on a polythene sheet and required quantity of biopesticides can
be sprinkled on seed lot and mixed mechanically by the farmers.
6.1.1.2 Advantages of seed treatment
1. Protects germinating seeds and seedlings against soil and seed borne pathogens
2. Seed germination enhancement.
3. Early and uniform establishment and growth
6.1.1.3 Seed treatment procedure
1) The powder based or liquid based formulation of the product Trichoderma spp. or
Pseudomonas fluorescens is used @ 10 gm or 10 ml per kg of seed material.
2) Mix the formulation with 250 ml starch/ jaggery solution (5%) to make it sticky.
3) Uniformly coat the seeds with the formulation
4) Dry the coated seeds under shade.
5) Sow the dried seeds on the same day.
6.1.1.4 Recommendation of Trichoderma & Pseudomonas for seed treatment
Sr. No. Crop Disease
1 Sugarcane Root rot, wilt
2 Rice Root rot disease, Bacterial sheath blight
3 Chilli Anthracnose, Damping off
4 Pigeon pea Wilt, Blight and Root rot
5 Pea Root rot, White rot
6 Okra Root knot nematode
7 Tomato Soil borne fungal disease, Early blight
Damping off, Wilt

30
 8 Coriander Wilt
9 Brinjal Bacterial wilt
10 Leguminous Vegetables Soil borne infection, Root knot nematode
11 Wheat Bunt/False smut/loose smut/covered smut
12 Cruciferous vegetables Soil / Seed borne diseases (Damping off)
Root knot nematode
13 Gram Wilt, Damping off
14 Capsicum Root knot nematode

6.1.2 Seedling root dip treatment

a) Mix 20 g or 20 ml of powder based or liquid based formulation of the product
Trichoderma spp. or Pseudomonas fluorescens in one liter of water.
b) Immerse the seedlings root (brinjal, chili, tomato, cabbage, etc.) in the mixture for five
to ten minutes before transplanting.
6.1.3 Soil application: Trichoderma spp. suppresses the activity of soil borne fungal
pathogens, especially Rhizoctania solani and Pythium spp. and protects transplanted seedlings by
colonizing their roots. Pseudomanas fluorescens is common non-pathogenic saprophyte that
colonises in soil, water and on plant surfaces. It produces a soluble greenish fluorescent
pigment. P. fluorescens suppress plant diseases by protecting the seeds and roots from fungal
infections. Soil application of Trichoderma or Pseudomonas fluorescens is generally done
through FYM enriched with required bioagent.
6.1.4 Preparation of enriched FYM with Trichoderma or Pseudomonas
1) Uniformly mix 1 kg of Trichoderma or Pseudomonas fluorescens formulation in 100-
250 kg of farmyard manure
2) Cover the mixture and keep it under the shade for 7-8 days
3) Sprinkle the heap with water intermittently.
4) Turn the mixture in every 3-4 days interval
5) Mix the enriched FYM with 1000 kg FYM and broadcast in 1 ha area.
6.1.5 Foliar application: Mix 4-5 g of formulation (2x108cfu/g) in 1 litre of water and
spray in the early morning or late evening hours.

6.2 Method of application of Beauveria and Metarhizium

6.2.1 Soil application: Soil application of entomopathogens viz., Beauveria and Metarhizium
is done through FYM enriched with required entomopathogen.
6.2.2 Preparation of enriched FYM with Beauveria and Metarhizium
1) Uniformly mix 1 kg of formulation of Beauveria or Metarhizium in 100-250 kg of
farmyard manure
2) Cover the mixture and keep it under the shade for 7-8 days
3) Sprinkle the heap with water intermittently.
4) Turn the mixture in every 3-4 days interval

31
5) Mix the enriched FYM with 1000 kg FYM and broadcast in 1 ha area.
6.2.3 Foliar application: Mix 4-5 g of formulation (2x108cfu/g) in 1 litre of water and
spray in the early morning or late evening hours.
6.2.4 Bio-efficacy of different entomopathogenic fungi against insect pests
Fungus Pest & Crop
Beauveria bassiana Rice hispa (Dicladispa armigera)
Coffee berry borer (Hypothenimus hampei)
Tea looper caterpillar (Buzura suppressaria)
Sun flower Helicoverpa armigera
Green gram white grubs
Beauveria brongniarti Sugarcane white grubs Holotrichia serrata
Coconut Rhinoceros beetle (Oryctes rhinoceros)
Sugarcae white grub
Pigeon pod borer
Potato White grubs (Brahmina)
Metarhizium anisopliae Soyabean white grubs Holotricha longipennis
Lecanicillium lecanii Coffee green scale (Coccus viride)
Citrus green scale (Coccus viride)
Indian mustard and Rapeseed- Mustard aphid
(Lipaphis erysimi)
Nomuraea rileyi Castor Spodoptera litura
Soybean Spodoptera litura
Helicoverpa armigera, Thysanoplusa orichalccea

6.3 Method of application of Bacillus thuringiensis

6.3.1 Foliar application: Mix 4-5 g of formulation (2x108cfu/g) in 1 litre of water and
spray in the early morning or late evening hours.
6.3.2 Recommendation of Bacillus thuringiensis against insect pests
Crop Insect Pest
Bacillus thuringiensis var. galleriae
Cabbage & Cauliflower Diamond back moth (Plutella xylostella)
Tomato & Cotton Fruit borer (Helicoverpa armigera)
Okra Fruit borer (Earias spp.)
Chilli Fruit borer (Spodoptera litura)
Rice Leaf folder (Cnaphalocrocis medinalis)
Bacillus thuringiensis Serovar kurstaki (3a, 3b, 3c) 5% WP
Cotton American Bollworm, Spotted Bollworm
Red gram Pod Borer

32
 Cabbage Diamond back moth
Bacillus thuringiensis var. kurstaki, Serotype H-39, 3b, Strain Z-52
Cotton Bollworms, Spodoptera
Rice Stem borer, Leaf folder
Gram & Pigeon Pea Heliothis
Soyabean & Tobacco Spodoptera, Heliothis, Semilooper, Leafminer
Castor Hairy caterpillar

6.4 Field application NPV

6.4.1 Directions for use of NPV
1) The recommended dosage is 200 ml of NPV/acre or 500 ml/ha containing 100 and 250 larval
equivalent (LE) of NPV respectively as active infective material (one LE = 6 x 109 POBs).
2) 100 ml of NPV could be diluted in 200-400 litres of water when high volume sprayer is used
and in 50-70 litres of water in case of power sprayers.
3) Preferable to spray using high volume knapsack sprayer. Virus should be sprayed during
evening hours. Spray should be initiated as soon as few newly hatched larvae are observed or
three to five days after a trap catch of 5 moths per pheromone trap. Subsequent sprays should
be made at 7 to10 days intervals depending upon the pest population.
6.4.2 Compatibility of NPV with other insecticides
The viral pathogen seems to be less sensitive to chemical pesticides (organo phosphates).
When the combination of pathogen and pesticide is used, sometimes synergistic action is
noticed. But is recent years mixing of NPV with insecticides is not advisable due to cross
resistance problem.
6.5 Application methods of EPN
Production and application technology is critical for the success of EPNs in biological
control. Infective juveniles of EPNs are usually applied using various spray equipment and
standard irrigation systems. Nematodes require a film of water around soil particles to move
through the soil profile in search of a host. Therefore, it is important to ensure adequate agitation
during application„ Enhanced efficacy in EPN applications can be facilitated through improved
delivery mechanisms (e.g., cadaver application) or optimization of spray equipment.
Entomopathogenic nematodes can be applied with nearly all commercially available aerial or
groundspray equipment, including mist blowers, electrostatic sprayers and pressurized sprayers.
6.5.1 Soil application
Soil is the natural habitat for EPNs and thus use of these organisms offers great potential
of successful bioocontrol against subterranean insect pests. A series of technology is available
for application of EPNs to soil from simple watering cans for small pot or home garden and for
large fields or orchards through aerial application. Other methods used in soil application include
various irrigation systems such as microjet, overhead etc. Many formulations of EPNs may be
used is soil application including activated charcoal, alginate and polyacrylamide gels, baits,
clay, vermiculite, peat, polyurethane sponge and water dispersible granules.

33
 The wettable powder formulation containing EPNs can be applied to soil. Mix 2-4 kg WP
formulation in 100 kg of any organic manure and apply this mixture in one acre as spot
application or broadcast followed by a light irrigation in case soil is dry.
6.5.2 Foliar application
Environmental factors such as ultraviolet rays, desiccation are the key factors in
influencing the nematode efficacy on foliage. Conventional equipments are used to apply.
Antidesiccants are used to retard evaporation of the nematode suspension and reduce desiccation
(Eg. Glycerin 10%). Substantial progress has been made in recent years in developing EPN
formulations, particularly for above ground applications, e.g., water-dispersible granules,
nematodes on gel, micronized vermiculite, and an aqueous suspension of nematodes. Bait
formulations and insect host cadavers can enhance EPN persistence and reduce the quantity of
nematodes required per unit area.
Finally, superior bio control applications with EPNs can also be achieved through strain
improvement. Improved strains may possess enhanced levels of various beneficial traits such as
environmental tolerance, virulence, reproductive capacity, etc. Methods to improve EPNs
include strain or species discovery or genetic enhancement via selection, hybridization or
molecular manipulation. Many researchers were reported that EPNs can be applied in
combination with insecticides and other bio control agents.
6.6 Formulation of biopesticides: part of delivery system
Broadly formulations can be classified into solid (dusts, granules, and powders) and
liquid (termed suspensions; oil or water based and emulsions) formulations, Dusts are formulated
by the sorption of an active ingredient onto a finely ground solid inert such as talc, clay or chalk
with particle size ranging from 50-200 µm.
6.6.1 Wettable powders (WPs)
They consist of 50-80% technical powder, 15-45% filler, 1-10% dispersant and 3-5%
surfactant by weight to achieve a desired potency formulation. Fillers are hydrophilic and usually
contain silica which resists cake formation and friability during grinding. Dispersant must be
added to retain suspension in dispersion; surfactants to overcome surface tension at liquid-solid
interface. Among the dried formulations of biopesticides, much attention has been given to WPs
because of good miscibility with water and ease of application as sprays with conventional
equipment.
6.6.2 Flowables
They are the suspensions of particulates in liquids with 10-40% microorganisms, 1-3%
suspender ingredient, 1-5% dispersant, 3-8% surfactant and 35-65% carrier liquid (oil or water)
6.6.3 Additives
They are chemically and biologically active compounds that can alter the formulation
physics and kill the targeted species without harming other insects. Important adjuvants are
dispersants (amylose), wetting agent (Triton X), spreaders (molasses), stickers (gums), drift
control (glycerol), UV radiation screens (Congo red, optical brightener) and phagostimulants
(corn meal).

34
 7. METHODS OF QUALITY CONTROL AND TECHNIQUES OF
BIOPESTICIDES
7.1 Insecticide act and bio-pesticides
Regulation and policies of bio-pesticide usage and promotion in India
Nearly 500 biopesticides are available in the Indian market duly registered by Central
Insecticide Board, but quality control is a major issue in most of the products. Indian government
is promoting several rules, regulations, schemes and policies for the promotion of production,
research, adoption and registration of bio-pesticides. Insecticide Act 1968 has been amended so
as to simplify the registration process and allow quick development and invention of bio-
pesticides.
Indian government has made certain policies and acts for the promotion and regulation of
pesticides including bio-pesticides.
7.2 The Destructive Insects and Pests Act, 1914: an act to prevent the introduction into and the
transport from one state to another in India of any insects, fungus or other pest which is or may
be destructive to crops.
7.3 The Insecticide Act, 1968: This act regulates the import, manufacture, sale, transport,
distribution and use of insecticides with a view to prevent risk to human beings or animals, and
for matters connected therewith.
7.4 Insecticides Rules, 1971: The main objectives are the functioning of board, registration
committee and laboratory, registration of insecticides, grant of licenses, packaging and labeling,
appointment of insecticide analysts and insecticide inspectors, transport and storage of
insecticides in transit by road, water and rail. These rules also take into account the provisions
regarding protective clothing, equipment and other facilities for workers during the manufacture
of insecticides.
7.5 Promotion of Integrated Pest Management, 1991: promotion of use of bio-pesticides:
neem based pesticides, bacillus based bio-pesticides, insect pathogen as alternative to chemical
pesticides.
7.6 Biopesticides registered under section of 9(3) of the insecticides act, 1968 for the use in
country (as on 29.02.2020)
Sr.No Biopesticide Registered as

1. Azadirachtin (Neem Product) Botanical

2. Pyrethrin (Pyrethrum)
3. Trichoderma viride Antagonistic fungi
4. Trichoderma harzianum
5. Ampelomyces quisqualis
6. Pseudomonas flourescens Antagonistic bacteria
7. Bacillus subtilis

35
8. Metarhizium anisopliae Entomopathogenic fungi
9. Beauveria bassiana
10. Lecanicillum (Verticillium) lecanii
11. Bacillus sphericus Entomotoxic bacteria
12. Bacillus thringiensis var. kurstaki
13. Bacillus thringiensis var. galleriae
14. Bacillus thringiensis var. israelensis
15. NPV of Helicoverpa armigera Baculovirus
16. NPV of Spodoptera litura

7.7 Indian standards for different biopesticides available in India

Parameters to be Parameter Biopesticides
Biopesticide
tested specification Registered
Antagonistic Fungi Moisture content 8 % maximum Trichoderma viridae
pH 6.5 to 7.5 Trichoderma
CFU/g of the product 2 X 106 minimum harzianum
by serial dilution Ampelomyces
quisqualis
Antagonistic ability Minimum 60 %
Pathogenic Should be nil.
contaminants such as
gram negative
bacteria Salmonella,
Shigella, Vibrio and
such other microbials
Other microbial 1 X 104 maximum
contaminants
Chemical/botanical Should be nil.
pesticide
contaminants
Bioassay based on >70% germination
diseased severity and < 30 % disease
root colonization severity.
Antagonistic Colony Forming 1 X 10 8 CFU/g Pseudomonas
Bacteria Unit (CFU) flourescens

36
 Pathogenic Nil Bacillus subtilis
contaminants such as
gram negative
bacteria Salmonella,
Shigella, Vibrio and
such other microbials
Other microbial 1 X 104 CFU/g
contaminants max.
Chemical/botanical Absent
pesticide
contaminants
Antagonistic At least 35 %
capability on target
organism
Bioassay for diseased >70% germination
severity and root < 30 % disease
colonization severity.
Entomopathog-enic Colony Forming Unit 1 X 108 CFU/g Beauveria bassiana
Fungi (CFU) minimum
Pathogenic Nil Metarhizium
contaminants such as anisopliae
gram negative
bacteria Salmonella, Lecanicillium
Shigella, Vibrio and (Verticillium) leccanii
such other microbials
Other contaminants 1 X 104 maximum
Chemical/botanical Nil
pesticide
contaminants
Entomopathogenic LC-50: Not more than
capability on target 2.00X106 spores/ml
insects by bioassay. (3.0X103
spores/mm2) against
S.litura.
LC50: Not more than
4.00X106 spores/ml
(6.0X103
spores/mm2) against
H. armigera

37
Entomotoxic Moisture content <5% Bacillus sphaericus
bacteria
Beta exotoxin content Negative Bacillus thuringiensis
by housefly bioassay var. israelensis
Potency of product by 15,000 ITU/mg for
bioassay (LC50) Bacillus Bacillus thuringiensis
thuringiensis var. var. kurstaki
israelensis,
1,700 ITU/mg for Bacillus thuringiensis
Bacillus sphaericus var. galleriae
16,000 ITU/mg for
Bacillus
thuringiensis var.
kurstaki
Bt Delta endotoxin Minimum 2 %
content by ELISA
Pathogenic Should be nil
contaminants
Other microbial 1 X 104
contaminants
Chemical/botanical Should be nil
pesticide
contamination
Baculoviruses – Viral Unit: 1x109 POB/ml or gm Helicoverpa
Nuclear NPVs (Helicoverpa & Nuclear Polyhedrosis
Polyhedrosis Virus Spodoptera) Virus
(NPV) Bioassay of NPV for HaNPV <0.5 (Ha NPV)
determining LC50 POB/mm2
against S NPV <20 Spodoptera
Helicoverpa or POB/mm2 Nuclear Polyhedrosis
Spodoptera Virus (SNPV)
Pathogenic Nil
contaminants
Other microbial 1 X 104 maximum
contaminants
Chemical/botanical Nil
pesticide
contaminants

38
 8. IMPEDIMENTS AND LIMITATION IN
PRODUCTION AND USE OF BIOPESTICIDES

Biopesticides control pests in an ecofriendly and nontoxic manner. These offer the
following advantages:
1) Zero or little residual effect: Residues of microbial bio-pesticides produce no adverse
effects on living beings.
2) Applicability in organic farming and potential to be used as a part of Integrated Pest
Management (IPM).
3) Fewer chances to develop resistance by pests: As alternatives to conventional chemical
pesticides, they can help reduce the selection pressure for the evolution of pesticide
resistance in pest populations.
4) Biodegradable in nature.
5) Effective in low concentration and provide pest control over generations.
6) These do not affect ecofriendly insects such as pollinators and soil micro flora.
7) Biopesticides often have good compatibility with biological pest control agents (natural
enemies) and with few conventional chemical pesticides, so they can be readily
incorporated into IPM programmes.
Limitations and constraints of using bio-pesticides
1) Biopesticides show slow rate of control as compared to chemical insecticides Most of the
microbial bio-pesticides cannot be used alone for the complete replacement of
conventional pesticides. Therefore, these bio-pesticides are used as components of
Integrated Pest Management (IPM).
2) Lower efficacy and shorter persistence.
3) Specific to the life cycle of the pest.
4) Greater susceptibility of biopesticides to adverse environmental conditions.
5) Lack of highly virulent strains.
6) As bio-pesticide is totally a live organism, it is important to maintain the vigor and
microbial load.
7) Sophisticated equipments are required for the production of quality biopesticides.
8) Their market performance is very poor as the quality of bio-pesticides produced is not
good.
9) In many of the cases, bio-pesticides that are sold in the market are contaminated and
microorganism count is also low which results in poor performance.
10) The market for bio-pesticides is not well established like markets for chemical pesticides.
11) Due to lack of wide spread studies and consistent results, farmers get confused whether
they should adopt bio-pesticides or not.
12) Pesticide storage requires specific instruments and environmental conditions which are
very costly and cannot be afforded by farmers, shopkeepers and sellers.
13) Budget required for bio-pesticides production is high.

39
 14) Import and export of bio-pesticides is much difficult as compared to chemical pesticides/
Regulatory and marketing constraints.

The registration of biopesticides often poses a particular challenge to regulatory

authorities and the special biological properties of these natural control agents should be taken
into account. For small biopesticides companies aiming to development a range of niche
products, the cost, represent a serious constraint to registering new biopesticide products.
The development of a more balanced regulatory system for biopesticides production
would be beneficial for natural enemies and other biopesticides for agricultural development
without risk to people or environment, and the process of registration could be speeded up and
the cost might be cut down. The regulatory system should be more flexible and it must have the
full and justified trust of the public.

Remedies for bio-pesticide production, usage and marketing

1) Adulteration should be avoided during packaging of bio-pesticides.
2) Lyophilized and dried preparations of bio-pesticides should be used to achieve viability
and stability of biological products.
3) The registration of bio-pesticides is expensive compared to their production which poses
major hurdle in their development. The problems related to their registration should be
addressed.
4) The developed bio-pesticide strain should be monitored extensively to access its threats
to the consumer and environment.
5) Microbial bio-pesticides should be protected from contamination in order to improve the
shelf life.
6) Sustainable and controlled release of biopesticide is also necessary.
7) Pathogenicity and virulence of some microbial strains can be improved using
biotechnological tools.
8) There are some aspects such as resistance, potential of dispersion and persistence should
be studied thoroughly.

40
41
 PART II:
BIOFERTILIZERS

42
1. BIOFERTILIZER: INTRODUCTION, STATUS AND SCOPE

1.1 Introduction
The population in India as well as in the World is increasing day by day and it puts
considering pressure on the agricultural lands and other resources to fulfill the need for food of
this huge population.
Generally, in conventional agriculture there are two major inputs necessary for crop
production, viz., fertilizer and pesticide; in other words, it can be said that fertilizer is food and
pesticide is medicine for plants.
Though the production in India was remarkably high during periods of Green Revolution
due to increased use of chemical fertilizers and pesticides. High utilization of chemical fertilizers
caused poor soil health due to lack of organic matter, loss of inherent fertility by affecting soil
micro flora and fauna. Even it also impaired the health of human beings and animals.
Moreover, plants cannot uptake all the nutrients applied through chemical fertilizers so,
some amount of nutrients are either fixed in the soil or leached out and ultimately mixed with
water bodies. In order to make agriculture sustainable it is necessary to implement a balanced
and reasonable use of nutrients which are cost effective and ecofriendly; in that case biofertilizer
could be a suitable option.
1.2 Biofertilizer
Biofertilizers may be defined as “substances which contain living microorganisms that
colonize the rhizosphere or the interior of the plants and promote growth by increasing the
supply or availability of primary nutrients to the target crops, when applied to soils, seeds or
plant surfaces”.
Biofertilizers can fix atmospheric nitrogen through the process of biological nitrogen
fixation (BNF) and solubilize plant nutrients like phosphates, potash; in addition, it also
stimulates plant growth through synthesis of different growth promoting substances and
has C: N ratio 20:1 indicating its stability.
Biofertilizers can be categorized into five groups based on their nature and activity as
described below. Recently, the potash mobilizers like Frateuri aaurentia, zinc and sulphur
solubilizers like Thiobacillus sp. and manganese solubilizer fungal culture like Pencillium
citrinum have also been identified for commercial operations.
Only a few prokaryotic organisms are able to fix nitrogen directly through a biological
process. Annual biological nitrogen fixation (BNF) is estimated to be around 175 million tones
of which close to 79 % is accounted for by terrestrial fixation.
1.2.1 History of Biofertilizers
 In the 19th century scientists had understood the value of mineral nutrition of plants and few
others suspected that plants could obtain nitrogen from the atmosphere.
 Sir Humphrey Davy, in 1813, became the first person to propose that legumes obtain their
nitrogen requirements from the atmosphere.
 Boussingault first demonstrated the nitrogen present in leguminous plants

43
 Two German chemists, Hellriegel and Wilfarth (1988), showed innate ability of legumes to
fix elemental nitrogen in the atmosphere. Since, neither plants nor microorganisms can fix
nitrogen independently; the process has been called symbiotic nitrogen fixation.
 The root nodule bacteria were isolated by Beijerinck in 1888 and were grouped in the genus
Rhizobium (“Rhizo” means “root” in Greek). These are gram negative, rod shaped, motile,
aerobic, non spore forming bacteria.
 Malpighi, in 1679, became the first person to provide the diagrams and descriptions of root
nodules of plants but he considered them as insect galls.
 In 1866, Woronin saw fungal hyphae in root nodules which looked like threads, probably the
'infection threads' as they are known today. Subsequent observations led to the belief that
root nodules were caused by fungi.
 Ward (1887) studied the development of root nodules from the root hair to the origin of
nodule protrusions.
 American scientists Fred, Baldwin, and McCoy (1932) put forth their exhaustive work on
root nodule bacteria.
 Jensen devised a method in 1942 for studying nodulation of plants on agar in test tubes,
which helped in further investigation in this field.
 Thornton, an English scientist, in 1947 studied nodule forming bacteria from clovers.
 Bortels (1936)- demonstrated the role of molybdenum in accelerating nitrogen fixation by
nodulating legumes.
 Kubo (1939) discovered role of a red pigment in making root nodules of legumes effective
for nitrogen fixation. This red/pink pigment was later characterised as leghemoglobin.
 Virtanen and others (1947) at Helsinki extensively studied leghemoglobin and the
chemistry and mechanism of nitrogen fixation.
 Carnahan and co-workers (1960) at the Du Pont Laboratory in the USA - isolation of cell-
free extracts of the enzyme „nitrogenase‟ from „Clostridium pasteurianum‟.
 Burris and Wilson (1957)- discovery of isotopic method to quantify nitrogen fixation by the
use of 15N and mass spectrometer.
 Bergersen (1971) elaborated the biochemistry of nitrogen fixation in legume root nodules
using the knowledge of asymbiotic nitrogen fixation.
 Nutman (1949) in England gave an insight into the hereditary mechanisms behind
nodulation in legumes. Nutman proposed the theory of microinvagination of root hairs as an
explanation for the origin of infection threads in root hairs of clovers.
 Bond in United Kingdom and Quispel, Silver and Becking in Europe made considerable
headway on nitrogen fixation in non-leguminous nodulated plants between 1950 and 1970.
 Becking identified the bacterial nature of the microsymbiont and named it Frankia sp., and
classified it as the only member of the family Frankiaceae in the order Actinomycetales.
 Fogg and Stewart intensified their work on nitrogen fixing blue green algae during the same
period in United Kingdom.

44
  Callaham, Del Tredici and Torrey made significant advances with regard to non-
leguminous root nodulation by isolating Frankia (an actinomycetous endophyte) from the
root nodules of Comptonia peregrina in 1978. This bacterium was slow growing.
 Trinick (1973) isolated Rhizobium strain from the root nodules of a non-leguminous plant
called Parasponia. This finding highlighted the fact that Rhizobium-plant symbiosis is not
limited to the leguminous plants.
 Kransilnikov and Mishustin in 1937 worked on problems relating to interaction between
plants and soil microorganisms.
 Vincent and Waters (1954) studied all aspects of nodulation, particularly understanding the
environmental factors in legume root nodulation.
 Date, Brockwell and Roughley worked on the development of techniques involved in
inoculant production and its application to seed, referred to as “Bacterization”.
 Burtan, was chiefly responsible for the establishment of legume inoculant research and its
industrial application.
 Hardy and his group (1968) at the Du Pont Laboratory of USA, discovered and popularized
acetylene reduction assay (ARA) for estimation of N2 fixation.
 Pate and his associates (1993) have also quantified the interrelationship between
photosynthates/energy and nitrogen fixation in legumes.
 The Brazilian group of workers was headed by Dobereiner who introduced the concept of
„associative symbiosis‟ or „diazotrophic biocoenosis‟ while working with the association
between Azospirillum and many graminaceous plants.
 Dobereiner further showed that Azotobacter paspali could significantly inhabit roots of the
grass Paspalum notatum and fix abundant amount of nitrogen. This is the only known
species of Azotobacter which shows symbiotic association (associative) with a plant whereas;
all other known species exist as free-living.
1.2.2 History of biofertilizer use in India
In India, systematic study on biofertilizers was started by N. V. Joshi in 1920. Rhizobium
was the first isolated from various cultivated legumes and this was followed by extensive
research by Gangulee, Sarkaria and Madhok on the physiology of the nodule bacteria along
with its inoculation for better crop production. The milestones in research, production and
promotion of biofertilizer in India are given below.
1920- First study of legume-Rhizobium symbiosis by N.V. Joshi.
1939- Discovery of Blue-green algae (BGA) in rice fields by P.K. Dey
1960-First isolation of non symbiotic N-fixing bacteria Derxia gummosa in the World by P.K.
Dey and R. Bhattacharya
1969- Use of Indian peat as a carrier by V. Ishwaran
1979- Initiation of All India Coordinated project on Biological Nitrogen Fixation.
1983- Setting up of National Project on Development and use of Biofertilizer by Ministry of
Agriculture, Govt. of India.
1988- Setting up of National Facility Centre of BGA at IARI.

45
1.2.3 Different groups of biofertilizers

1.3 Status of Biofertilizer in India

Estimated annual requirement of Rhizobium inoculum varies from 1,250 to 15,000t.
Based on crop area in India, the present requirement of biofertilizers is around 5,50,000 metric
tons and there is an ample potential to increase it to 50,000-60,000 tons by 2020; however, the
total production in our country is much less than requirement which points out the inevitability
of increase in biofertilizer production.
Now, the government of India is boosting the biofertilizer industries by providing
subsidies to a maximum of 20 lakh rupees and awarding a national productivity award to the
efficient biofertilizer production unit. Agro Industries Corporation has the maximum production
capacity which is followed by State Agriculture Departments, National Biofertilizers
Development Centers, State Agricultural universities and private sectors.
1.3.1 Liquid biofertilizers - A step forward to biofertilizer technology
Liquid biofertilizers are suspension containing desired microorganisms and special cell
protectants or chemicals that encourage formation of latent spores or cysts for longer shelf life
and tolerance to adverse environments. The advantages of liquid biofertilizers over powder
based are that:
 Microorganisms have longer shelf life up to 2 years,
 generally they circumvent the effect of high temperature,
 Maintain high CFU more than 109ml-1 up to 12 months and
 Better survive on seeds and soil,
 Liquid biofertilizers are easy to use, handling and storage by farmers,
 The dosage is ten times less than that of powder form; it can be packed in different
volumes and save carrier materials.

46
1.3.2 Advantages/Disadvantages of the production technology of biofertilizers
Carrier-based Liquid-based
Advantages
Cheap Longer shelf-life
Easier to produce Easier to produce
Less investment Temperature tolerant
High cell counts
Contamination-free
More effective
Product can be 100% sterile
Disadvantages
Low shelf-life High cost
Temperature sensitive Higher investment for production unit
Contamination prone
Low cell counts
Less effective
Automation difficult

1.3.3 Marketed biofertilizers in India

 Nitrogen fixer, e.g. Rhizobium, Bradyrhizobium, Azospirillum, Azotobacter, Acetobacter,
Azolla and BGA.
 Phosphorus solubilizer, e.g. Bacillus, Pseudomonas and Aspergillus.
 Phosphate mobilizer, e.g. VA-mycorrhiza (VAM) like Glomus.
 K-solubilizer, e.g. Frateuria aurantia.
 Silicate solubilzer, e.g. Thiobacillus thiooxidans.
 Plant growth promoting biofertilizers, e.g. Pseudomonas sp.
1.3.4 Environmental Limitations for Application of Bio-fertilizer
1. Unavailability of suitable carrier Resource constraint
2. Market level constraints and lack of awareness of farmers
3. Lack of quality assurance and limited resource generation for Biofertilizers production
4. Seasonal and un assured requirement
5. Soil and climatic factors and inadequate experienced staff
6. Native microbial population, faulty inoculation techniques and mutation during
fermentation.
1.4 Scope of Biofertilizer
Biofertilizers make nutrients available that are naturally abundant in soil and atmosphere
to plants. These to be effective and cheap inputs, free from any environmental hazards. In a
nutshell, it provides "ecofriendly" organic agro-input which has the ability to convert
nutritionally important elements from unavailable to available form through biological processes.

47
So, it can be expected to reduce the use of chemical fertilizers and pesticides by introducing
biofertilizers.
The microorganisms in biofertilizers reestablish natural nutrient cycle, maintain optimum
nutrient level in soil and also increase soil organic matter content as a result healthy plants can be
grown, while upholding sustainability and fertility of the soil. Therefore, they are extremely
advantageous in enriching soil fertility.
Different biofertilizers provide growth-promoting factors to plants through secretion of
different vitamins, phytohormones and by successfully facilitating composting and controlling
attack of pest and soil borne diseases. It not only saves chemical fertilizers but also help in its
effective utilization and results in higher yield rates.
Dryland agriculture constitutes a very large part of agricultural area in and more than
90% of coarse cereals, 80% of groundnut and 85% of pulses come from these regions. Dryland
agriculture is characterized by low productivity, unpredictable climatic swings and low dosage of
chemical fertilizers and in this situation biofertilizers, particularly Rhizobium, Azotobacter, and
PSB could be a bridge between removals and additions to soil nutrients where farmers can
scarcely afford costly inputs.
It is an established fact that due to fixation in acidic and alkaline soils, the efficiency of
phosphatic fertilizers is very low (15-20%) and unfortunately both soil types are prevailing in
India. On that account, the inoculation of phosphate solubilizing bacteria in soils is needed to
restore and maintain the effective microbial populations for solubilization of chemically fixed
phosphorus as well as availability of other macro and micronutrients to harvest good sustainable
yield of various crops.

48
 2. STRUCTURE AND CHARACTERISTIC FEATURES OF
BACTERIAL BIOFERTILIZERS
2.1 Bacteria:
 Symbiotic nitrogen fixers.: Rhizobium, Azospirillum spp
 Free living nitrogen fixers. Azotobacter, Klebsiella etc.,
 Algal biofertilizers: BGA in association with Azolla Anabena, Nostoc, Ocillatoria
 Phosphate solubilising bacteria: Pseudomonas, Bacillus megaterium
2.1.1 Azospirillum
 Azospirilla are one of the earliest discovered and best characterized associative N2-fixing
bacteria.
 The genus Azospirillum owes its name to its N2-fixing capability (Azo-) and the spiral
movements of the cell (-spirillum).
 These short, rod-shaped, slightly curved Gram-negative bacteria were first isolated from soil
in the Netherlands in 1925.
 Not many reports followed this first isolation until the group of Dr. Johanna Döbereiner
„rediscovered‟ Azospirillum in the mid-1970s.
 It mainly present in cereal plants. Inhabits both root cells as well as surrounding of roots
forming associative symbiotic relation and increasing nitrogen fixing potential of the cereal
plant. Azospirillum is recognized as a dominant soil microbe. It fixes nitrogen in the range of
20- 40 kg/ha in the rhizosphere in non-leguminous plants such as cereals, millets, Oilseeds,
cotton etc. Considerable quantity of nitrogen fertilizer up to 25-30 % can be saved by the use
of Azospirillum inoculant. These species have been commercially exploited for the use as
nitrogen supplying Bio-Fertilizers.
 Presently eight Azospirillum species have been described: A. brasilense, A. lipoferum, A.
halopraeferens, A. irakense, A. largimobile, A. doebereinerae, A. oryzae and A. amazonense.
 Bacteria belonging to the genus Azospirillum are highly motile.
 A. brasilense, A. lipoferum and A. irakense display a mixed pattern of flagellation. One polar
flagellum is synthesized during growth in liquid medium and is primarily used for
swimming. Additional lateral flagella are induced during growth on solidified media and are
responsible for swarming of the bacteria over solid surfaces.
 A. halopraeferens and A. amazonense only display the polar flagellum. Motility offers the
bacterium the advantage of moving towards favorable nutrient conditions.
 Azospirilla exhibit positive chemotaxis towards organic acids, sugars, amino acids and
aromatic compounds as well as towards root exudates.
 Another feature of azospirilla is the directed movement towards optimal oxygen
concentrations, called aerotaxis. This behavioral response can be advantageous to guide the
bacteria to optimal niches for nitrogen fixation.
 Azospirillum has been shown to positively influence plant growth, crop yields and N content
of the plant. This plant stimulatory effect exerted by Azospirillum has been attributed to

49
 several mechanisms, including biological N2 fixation and production of plant-growth-
regulating substances, in particular auxins, which increase the number of lateral roots and
enlarge the root hairs. Given that the concentration of auxin and other phytohormones is
suboptimal in roots, this could result in a higher nutrient uptake and an improved water status
of the plant under suboptimal conditions for normal plant root development. The potential
sustainable yield-enhancing capacity of A. lipoferum and A. brasilense strains have been
exploited by using them in commercial inocula.
Bacterial species Origin of isolation
Azospirillum brasilense Rhizosphere soil of Digitaria decumbens, Brazil
Azospirillum lipoferum Roots of wheat and maize, Brazil
Azospirillum amazonense Roots and rhizosphere soil of Gramineae, Amazon region, Brazil
Azospirillum halopraeferens Roots of Kallar grass, grown in saline soils in Pakistan
Azospirillum irakense Roots and rhizosphere of rice, Iraq
Acetobacter diazotrophicus Roots and stems of sugarcane, Brazil
Herbaspirillum seropedicae Cereal roots (maize, sorghum, rice), Brazil
Azoarcus Roots of Kallar grass, grown in saline soils in Pakistan

2.1.2 Azotobacter
Azotobacter spp. are Gram negative, heterotrophic free-living/non-symbiotic (does not
form nodules but makes association by living in the rhizosphere), obligate aerobe, present in
alkaline and neutral soils, oval or spherical bacteria that form thick-walled cysts (means of
asexual reproduction under favorable condition). There are around six species in the genus
Azotobacter some of which are motile by means of peritrichous flagella, others are not. They are
typically polymorphic and their size ranges from 2-10 µm long and 1-2 µm wide. The
Azotobacter genus was discovered in 1901 by Dutch microbiologist and botanist Beijerinck et
al. (founder of environmental microbiology). A chroococcum is the first aerobic free-living
nitrogen fixer discovered.
Apart from its ability to fix atmospheric nitrogen in soils, it can also synthesize growth
promoting substances such as auxins and gibberellins and also to some extent the vitamins.
Many strains of Azotobactor also exhibit fungicidal properties against certain species of fungus.
Response of Azotobactor has been seen in rice, maize, cotton, sugarcane, pearl millet, vegetable
and some plantation crops. It improves seed germination and plant growth. Azotobacter is
heaviest breathing organism and requires a large amount of organic carbon for its growth.
These bacteria utilize atmospheric nitrogen gas for their cell protein synthesis. This cell
protein is then mineralized in soil after the death of Azotobacter cells thereby contributing
towards the nitrogen availability of the crop plants. Azotobacter spp. is sensitive to acidic pH,
high salts, and temperature. Azotobacter has beneficial effects on crop growth and yield through,
biosynthesis of biologically active substances, stimulation of rhizospheric microbes, producing
phyopathogenic inhibitors. Modification of nutrient uptake and ultimately boosting biological
nitrogen fixation.

50
Azotobacter is a genus of bacteria that live in soil and have the following characteristics:
 They are bacilli.
 They are gram-negative.
 They are obligate aerobes.
 They can fix nitrogen. (Unlike some other nitrogen-fixing bacteria, which associate with
the roots of plants, Azotobacter species are free-living)

The various species of Azotobacter:

 Azotobacter vinelandii,
 Azotobacter chroococcum,
 Azotobacter paspali
 Azotobacter beijerinckii
 Azotobacter armeniacus
 Azotobacter macrocytogenes
 Azotobacter nigricans subsp. Nigricans and subsp. Achromogenes

2.1.3 Bacillus
 In 1972, recognized by Ferdinand Cohn and named as Bacillus subtilis.
 Characteristics:
- Gram Positive
- Growth in Presence of Oxygen
- Formed unique type of resting cells called Endospore (Central, Sub - terminal of Terminal)
- 1 X (3-4) µ in size
- Spores are resistant to heat
- The cells are often arranged in pairs or chains with rounded or square ends and usually have
a single endospore. Endospores are generally oval or sometimes round or cylindrical and
are very resistant to adverse conditions.
 In 1976, Koch provided the first proof that a specific microorganism could cause a specific
disease (Bacillus anthracis)
 Traditionally, Bacillus species was broadly divided in to three groups based on the
morphology of the spore and sporangium. The groups are:
Group 1 – Gram positive, produce central or terminal, ellipsoidal or cylindrical spores that
do not distend the sporangium. It comprises of Bacillus anthracis, Bacillus cereus,
Bacillus mycoides, Bacillus thuringiensis,Bacillus megaterium, Bacillus pumilus, Bacillus
subtilis and Bacillus licheniformis.
Group 2 – Gram variable with central or ellipsoidal spores and swollen sporangia: Bacillus
circulans and Bacillus coagulans. Bacillus alvei, Bacillus brevis and Bacillus macerans
belonged to this group but have since been re-classified to other genera.
Group 3 – Gram variable, sporangia swollen with terminal or subterminal spores: B.
sphaericus

51
 Some former members of the genus Bacillus were gathered into new Families, including -
Acyclobacillaceae, - Paenibacillaceae
Structure of Bacillus
 The surface of the Bacillus -The surface of the Bacillus is complex and is associated with
their properties of adherence, resistance and tactical responses.
 The vegetative cell surface is a laminated structure that consists of - a capsule, - a
proteinaceous surface layer (S-layer), - several layers of peptidoglycan sheeting, & - the
proteins on the outer surface of the plasma membrane.
 The capsules of many bacilli, including B. anthracis, B. subtilis, B. megaterium,and B.
licheniformis, contain poly-D- or L-glutamic acid. Other Bacillus species, e.g., B.
circulans, B. megaterium, B. mycoides and B. pumilus, produce carbohydrate capsules.
Dextran and levan are common, but more complex polysaccharides are produced, as well.
 The vegetative cell wall of almost all Bacillus species is made up of a peptidoglycan
containing meso-diaminopimelic acid (DAP). (The cell walls of Sporosarcina pasteurii and
S. globisporus, contain lysine in the place of DAP.)
 In all bacillus species, peptidoglycan in the cell wall, contain large amounts of teichoic acids
which are bonded to muramic acid residues. The types of glycerol teichoic acids vary greatly
between Bacillus species and within species. As in many other Gram-positive bacteria,
lipoteichoic acids are found.
 STRUCTURE FLAGELLA: Most aerobic spore formers are motile by means of
peritrichous flagella.
Endospore: First discovered by Cohn in Bacillus subtilis and later by Koch in pathogen
Bacillus anthracis. • Cohn demonstrated heat resistance of endospores. • Koch described the
developmental cycle of spore formation in B. anthracis.
 Endospores are formed intracellularly, although they are eventually released from this
mother cell or sporangium as free spores
 Endospores do not form normally during active growth and cell division.
 Their differentiation begins when a population of vegetative cells passes out of the
exponential phase of growth, usually as a result of nutrient depletion.
 They can remain viable for extremely long periods of time, perhaps millions of years.
 Endospore Staining: • When viewed unstained, endospores of living bacilli appear edged in
black and are very bright and refractile. • Endospores strongly resist application of simple
stains or dyes and hence appear as non-staining entities in Gram-stain preparations. • Once
stained, endospores are quite resistant to decolorization. • Schaeffer-Fulton staining
method differentiates the spores from sporangia and vegetative cells.
• Spore stain of a Bacillus species by Schaeffer-Fulton method. A fixed smear is flooded with
a solution of malachite green and placed over boiling water for 5 minutes. After rinsing, the
smear is counterstained with safranine. Mature spores stain green, whether free or still in the
vegetative sporangium; vegetative cells and sporangia stain red.

52
Structure of endospore: Drawing of a cross-section of a Bacillus endospore

The spore protoplast (core) is surrounded by the core (cell) wall, the cortex, and then the spore
coat. Depending on the species, an exosporium may be present. The core wall is composed of the
same type of peptidoglycan as the vegetative cell wall. The cortex is composed of a unique
peptidoglycan that bears three repeat subunits, always contains DAP, and has very little cross-
linking between tetrapeptide chains. The outer spore coat represents 30-60 percent of the dry
weight of the spore. The spore coat proteins have an unusually high content of cysteine and of
hydrophobic amino acids, and are highly resistant to treatments that solubilize most proteins.
Ecophysiological groups of Bacillus

Bacillus as pathogens of insects

53
Role of Bacillus in Agriculture:
 Bacillus spp. promotes plant growth by: (1) excreting cytokinins into rhizosphere and (2)
stimulating the synthesis of phytohormones, such as IAA and GA3.
 Bacillus spores act as biological control agents by inhibiting the growth of various
pathogenic microbes.
 Studies have shown that the impact of Bacillus spp. varies among crop species and that the
application of Bacillus can improve agronomic traits of crop plants and impart enhanced
tolerance to some pathogens.
 Treatment with Bacillus spp. elicited ISR in most of the plant species evaluated and also
altered secondary metabolite biosynthesis in plants; both effects contributed to protection
against plant diseases.
 In contrast to Pseudomonas, using Bacillus strains to trigger the ISR pathway in plants is
dependent on the ethylene and jasmonate pathways.
 To date, studies on Bacillus spp. as a biocontrol agents and elicitors of ISR have mainly
focused on aspects of microbial ecology, the resilience of plants with activated ISR and
direct plant growth promotion.

2.1.4 Pseudomonas
 Pseudomonas is Gram-negative, aerobic, motile (one or several polar flagella), non-spore-
forming straight or slightly curved rods. In addition to the polar flagella, some species (P.
stutzeri, P. mendocina) have shorter lateral flagella.
 The number of flagella has taxonomic importance. Most P. aeruginosa cells carry only one
flagellum, although some cells hold two or three flagella. P. alcaligenes, P. mendocina, P.
pseudoalcaligenes, and P. stutzeri are also characterized by a single flagellum. The majority
of species possess more than one flagella.
 Some Pseudomonas species also form pili (P. aeruginosa, P. alcaligenes, P. syringae). Pili
are involved in cell adhesion, development of biofilms, functions as receptors for
bacteriophage binding and in P. syringae serve as a conduit for long-distance translocation of
effector proteins in plant cells. The adhesive region is located at the tip of the pilus.
 The bacterial cells don‟t produce prosthecae and aren‟t surrounded by sheaths, but they can
form biofilms that provide attachment of cells to the substrate and increase stability under
adverse conditions.
 Another important Pseudomonas feature is production of variety of pigments. Character of
pigmentation is significant factor as diagnostic traits of Pseudomonas. Pigments may be
soluble in water and diffusible into the medium or may be associated with the cells.
Pseudomonads can produce diffusible pigments that fluoresce in short wavelength (254 nm)
ultraviolet light. Some of these pigments, like yellow-green pyoverdine (fluorescein), are
siderophores that play an important physiological role in satisfying the iron requirement. The
synthesis of pyoverdine is strongly related to iron starvation. It can be demonstrated by
cultivating the bacteria in media such as King‟s medium B. Pyoverdine binds iron (III) ions

54
 very tightly, and that ferripyoverdine complex is actively transported into the bacterial cell.
Pyoverdine also can be a tool for identification of Pseudomonas because each genomic group
is characterized by a specific pyoverdine. Other pigments produced by species of
Pseudomonas include pyocyanin (P. aeruginosa, blue color), pyorubin (P. aeruginosa, red
color), chlororaphin (P. chlororaphis, green color), pyomelanin (P. aeruginosa, brown/black
color). P. mendocina is able to produce carotenoid pigment.
 Pseudomonas are aerobic bacteria, but in some cases they can use nitrate as alternate electron
acceptor and carry out denitrification (P. aeruginosa, P. stutzeri, and some P. fluorescens
biovars), reducing nitrate to N2O or N2. Additionally, P. chloritidismutans can utilize
chlorate (ClO3–) as an alternative energy-yielding electron acceptor.
 Pseudomonas can grow in minimal media with ammonium ions or nitrate as N-source and a
single organic compound as the sole C and energy source, not requiring organic growth
factors.
 Optimal temperature for growth is approximately 28ºC, although some species can grow at
4ºC or 41ºC. Most species can‟t tolerate acid conditions (pH 4.5 or lower).
 P. fluorescens is a rod-shaped bacteria and cells are single in arrangement, not linked
together in chains. It is also a motile bacteria, which means it has flagella present. Flagella
are whip-like structures on the bacterial cells that can allow the cells to move, and serve as a
helpful characteristic when it comes to identification.

Pseudomonas in plant growth and disease management

 In general, Pseudomonas spp. show good colonization in numerous ecological niches
including soil, water, and plant surfaces and can inhibit the growth of plant pathogens and
promote plant growth.
 Pseudomonas strains can promote plant growth by producing plant hormones such as IAA
and ACC deaminase and function as biocontrol agents by producing various pathogen-
deterrent compounds, including antibiotics, polysaccharides and siderophores.
 Pseudomonas can induce ISR and strains of Pseudomonas can significantly reduce disease
caused by the many plant pathogens.

55
2.1.5 Rhizobium
 Rhizobia (fast-growing Rhizobium spp. and slow-growing Bradyrhizobium spp.) or root
nodule bacteria are medium-sized, rod-shaped cells, 0.5-0.9 µm in width and 1.2-3.0 µm in
length.
 They do not form endospores, are Gram-negative, and are mobile by a single polar flagellum
or two to six peritrichous flagella.
 Uneven Gram staining is frequently encountered with rhizobia, depending on the age of the
culture.
 Cells from a young culture and nodule bacteroids usually show even Gram staining while
older and longer cells give a banded appearance with unstained areas. These unstained areas
have been identified to be large granules of polymeric beta-hydroxybutyric acid (PHBA).
 Rhizobia are predominantly aerobic chemoorganotrophs and are relatively easy to culture.
They grow well in the presence of O2 and utilize relatively simple carbohydrates and amino
compounds.
 Normally, they have are not found to fix N in free-living form. Some strains of rhizobia
require vitamins for growth. Optimal growth of most strains occurs at a temperature range of
25-30°C and a pH of 6.0-7.0.
 Despite their usual aerobic metabolism, many strains are able to grow well under
microaerophilic conditions at O2 tensions of less than 0.01 atm.
 Most rhizobia produce white colonies when cultured in yeast-mannitol (YM) medium.
 Most rhizobia only weakly absorb congo red (diphenyldiazo-bis-a-naphthylaminesul-fonate)
dye, which is included in culture media for isolating rhizobia.
Free-living rhizobia in the soil
 Rhizobia are facultative microsymbionts that live as normal components of the soil microbial
population when not living symbiotically in the root nodules of the host legume.
 Outside the root nodule, rhizobia are mostly found on the root surface (rhizoplane), soil
around and close to the root surface (rhizosphere), and, to a lesser extent, nonrhizosphere
soil.
Rhizobia as symbionts
 The free-living rhizobia in the soil enter the root hairs of the susceptible host legume by a
complex series of interactions known collectively as the infection process.
 This begins with adhesion of specific rhizobia to the surface of the root hair. Adhesion is
followed by deformation, and curling of the root hair, which results in the characteristic
shepherd's crook appearance. The hypha-like infection thread develops gradually in the root
hair as a tubular structure that is actually an invagination of the root hair wall. The infection
thread contains large numbers of rhizobial cells, and the thread branches through the root
cortex passing close to the host cell nuclei. The rhizobia are released from the tip of the
infection thread into the cytoplasm of the host cells, where multiplication takes place. Before
the release of the rhizobia, rapid host cell division takes place. The dividing host cells are

56
 tetraploid. The final structure is a central core containing the rhizobia and a cortical area that
becomes occupied by the vascular system, which connects to the young root.
 Host cell membrane, which enclose the infection thread, buds off vesicles containing the
rhizobia. The rhizobia divide and differentiate into the form known as bacteroids.
 The host cell membrane, now referred to as the peribacteroid membrane and the bacteroids,
together form the peribacteroid unit. The peribacteroid membranes effectively separate the
bacteroids from the plant cytoplasm and provide the plant with a means of regulating nutrient
exchange with the bacteroids. An infected cell from a nodule of a mature soybean plant may
contain up to 10,000 peribacteroid units. The forms of bacteroids encountered in the nodules
of legumes vary considerably. The bacteroids may be X-and V-shaped, pear-shaped, or
spherical depending upon the type of species.
 The presence of leghemoglobin gives a pink/red color to the nodule interior.
 The enzyme nitrogenase is a complex of two enzymes, an Fe-containing protein and an Fe-
Mo protein. It is responsible for the conversion (reduction) of atmospheric N into NH4+, and
is synthesized in the cytosol of the bacteroids.
 Legume utilizes NH4+ to convert certain precursor metabolites (e.g., α-ketoglutarate,
phosphoenopyruvate) into amino acids, which, in turn, are synthesized into proteins.
 The complex biochemical reactions whereby the inert atmospheric nitrogen is enzymatically
reduced into a utilizable form for the plant by the nitrogenase enzyme complex of the
bacteroids is called biological nitrogen fixation (BNF).
Classification of the rhizobia
 Rhizobia are a genetically diverse and physiologically heterogeneous group of bacteria that
are nevertheless classified together by virtue of their ability to nodulate members of the
Leguminosae.
 The ability of certain rhizobia to infect and nodulate particular group(s) of legume species is
important in the classification of rhizobia.
 Rhizobia are generally classified according to a host-based system. In this host-based system,
legume(s) have been assembled into cross-inoculation groups, which are useful in organizing
the diverse legumes and their rhizobial partners.
 Essentially, a cross-inoculation group consists of a collection of legume species that will
develop effective nodules when inoculated with the rhizobia obtained from the nodules from
any member of that legume group.
 Rhizobia belong in the family Rhizobiaceae, which consist of the following genera: Genus I-
Rhizobium; Genus II-Bradyrhizobium; Genus III-Agrobacterium; and Genus IV-
Phyllobacterium. Only Genera I and II fix N symbiotically in the root nodules of legumes.
The species of rhizobia in Genera I and II, and the cross-inoculation groups of legumes
nodulated by these rhizobia are summarized in Table.
 In Genus I are the fast-growing acid producers that develop pronounced turbidity in liquid
media within 2-3 days and have a mean doubling time of 2-4 h. The cells are motile by two
to six peritrichous flagella. They can grow on a wide range of carbohy-drates, but usually

57
 grow best on glucose, mannitol, or sucrose. Rhizobia of this group are generally infective on
temperate legumes.
Table: Cross-inoculation grouping of Rhizobium
Rhizobium spp. Cross-inoculation Legume types
grouping
Genus I - Rhizobium
R. leguminosarum bv. viceae Pea group Pisum, Vicia, Lathyrus, Lens
R. leguminosarum bv. trifolii Bean group Trifolium
R. leguminosarum bv. Cover group Phaseolus vulgaris
phaseoli
R. meliloti Alfalfa group Melilotus, Medicago, Trigonella
R. loti Lotus group Lotus, Lupinus, Orinthopus
R. galagae Galagae group Galagae orientalis
R. fredii Soybean group Glycine max
R. sp. Chickpea group Cicer arietunum
R. spp. - Leucaena, Glyricidia, Sesbania,
Prosopis, Acacia
Genus II - Bradyrhizobium
Bradyrhizobium japonicum Soybean group Glycine max
B. spp. Cowpea group Arachis hypogaea, Cajanus cajan,
Vigna unguiculata, Vigna mungo,
Vigna radiata, Acacia, Cyamopsis
tetragonoloba, cowpea, Dalbergia,
Dolichos, Aeschynomene,
Indigofera, Tephrosia

 In Genus II are the slow-growing, alkali-producing rhizobia, collectively known as

bradyrhizobia. They require 3-5 days to produce moderate turbidity in liquid media and have
a mean doubling time of 6-8 h. Most strains in this group grow best with pentoses as their C
source. The cells are motile by a single polar or subpolar flagellum. A large genera of
tropical legume species are nodulated by bradyrhizobia.
 Rhizobia are somewhat unique among soil microorganisms in their ability to form N2-fixing
symbioses with legumes and, exceptionally, a nonlegume (Parasponia).
 Classification of rhizobia is becoming increasingly complex and is revised periodically
because of new findings that propose new genera and new species.

2.1.6 Frankia
 Frankia comprises Gram-positive and Gram-variable, N-fixing bacteria actinomycetes that
grow in hyphal form and live in symbiosis with actinorhizal plants. Frankia also initiate the
forming of root nodules.
58
 This genus was originally named by Jørgen Brunchorst, in 1886 to honor the German
biologist Albert Bernhard Frank. Brunchorst considered the organism he had identified to be
a filamentous fungus.
 Hendrik Beking redefined the genus in 1970 and created the family Frankiaceae within the
Actinomycetales. He retained the original name of Frankia for the genus.
 During growth, Frankia produces three cell types: sporangiospores, hyphae, and diazo-
vesicles (spherical, thick walled, lipid-enveloped cellular structures). Diazo-vesicles are
responsible for supply of sufficient N to the host plant during symbiosis.
 Frankia strains can also fix N2 in the free-living state. Under fixed-N limitation and aerobic
conditions, special organs for N2-fixation (spherical vesicles) are formed at the ends of
hyphae. Vesicles are usually swollen structures formed at the ends of filaments under
conditions of low oxygen concentration. Where present they are the site of nitrogenase
activity and the thickness of their lipid-based walls is adjusted to prevent oxygen reaching the
enzyme. Frankia can also form multilocular sporangia.

Fig.: Actinorhizal nodules. (a) A young nodule as found in Alnus. Nodules branch repeatedly,
forming a woody coralloid structure up to 8–10 cm in diameter; (b) longitudinal section through
the tip of a lobe of a generalized nodule.
 Actinorhizal Plants: Plants nodulated by Frankia are collectively referred to as actinorhizal
plants. The nodules formed by Frankia show indeterminate growth, most are perennial, and
older parts may become very woody, with the active regions confined to the tips of nodule
lobes. It is now generally agreed that nodulation with Frankia has evolved on a number of
different occasions.
 Actinorhizal plants are mainly woody in nature, most being shrubs or trees. They are found
worldwide from the arctic (Dryas) to the tropics (members of the Casuarinaceae), on high
mountains (Himalayan alder) and in lowland bogs (Myrica gale). The amounts of nitrogen
fixed by them in their natural environment are related to soil and climatic conditions (up to
approximately 100 kg N ha−1 per year).

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 3. STRUCTURE AND CHARACTERISTIC FEATURES OF
CYANOBACTERIA (BLUE-GREEN ALGAE BIOFERTILIZERS
Cyanobacteria (blue-green algae) are a diverse group of prokaryotes, are
photosynthetic, Gram negative prokaryotes and have the ability to grow in a variety of aquatic
and terrestrial environments. They occur freely or in symbiotic associations with a wide range of
lower and higher plants or in microbial mats. Cyanobacteria are well known for having nitrogen
fixing ability and other agriculturally important potentialities.
These organisms are mainly used as biofertilizers in agricultural field crops (especially
paddy crop). These organisms may be exploited for boosting agriculture as they can fix nitrogen;
produce various plant growth regulators (auxins, gibberellins, cytokinins etc.); enhance soil
fertility by adding organic matter, nitrogen and phosphorus to soil; degrade various
agrochemicals (pesticides and herbicides) and control pathogenic effects of other
microorganisms and plants.
They are ubiquitous in their distribution and grow in all sorts of aquatic and terrestrial
environments exhibiting wide range of temperature, salinity, water potential, pH and
irradiance. Their widespread distribution reflects a broad spectrum of physiological
properties and tolerance to environmental stresses. The economic importance of cyanobacteria
primarily lies in their agronomic importance as biofertilizers due to their N2-fixing ability
that helps them to grow successfully in habitats where little or no combined nitrogen is
available.

Important nitrogen fixing cyanobacterial genera

Form of Cyanobacteria Cyanobacterial members
Unicellular Aphanothece, Synechococcus, Gloeocapsa, Gloeothece*,
Myxosarcina, Pleurocapsa*, Xenococcus
Filamentous Anabaena*, Anabaenopsis, Aulosira, Calothrix*, Chlorogloea,
heterocystous Cylindrospermum, Fischerella*, Gloeotrichia, Hapalosiphon,
Mastigocladus, Nodularia, Nostoc*, Rivularia, Scytonema*,
Stigonema, Tolypothrix, Westiellopsis
Filamentous non- Lyngbya, Microcoleus, Myxosarcina, Oscillatoria, Plectonema,
heterocystous Pseudanabaena, Schizothrix, Trichodesmium
*These Cyanobacteria may exist in symbiotic associations also.
Cyanobacteria as Bio-fertilizers
Cyanobacteria fix atmospheric N2 by forms,i.e.,free-living and symbiotic associations with
partners such as water fern Azolla, cycads, Gunnera, etc.
Some Cyanobacterial members are endowed with the specialized cells known as
heterocyst–thick-walled modified cells, which are considered site of nitrogen fixation by
nitrogenase enzyme. The enzyme is a complex, catalyzes the conversion of the molecular N2
into reduced form like ammonia.

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 Cyanobacteria can contribute to about 20–30 kg N ha-1 as well as the organic matter to
the soil. Many Asian countries like China, Vietnam, India, etc., have been utilizing
Cyanobacteria in paddy cultivation as the alternative to nitrogen fertilizers.
The following benefits to the agro-ecosystem are offered through use of cyanobacteria:
 Enhanced solubilization and mobility of nutrients of limited supply.
 Complexing ofheavy metals and xenobiotics to limit their mobility and transport in plants.
 Mineralization of simpler organic molecules such as amino acids for direct uptake.
 Protection of plants from pathogenic insects and diseases as bio-control agents.
 Stimulation of the plant growth due to their plant growth promoting attributes.
 Improving the physico-chemical conditions of soils.
Cyanobacteria as Plant Growth Promoters
Cyanobacteria release extracellular plant growth promoting substances; some described
as hormones like gibberellins, cytokinin, auxin, or abscisic acids. Others are explained as
vitamins, particularly vitamin or amino acids, antibiotics and toxins. Cyanobacteria related to
paddy crop revealed that cyanobacterial inoculation could enhance rice seed germination,root
and shoot growth.
The fast cyanobacterial cell growth and simple nutritional requirements mainly water,
sunlight, and CO2 provides a wide scope for the commercial application of cyanobacterial
species as plant growth promoters.

3.1 Anabaena (Cyanobacteria)

Structure of Anabaena
 It has filamentous structure. Its filament resembles the filament of Nostoc. The filaments of
Nostoc are covered by mucilage and form a colony which is absent in Anabaena.
 The filament of Anabaena consists of string of beaded cells.
 Several intercalary heterocysts are present in the trichome. Heterocysts are of same shape
as of vegetative cell.
 The filaments are ordinarily straight. But they may be circinate or irregular. • Filaments
occur singly within a sheath. Sheaths are always hyaline and watery gelatinous.
Akinetes formation: Akinetes are formed during unfavorable conditions. Akinetes are thick
walled spores with a large amount of reserved food material. Their wall is two to three
layers thick. They have granular protoplasm. Akinetes are capable of forming new filaments.
The Akinetes can survive dry conditions.
Endospore formation: Endospores formation is rare in Anabaena.
Structure of cells of Anabaena
 The cells are spherical or barrel shaped. They are rarely cylindrical and never discoid.
 The majority of the cells of a colony are similar in size.
 Its cells have following components: Each cell has outer cell wall. This wall consists of
three layers. The inner layer is thin cellular layer, medium is pectic layer and outer is
mucilage layer.

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  The peripheral of the protoplasm is composed of part called chromoplasm. It contains
pigment. Hence it is colored. The central colourless part of protoplasm contains nucleus like
material called central body or chromatin granules.
 Heterocysts are of same shape as of vegetative cell.
 Golgi bodies, encoplasmic reticulum and mitochondria are absent in their cells.
 Heterocysts are the point at which the filament breaks into hormogones. • Heterocysts
convert nitrogen into ammonia. • Hormogones may also formed by the breaking of filament
or decay of filament.
Nitrogenases are kept isolated from oxygen. Therefore, heterocysts have developed
elements to maintain a low level of oxygen within the cell. The developing heterocyst builds
three additional layers outside the cell wall. These layers prevent the entrance of oxygen into
the cell. It gives heterocyst its characteristic enlarged and rounded appearance. Due to these
adaptations, the rate of oxygen diffusion into heterocysts is 100 times lower than of
vegetative cells. One layer creates an envelope polysaccharide layer. The nitrogen is fixed in
this oxygen-restricted envelope. To lower the amount of oxygen within the cell, the
heterocysts are adapted to not have photosystem II inside them.
Occurrence of Anabaena
 Anabaena is found as plankton.
 They form symbiotic relationships with certain plants, such as the Azolla fern.
 Some species of Anabaena are endophytes. They live in the roots of Cycas and Azolla.
 Anabaena is found in all types of water. • Blooms or massive growths can occur in waters
with a lot of nutrients. These blooms discolor the water and give it a bad odor when the
cells die and decay.
Characteristics of Anabaena
 Anabaena is a genus of filamentous cyanobacteria that exist as plankton.
 They perform oxygenic photosynthesis.
 They are heterocyst forming and photoautotrophic.
 During times of low environmental nitrogen, about one cell out of every ten will
differentiate into heterocyst. • They are known for nitrogen-fixing abilities, and they form
symbiotic relationships with certain plants, such as the Azolla
 They are one of four genera of cyanobacteria that produce neurotoxins, which are harmful
to local wildlife, as well as farm animals and pets.
Importance of Anabaena:
 It is believed that cyanobacteria on Earth are responsible as being the producer for most of
the oxygen in the atmosphere. • Plants also produce oxygen, however, they depend on
chloroplasts. These plant organelles are believed to have been derived from the
cyanobacteria.
 The Anabaena has been extensively studied because it also undergoes a process where it
produces hydrogen gas by using sunlight. This product could provide a reusable source of

62
 energy. By further understanding the process, the hydrogen gas could be mass produced
and used as fuel or energy.
 Certain species of Anabaena have been used on rice, paddy fields. They act as natural
fertilizer. • The cyanobacteria seem to have been the foundation of changing the Earth‟s
atmosphere because it takes care of half of the Earth‟s photosynthesis.

3.2 Nostoc (Cyanobacteria)

Taxonomic position- • Kingdom : bacteria • Phylum : cyanobacteria • Class: cyanophyceae •
Order : nostocales • Family: nostocaceae • Genus : Nostoc
Occurrence- Common in fresh water ponds. The large colonies are free floating. Appears as
circular balls. They may be attached or submerges. It can also be found in soil, or moist rocks, at
the bottom of lakes and springs (fresh and salt water), but rarely in marine habitat. It can also
show symbiosis, with plant tissues like in hornworts, and provide nitrogen to its host through
heterocyst. It may also found as a part of lichen.
Colony- Many twisted trichomes arrogate in a gelatinous matrix to form a ball like globular
colony which ranges from greenish to bluish green in color. The colony is externally bounded by
a tough, firm and pellicle membrane, that provides a definite shape to this.
Cells of Nostoc spp. are spherical, barrel-shaped, or oval forming unbranched filaments. The
filaments (trichomes) may contain both heterocysts (thick walled, specialized N-fixing cells) and
akinetes (thick walled cell which functions as a resting cell).
Cell structure- It is prokaryotic type. The cells are without any mitochondria, endoplasmic
reticulum and Golgi apparatus. It has following characteristics: Cell wall, pigments, nucleoplasm
Cell wall- It is made up of cellulose and it surrounds the proplast.
Nucleoplasm- A definite nucleus is absent and nuclear material is present as central body. It
lacks nucleolus. Protoplast shows the typical Myxophycean structure, i.e., inner colourless
centroplasm and outer pigmented chromoplasm. In the chromoplasm are present pigments,
proteinaceous cyanophycin granules and cyanophycin starch granules while in the centroplasm is
present the incipient nucleus.
Pigments- Plastids are absent. The pigments are present inside chromatoplasm. The cells are
blue in color due to presence of phyccocyanine (blue pigment) Other pigments include :
chlorophyll , carotene and phycorythrin.

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Reserve food material- It is in the form of sugars , glycogens, and proteinaceous material called
cyanophycin.

Fig. A single Nostoc filament

Reproduction: It reproduces by following methods:
A: colony fragmentation
B: hormogonia formation
C: Akinites
D: arthrospores
E: endospores
Colony fragmentation The colony breaks down in to two or more fragments accidently or due
to some physiological reason. Each fragment grows into a new colony.
Hormogonia- It is the most common method in Nostoc. The filament is broken down into many
short length pieces these are called as hormogonia. These fragments are formed due to
development of heterocyst or death of any vegetative cell. These hormogonia come out of the
colony by piercing the colonial sheath Each develops its own sheath and aggregate to form a new
colony.

Akinites- These are also called resting spores. A vegetative cell enlarges and secretes a thick
wall around. Then stores a large amount of food and functions as Akinites They are usually
present adjacent to the heterocyst, singly or in chains They help plant during unfavorable
conditions When conditions become favorable these Akinites release their contents through a
pore that germinate into a new filament .
Arthrospores- The resting Akinites are called arthrospores.

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Endospores- In some species like Nostoc microscopium and N. commune , heterocyst contents
divide to produce endospores. These spores are released into new filaments.

3.3 Hapalosiphon (Cyanobacteria)

 Thallus- non-mucilaginous, aggregations mostly brown, rarely green specially at the first
days after inoculation, creeping, branched, uniseriate trichomes.
 Sheath- not permanent, thin to moderately thick, hyaline to yellowish brown.
 Trichome- the uniseriate trichome, bearing true branches, branching usually at right angles,
usually on one side of the filament.
 Cells- cylindrical, subcylindrical and obovoid; sometimes elliptical and constricted at the
cross walls. Cell content granulated, pale green to pale blue-green.
 Heterocysts- frequent, quadrate–cylindrical, the contents homogeneous.
 Highest growth rate in alkaline conditions, pH 9.
 Spores- spherical and subspherical with bilayer envelopes, numerous.
Branching in cyanobacteria
 False branching = outgrowth of filaments adjacent to dead or specialized cells; filament
curves. Ex. Scytonema, Tolypothrix.
 True branching = outgrowth from cells that change their axis of division, 90 degrees from
axis of trichome. Ex. Hapalosiphon, Fischerella.
 This cyanobacterium is well known to produce hepatotoxin, in particular, microcystins.

False branching True branching Hapalosiphon

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 4. FUNGAL BIOFERTILIZER- AM MYCORRHIZA AND
ECTOMYCORHIZA
Introduction: Mycorrhizae are mutualistic symbiotic associations formed between the roots of
higher plants and fungi. It is an Greek word, mykes: mushroom or fungi; rhiza: root. Fungal
roots were discovered by the German botanist A. B. Frank in the last century (1855) in forest
trees such as pine. In nature approximately 90% of plants are infected with mycorrhizae. Convert
insoluble form of phosphorous in soil into soluble form.
Types of mycorrhizae : On the basis of morphological and anatomical features, mycorrhizae are
divided into the three types. 1. Endomycorrhizae 2. Ectomycorrhizae 3. Ectendomycorrhizae.
Endomycorrhiza: Endomycorrhizae is a mycorrhizal association in which the fungal hyphae are
present on root surface as individual threads that may penetrate directly into root hairs, other
epidermal cells & into cortical cells.
Endomycorrhizae further classified in to five types. 1. VAM fungi (vesicular arbuscular
mycorrhizae) 2. Orchidoid mycorrhizae 3. Monotropoid mycorrhizae 4. Ericoid Mycorrhizae 5.
Arbutoid mycorrhizae
1. VAM fungi (Vesicular Arbuscule mycorrhizae) : Fungi formed VAM association with plants
may belongs to ascomycetes , basidiomycetes and zygomycetes. All VAM fungi are obligate
biotrophic, as they are completely dependent on plants for their survival.
2. Orchidoid Mycorrhizae : Fungi belongs to basidiomycotina and colonize only member of
family orchidaceae. This association is probably pseudomycorrhizal but play an important
role in establishment of orchid seedlings.
3. Ericoid Mycorrhizae : Fungal members are usually basidiomycetous and Ascomycotina. This
is found in roots of plants belonging to order ericales. Rootlets are covered by a loosely
woven mesh of dark brown septate hyphae from which branches penetrate the cortical cells.
4. Arbutoid Mycorrhizae: Arbutoid mycorrhizal associations are variants of ectomycorrhizae
found in certain plants in the ericaceae characterized by hyphae coils in epidermal cells. A
major difference between the arbutoid and ectomycorrhizal association is that the hyphae
of the former actually penetrate the outer cortical cells and fill them with coils.
5. Monotropoid Mycorrhizae: The fungi belong to basidiomycotina, colonizing achlorophyllous
members of angiosperms belonging to family monotropaceae. Fungal sheath present.
Ectomycorrhizae : Ectomycorrhizae (ECM) are association, where fungi form a mantle around
roots. There is no hyphal penetration of cells. Fungal hypha is generally separate. A distinct
Hartig‟s net is present between the cells.
Ectendomycorrhizae : The fungi belong to Basidiomycotina, which covers both gymnosperms
and Angiosperms plants. Ectendomycorrhizae show many of the same characteristics‟ of
Endomycorrhizae but also show extensive intercellular penetration. The formation of
Ectendomycorrhizae begins with formation of a hartig‟s net, which grows behind the apical
meristem of the growing root. The hartig net penetrates between the epidermal and outer cortical
cells and later extends to the inner cortex.

66
Applications of Mycorrhizae :
1. Significant role in nutrient recycling. Increase nutrient uptake of plant from soil.
2. Increases absorption of phosphate by crops, uptake of zinc also increases. Increases
uptake of water from soil. Increases uptake of sulphur from the soil. Increases the
concentration of cytokinins and chloroplast in plants. They protect plants during stress
condition.
3. Increase diversity of plant. Produce uniform seedling.
4. More tolerant to adverse soil chemical constraints which limit crop production.
5. Increase plant resistance to diseases and drought.
6. Stimulate the growth of beneficial microorganisms.
7. Improve soil structure. Stable soil aggregate – hyphal polysaccharides bind and aggregate
soil particles.
Preparation of Mycorrhizal biofertilizer
1.Isolation: A) Sieving method & B) Floatation method
A) Sieving method: Soil sample + sterile water → Hot water → Filter and sieve ( 719μm
→ 250μm → 50μm → 45μm ) → Spores separated from soil particles→ Mix with
carrier material→ Use when required as biofertilizer.
B) Floatation method: Soil sample + sterile water → Separate the soil particles using
membrane filter→ Centrifuge (Density gradient centrifuge = at 3000rpm for 30 min) →
Spores separated from soil particles → Mix with carrier material → Use when required
as biofertilizer.
2. Mass production: Spores + antibiotic solution ( streptomycin of 220 ppm concentration
for 15 min ) → Wash spores with mercuric chloride → Wash with distilled water →
Inoculate the plant pots [Guinea grass (Panicum maximum) or Bahiya grass (Paspalum
notatum)] → Keep in green house for 3 - 4 weeks → Uproot the plants → Check for
colonization → Again keep for field growth ( 1 – 1½ months ) → Macerate the root →
Check for moisture content ( only 5 % should be there) → Use as biofertilizer.
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 5. MECHANISM OF NITROGEN FIXATION
Biological Nitrogen Fixation: The biological nitrogen fixation is carried out by some bacteria,
cyanobacteria and symbiotic bacteria. In symbiotic association, the bacterium provides fixed
nitrogen (NH3) to the host and derives carbohydrates and other nutrients from the latter.
Mechanism of biological N-fixation
Biological nitrogen fixation occurs in the presence of the enzyme nitrogenase which is
found inside the nitrogen fixing prokaryote. In addition to this enzyme, a source of reducing
equivalents (ferredoxin (Fd) or flavodoxin in vivo), ATP and protons are required.
The overall reaction of biological nitrogen fixation (BNF) is represented by the following
equation:
N2 + 8H+ + 8e– + 16 ATP → 2NH3 + H2 + 16 ADP + 16 Pi
The enzyme nitrogenase is in-fact an enzyme complex which consists of two metallo-proteins.
(i) Fe-protein or iron-protein component (previously called as azo ferredoxin) and
(ii) Fe Mo-protein or iron-molybdenum protein component (previously called as
molybdoferredoxin). None of these two components alone can catalyse the reduction of N2 to
NH3.
The Fe-protein component of nitrogenase is smaller than its other component and is an
Fe-S protein which is extremely sensitive to O2 and is irreversibly inactivated by it.
The MoFe-protein component of nitrogenase is larger of the two components and consists of
two different peptide chains which are associated as a mixed (α2β2 ) tetramer and contains two
Mo atoms. This component is also sensitive to O2.
1. Biological nitrogen fixation requires very low level of O2 around the enzyme nitrogenease.
2. Apart from N2, enzyme nitrogenase can also reduce other substrates such as N2O (nitrous
oxide), N3– (azide), C2H2 (acetylene), protons (2H+) and catalyse hydrolysis of ATP.
3. Direct measurement of nitrogen fixation is done by mass spectroscopy. However, for
comparative studies reduction of acetylene can be measured rather easily by gas
chromatography method (Acetylene reduction assay).
The electrons are transferred from reduced ferredoxin or flavodoxin or other effective
reducing agents to Fe-protein component which gets reduced. From reduced Fe-protein, the elec-
trons are given to MoFe-protein component which in turn gets reduced and is accompanied by
hydrolysis of ATP into ADP and inorganic phosphate (Pi). Two Mg++ and 2 ATP molecules are
required per electron transferred during this process.
Binding of 2 ATPs to reduced Fe-protein and subsequent hydrolysis of 2 ATPs to 2 ADP
+ 2 Pi is believed to cause a conformatorial change of Fe-protein which facilitates redox
(reduction-oxidation) reactions. From reduced MoFe-protein, the electrons are finally transferred
to molecular nitrogen (N2) and 8 protons, so that two ammonia and one hydrogen molecule are
produced.

68
4. Six electrons and six protons would be required for reduction of one N2 molecule to two
molecules of ammonia. But, the reduction of N2 is obligatorily linked to the reduction of two
protons to form one H2 molecule also.
5. The electrons for regeneration of reduced electron donors (ferredoxin, flavodoxin etc.) are
provided by the cell metabolism e.g., pyruvate oxidation.
6. Substantial amount of energy is lost by the micro-organisms in the formation of H2 molecule
during nitrogen fixation. However, in some rhizobia, hydrogenase enzyme is found which
splits H2 to electrons and protons (H2 → 2H+ + 2e–). These electrons may then be used again
in reduction of nitrogen, thereby increasing the efficiency of nitrogen fixation.

5.1 Symbiotic N-fixation: Formation of Root Nodules in Leguminous Plants

The rhizobia occur as the free-living organisms in the soil before infecting their respec-
tive host plants to form root nodules. The symbiosis between rhizobia and leguminous host plant
is not always obligatory. However, under conditions of limited nitrogen supply in the soil, there
is elaborate exchange of signals between the two symbionts for development of symbiotic
relationship.
There are separate host specific genes and rhizobial specific genes which are involved in
nodule formation. The host plant genes are called as nodulin genes while rhizobial genes are
called as nodulation or nod genes. Some Nod factors produced by rhizobia act as signals for
symbiosis.
The rhizobia migrate and accumulate in the soil near the roots of the legume plant in response to
the secretion of certain chemicals such as flavonoids and betaines by the roots. Root hairs of
legume produce specific sugar binding proteins called as lectins. These lectins are activated by
Nod factors to facilitate the attachment of rhizobia to the root hairs whose tips in turn become
curved.
Rhizobia now secrete enzymes which degrade the cell walls of root hairs at the point of
their attachment for entry into the root hair. From root hairs, the rhizobia enter into the cells of

69
inner layers of cortex through infection threads (tubular extensions of the in-folded plasma
membrane produced by fusion of Golgi-derived membrane vesicles).

The rhizobia continue to multiply inside infection thread and are released into cortical
cells in large numbers, where they cause cortical cells to multiply and ultimately result in the
formation of nodules on the upper surface of the roots. After their release into cortical cells, the
rhizobia stop dividing and enlarge.
Electron microscopic studies have shown groups of rhizobia to the surrounded by single
membranes which originate from host cell plasma membrane. The enlarged and non motile
groups of bacteria inside the membranes are called as bacteroids and the membrane surrounding
them as peribacterioid membrane.
The space between bacteroids and peribacteroid membrane is called as peribacteroid
space. These bacteroids are aerobic and the nitrogenase enzyme is found inside them. The
bacteroides lack a firm wall and are osmotically labile. In root nodule cells, often groups of 4 – 6
bacteroids are enclosed inside the peribacteroid membranes.

70
 The number of chromosomes in cortical cells infected by rhizobia which later develop
into nodule is double the number of chromosome in other somatic cells of the legume (i.e., they
are tetraploid) and seems to be pre-requisite for nodule formation. Apart from infected cells
which are tetraploid, some unifected diploid cells are also found in nodule. The nodule has its
own vascular system which is connected with vascular system of the root to facilitate transfer of
fixed nitrogen i.e., NH3 to the host and carbohydrates and other nutrients from the host to the
bacteroids.
In root nodules of leguminous plants, a red pigment- an oxygen binding heme protein
which is very much similar to hemoglobin of red blood corpuscles is found. This pigment is
called as leghemoglobin and occurs in cytosol of infected nodule cells. Leghemoglobin gives
pinkish-red colour to the nodules. The globin part of this pigment is synthesized in host plant
genome in response to the bacterial infection, while its heme portion is synthesized by bacterial
genome.
Although a correlation has been found between the concentration of hemoglobin and the
rate of nitrogen fixation, but this pigment does not play a direct role in nitrogen fixation. It (i)
protects the nitrogenase inside the bacteroids from deterimental effect of oxygen and (ii) main-
tains adequate supply of oxygen to the bacteroids, so that through respiration ATPs continue to
be generated which are required for nitrogen fixation.
After its formation inside bacteroids, ammonia (or NH4+) is released into cytosol of
infected nodule cells where it is converted into amides (chiefly asparagine and glutamine) or
ureids (chiefly allantoic acid, allantoin and citrulline). These amides or ureids are then
translocated to shoots of host plant through xylem, where they are rapidly catabolized to NH4+
for entry into mainstream of ammonium assimilation.

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5.2 Asymbiotic N2-fixation
Some of the bacteria and most of the cyanobacteria comprise this class of
microorganisms. They are also called free-living diazotrophs. Among cyanobacteria unicellular,
filamentous non-heterocystous and filamentous heterocystous fix nitrogen independently. Both
aerobic and anaerobic bacteria are free-living diazotrophs. Water, oxygen, nutrients are required
in optimum amount, so that, the microorganism can grow. Cyanobacteria grow mainly in the
crop fields. The site of nitrogen fixation in the cyanobacteria is the heterocyst because the
enzyme (nitrogenase) required for nitrogen fixation acts under anaerobic condition. Then the
question arises how unicellular and non-heterocystous cyanobacteria fix nitrogen? Some cells in
these microorganisms become specialized i.e. have oxygen level reduced. Typically, they fix
nitrogen in dark and photosynthesize in light.
Bacterial types fixing nitrogen asymbiotically
Aerobic bacteria Anerobic bacteria Facultative Photosynthetic
anerobic bacteria bacteria
Azomonas Clostridium, Bacillus, Chlorobium,
Azotobacter, Desulfovibrio Enterobacter, Chromatium,
Beijerinckia, Derxia, Klebsiella Rhodomicrobium,
Methylomonas, Rhodopseudomonas,
Mycobacterium Rhodospirillum

Many free-living/non-symbiotic heterotrophic bacteria live in the soil and fix significant
levels of nitrogen without the direct interaction with other organisms. Examples of this type of
nitrogen-fixing bacteria include species of Azotobacter, Bacillus, Clostridium, and Klebsiella. As
previously noted, these organisms must find their own source of energy, typically by oxidizing
organic molecules released by other organisms or from decomposition or may be
chemolithotrophs which utilize inorganic compounds as a source of energy.
Because nitrogenase can be inhibited by oxygen, free-living organisms behave as
anaerobes or microaerophiles while fixing nitrogen.
It has been demonstrated that free-living microorganisms contributed 20 kilograms per
hectare per year to the long-term nitrogen needs of this cropping system (30-50% of the total
needs).
The biological fixation of dinitrogen is the most important way to access N to organisms,
this process requires a fairly high proportion of the ATP; which is generated in the course of
respiratory electron transport reactions with O2 as electron acceptor. The Nitrogenase enzyme
complex (the nitrogen. fixing enzyme) is sensitive to O2, that irreversible inactivates the enzyme.
Diazotrophs must employ mechanisms which, on the other hand, permit the supply of O2
required for energy regeneration and protect Nitrogenase from the deleterious effect of O2. They
have developed several strategies for limiting O2 access to Nitrogenase:
1) It could avoid O2 and live in environments which are permanently anaerobic (Clostridium).
2) Alternatively, it could generate a physical barrier (heterocyst) around its nitrogenase and in
this way prevent O2 from diffusing to the enzyme (cyanobacteria).
72
3) The microorganism could, by its metabolism, reduce the concentration of O2 within the
vicinity of nitrogenase (Azotobacter).
4) They could modify its nitrogenase in such manner as to render it resistant to inactivation by
O2 (conformational protection) (Azotobacter).
5) Segregation of Nitrogen Fixation and Oxygenic Photosynthesis in the Marine
Cyanobacterium (Trichodesmium)

Differences in Symbiotic and Asymbiotic Nitrogen Fixation

Symbiotic nitrogen fixation Asymbiotic nitrogen fixation
Glucose-6-phosphate reduces the ferredoxin Pyruvic acid is broken down in stepwise
in the following manner: manner which is given below and releases
electron which is accepted by
1. Glucose-6-phosphate is converted into 6- ferredoxin.
phosphogluconic acid. This step is
catalyzed by glucose-6- phosphate 1. Pyruvic acid is converted into acetyl CoA
dehydrogenase. with the release of one CO2 and H2. H2 is
The electrons released by converted into protons and electrons, the
glucose-6-phosphate are accepted by reaction is catalysed by hydrogenase.
+
NADP and it is reduced to NADPH+
H+. Pyruvic acid + CoA → Acetyl CoA + CO2 +
H2 ↔ 2H+ + 2e-
Glucose-6-phosphate + NADP + + H2O
→ 6-phosphogluconic acid + NADPH +
H+
2. The electron from NADPH is transferred 2. Acetyl CoA is converted into acetyl
to ferredoxin in the presence of enzyme phosphate with the release of CoA.
NADP-Fd-oxidoreductase and release
H+ to the medium. Acetyl CoA + H2PO4 → Acetyl phosphate +
CoA
3. Acetyl phosphate reacts with ATP and is
converted into acetate, as a result one
−− molecule of ATP is released
which plays important role in nitrogen
fixation.
Acetyl phosphate + ADP → Acetate + ATP

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 6. MECHANISMS OF PHOSPHATE SOLUBILIZATION AND
MOBILIZATION
 Bacteria are more effective in phosphorus solubilization than fungi. Among the whole
microbial population in soil, PSB constitute 1 to 50 %, while phosphorus solubilizing fungi
are only 0.1 to 0.5 % in P solubilization potential.
 Strains from bacterial genera Pseudomonas, Bacillus, Rhizobium and Enterobacter along
with Penicillium and Aspergillus fungi, and mycorrhiza are the most powerful P solubilizers.
6.1 The various Mechanisms of P Solubilization are as follows:
6.1.1. Lowering Soil pH
The principal mechanism for solubilization of soil P is lowering of soil pH by microbial
production of organic acids or the release of protons. In alkaline soils, phosphate can precipitate
to form calcium phosphates, including rock phosphate, which are insoluble in soil. Their
solubility increases with decreases in soil pH. PSMs increase P availability by producing organic
acids that lowers the soil pH. Strong positive correlation has been reported between phosphate
solubilization and organic acids produced. PSMs are also known to create acidity by evolution of
CO2, as observed in solubilization of calcium phosphates. Production of organic acid coupled
with the decrease of the pH by the action of microorganisms resulted in P solubilization. As the
soil pH increases, the divalent and trivalent forms of inorganic P, and HPO4-2, HPO4-3, occur in
the soil.

Schematic representation of the organic acids that may be produced by PSM and used to
solubilize inorganic forms of phosphate.
6.1.2. Chelation
Organic and inorganic acids produced by PSM dissolve the insoluble soil phosphates by
chelation of cations and competing with phosphate for adsorption sites in the soil. The hydroxyl

74
and carboxyl groups of the acids chelate the cations bound to phosphate, thereby converting it
into soluble forms. These acids may complete for fixation sites of Al and Fe insoluble oxides, on
reacting with them, stabilize them, and are called “chelates”. 2-ketogluconic acid is a powerful
chelator of calcium. Production of inorganic acids, such as sulphuric, nitric, and carbonic acid,
has been reported. Nitric and sulphuric acids react with calcium phosphate and convert them into
soluble forms.
6.1.3. Mineralization
Organic phosphate is transformed into utilizable form by PSM through process of
mineralization, and it occurs in soil at the expense of plant and animal remains, which contain a
large amount of organic phosphorus compounds such as nucleic acids, phospholipids, sugar
phosphates, phytic acid, polyphosphates, and phosphonates.
PSMs produce phosphatases like phytase that hydrolyze organic forms of phosphate
compounds, and release inorganic phosphorus that will be utilized by plants. Alkaline and acid
phosphatases use organic phosphate as a substrate to convert it into inorganic form.
The commonly reported phytase-producing fungus are: Aspergillus candidus, Aspergillus
fumigatus, Aspergillus niger, Aspergillus terreus, Penicillium rubrum, Penicillium
simplicissimum, Trichoderma harzianum, and Trichoderma viride.
Soil Bacillus and Streptomyces spp. mineralize complex organic phosphates through
production of extracellular enzymes like phosphoesterases, phosphodiesterases, phytases, and
phospholipases. Mixed cultures of PSMs (Bacillus, Streptomyces, and Pseudomonas) are most
effective in mineralizing organic phosphate.
Some PSM produces siderophores, which hydrolyze the organic P in the soil resulting in
P availability.

6.2 Mechanisms of Potassium Solubilization

The Rhizospheric microorganism that mainly solubilizes the insoluble K to soluble
forms of K are Frateuria aurantia, Bacillus mucilaginosus, B. edaphicus, B. circulans,
Acidothiobacillus ferrooxidans, Paenibacillus, Aspergillus terreus etc.
In general, the most important mechanisms known in K mineral solubilization by KSMs are: (i)
by lowering the pH; (ii) by enhancing chelation of the cations bound to K; and (iii) acidolysis of
the surrounding area of microorganism.
Organic matter after decomposition produces acids like citiric acid, formic acid, malic
acid, oxalic acid. These organic acids produced, enhance the dissolution of potassium
compounds by supplying protons and by complexing Ca2+ ions. • Organic acids produced by
micro-organisms such as acetate, citrate and oxalate can increase mineral dissolution in soil.
Solubilization of potassium occurs by complex formation between organic acids and metal ions
such as Fe2+, Al3+ and Ca2+.

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 7. PRODUCTION TECHNOLOGY OF BIOFERTILIZERS

Strain: According to the first edition of Bergey's Manual of Systematic Bacteriology `A strain is
made up of the descendants of a single isolation in pure culture and usually is made up of a
succession of cultures ultimately derived from an initial single colony '.
Criteria for strain selection: The following are the criteria for strain selection of biofertilizer
agents. A more efficient strain is that which fulfill most of these criteria:
 Efficient N-fixation/Phosphate solubilization in addition to several other beneficial PGPR
attributes.
 Should be able to adapt to a particular environment and soil type.
 It should be easy to mass multiply them under controlled cultural conditions in defined
media.
 It should maintain its viability during the tenure of storage.
 Should be able to colonise effectively and in more number in the rhizosphere and should
be able to compete with the natural microflora in the rhizosphere.
 The other beneficial PGPR attributes are:
 Production of plant growth promoting hormones like auxins, gibberellins, cytokinins,
should reduce the ethylene production by the production of enzyme ACC deaminase.
 Should increase the availability of other macro and micronutrients.
 Should inhibit the plant pathogens through production of various antibiotics,
siderophores, HCN etc.
 Should be able to induce ISR (Induced systemic resistance) in the plants against
infection from plant pathogens.
The efficient strains are selected and then multiplied on the nutritionally rich artificial medium
before inoculating in the seed and soil. In soil, the strain has to survive and multiply to compete
for infection site on roots against hostile environment in soil.

7.1 Sterilization techniques

Sterilization is defined as complete removal or killing of all living cells including their
spore from the material being sterilized.
1. Hot-air oven
It is equipment used for dry heat sterilization. It is most commonly used for sterilizing
glassware like Petri dishes, test tubes, pipettes and metal instruments that can tolerate prolonged
heat exposure. Sterilization is accomplished by exposure of materials / articles usually at 160o for
2.0 hr or at 180oC for 1.5 hr. An oven consists of an insulated cabinet, thermostat, thermometer
and a fan.
Exposure time is counted from when objects to be sterilized have reached the desired
temperature inside the oven. Glassware should be perfectly dried before placing in a hot air oven
since wet glassware may break. Objects, such as a glass petriplate, should be placed in sealable
metal box or double layered brown paper to prevent recontamination. After the sterilization

76
process, the oven and its contents should be allowed to cool before opening the doors to prevent
breakage and recontamination by cool air rushing into the chamber.
2. Autoclave
Moist heat is usually provided by saturated steam under pressure in an autoclave or pressure
cooker and it is the most reliable method of sterilization for most of the materials. It is an
apparatus in which saturated steam under pressure effects sterilization (autoclaving). Most of the
microorganisms are killed at 121oC (i.e., 15 lbs/in2) in 20 min. The autoclave or pressure cooker
should be equipped with pressure gauges, thermometer, automatic pressure control valves and
exhaust valves. It is used mostly for media sterilization.
3. Flame sterilization
Flame sterilization is used for metal objects, such as transfer needles and the tips of
forceps and glass objects, such as the lips of flasks and culture tubes, microscope slides and
cover slips. The object to be sterilized is held at a 45o angle in the upper portion of a flame from
a Bunsen burner or alcohol (spirit) lamp. Tempered metal can be heated to “red hot” and remains
sterile as long as it is hot.
4. Ultraviolet rays: It has lower energy content than ionizing radiation and is capable of
producing a lethal effect in exposed cells (range 210-300 nm). The most lethal wavelength is 265
nm, which corresponds to the optimal absorption wavelength of DNA. UV light induces
aberrant thymine dimer bonds between adjacent thymine nucleotide bases in the nucleic acid,
which results in a deletion mutation.
Because of the low penetration capacity, the UV light is used as a disinfecting agent and
has a very limited application as a sterilizing agent. It is effectively used to sterilize the air
(wavelengths of 250-265 nm) inside laminar flow, isolation chamber, and operating rooms in
hospitals.

7.2 Production technology of carrier based biofertilizers

A. Culturing in the small flasks containing broth
The isolated strain is inoculated in the small flasks containing suitable medium for
inoculums production. Now, the carrier is autoclaved at 15 psi at 121°C for 20 min. The culture
broth is mixed with the carrier at 30%, that is, for 1 kg carrier; 300 ml of culture broth is used.
The mixture is spread on a plastic sheet in a closed room for air drying. The biofertilizer is
packed in sterile plastic air tight bags and stored. For large scale production of inoculums,
culture fermenters are used.
B. Mass production
Isolated bacterial cultures are sub-cultured in to nutrient broth. The cultures are grown
under shaking condition at 30±2°C. The culture incubated until it reaches maximum cell
population of 1010 to 1011. Under optimum condition this population level could be attained
within 4-5 days for Rhizobium, 5-7 days for Azospirillum, and 6-7 days for Azotobacter. The
culture obtained in the flask is called Starter culture. For large scale production, inoculum from

77
starter culture is transferred in to large flasks / fermentor and grown until required level of cell
count is reached. The following media are used for mass multiplication of various biofertilizers.
Rhizobium: YEMA (yeast extract mannitol Agar + congo red)
Azospirillum: Dobereiner‟s (Semi-solid N-free bromothymol blue (Nfb) malate) medium
Azotobacter: Waksman‟s No. 77 broth medium
Phosbacteria (PSM): Pikovaskaya broth
Pseudomonas: Kings B broth
Trichoderma: Potato Dextrose Broth

Prepare appropriate media specific to bacterial inoculant in required quantity. The

medium is inoculated with specific bacterial strain under aseptic condition. Incubated at 30±2ºC
for 5-7 days in rotary shaker. Observe growth of the culture and estimate the population (starter
culture). The above the media is prepared in large quantities in fermentor. Sterilized and cooled
media in a fermentor is inoculated with the log phase of culture grown in large flask (usually 1-2
% of inoculum is sufficient).
C. Culturing in the Fermentor
Large scale multiplication of microorganisms often requires large vessels, commonly
called fermentors. Industrial fermentors are designed to provide the best possible growth. These
vessels must be strong enough, should be resistant to corrosion by the fermentation product and
should not contribute toxic ions to the growth medium.
It must have provisions for rapid incorporation of sterile air into the medium as the
fermentation of microorganism is to occur aerobically. The oxygen of the air is dissolved in the
medium and readily available to microorganisms and at the same time carbon dioxide resulting
from microbial metabolism is flushed out from the medium.
Some form of stirring should be available in the fermentor. The fermentor should provide
for the intermittent addition of antifoam agents as demanded by the foaming status of the
medium. A mechanism for detecting pH values of the culture medium and for adjusting these
values during growth is often required.
There must also be an outlet pipe in the bottom of the fermentor for removing the
completed fermentation broth from the tank.
Fermentors are available in varying sizes. These sizes are usually stated based on the
total volume/capacity of the fermentor. However, the actual operating volume in a fermentor is
always less than that of the total volume, because a “head space” must be left at the top of the
fermentor above the liquid medium to allow for splashing, foaming and aeration of the liquid.
This headspace usually occupies one fifth to one fourth or more of the volume of the fermentor.
Small laboratory fermentors have a total volume of one to two litres of medium with a
maximum of about 12 to 15 lit pilot plant fermentors, which are used in large scale studies of
fermentations.

78
 Pure culture fermentation usually requires that the medium be sterilized. In small
laboratory fermentors, the medium is placed directly in the fermentor and the fermentor is then
autoclaved.
Preparation of inoculants packet: Neutralized and sterilized carrier material is spread in a
clean, dry, sterile metallic or plastic. Bacterial culture drawn from the fermentor is added to the
sterilized carrier and mixed well by manual or mechanical mixer. Inoculants are packed in
polythene bags sealed with electric sealer.
Specification of the polythene bags
 Polythene bags should be of low density grade.
 Thickness of bag should be around 50-75 micron.
 Packet should be marked with the: Name of the manufacture, Name of the product, Strain
number, The crops to which recommended, Method of inoculation, Date of manufacture,
Batch number, Date of expiry, Price, Full address, storage instruction.

7.3 Production of liquid Formulations of biofertilizers

Liquid formulations use liquid materials as carrier, which is usually water, oil or some
solvents in form of suspension, concentrates or emulsions. Most popular liquid inoculant
formulations contain particular organism’s broth 10-40%, suspender ingredient 1-3%,
dispersant 1-5%, surfactant 3-8% and carrier liquid (oil and/or water) 35-65% by weight.
Viscosity is adjusted at equal to the setting rate of the particles, which is achieved by the use of
colloidal clays, polysaccharide gums, starch, cellulose or synthetic polymers.
Carrier material: Various types of material are used as carrier for seed or soil inoculation. For
preparation of seed inoculant, the carrier material is milled to fine powder with particle size of 10
- 40μm. The properties of a good carrier material for seed inoculation are:
(1) Non-toxic to inoculant bacterial strain or the plant.
(2) Good moisture absorption capacity.
(3) Easy to process and free of lump-forming materials.
(4) High organic matter content.
(5) Locally available.
(6) Inexpensive.
(7) Good adhesion to seeds.
(8) Good pH buffering capacity.
Essential criteria for carrier selection relating to survival of the inoculant bacteria should be
considered:
(1) Survival of the inoculant bacteria on seed. Seeds are not always sown immediately after seed
coating with the inoculant bacteria. The bacteria have to survive on seed surface against
drying condition until placed into soil.
(2) Survival of the inoculant bacteria during the storage period.
(3) Survival of the inoculant bacteria in soil. After being introduced into the soil, the inoculant
bacteria have to compete with native soil microorganisms for the nutrient and habitable

79
 niche, and have to survive against grazing protozoa. Such carrier materials that offer the
available nutrient and/or habitable micro-pore to the inoculant bacteria will be desirable
Sterilization of carrier: Carrier sterilization is autoclaving. Carrier material is packed in
partially opened, thin-walled polypropylene bags and autoclaved for 60 min at 121 ºC.

7.4 FCO specifications and quality control of Biofertilizer

Quality control
Till 2006 although BIS standards were followed for assessment of quality for four types
of biofertilizers, but it was voluntary in nature. The Government of India brought four
biofertilizers namely Rhizobium, Azotobacter, Azospirillum and PSB under the ambit of
Fertilizer (Control) Order 1985 (FCO) during 2006. With the picking up of mycorrhizal
biofertilizer production through tissue culture technique, the same was also brought under the
FCO with separate specifications. Recently, two more biofertilizers, namely potash mobilizing
and zinc solubilizing biofertilizers have also been incorporated under FCO.
Under the statutory provisions of FCO, biofertilizer production and its sales have been
regulated and is a mandatory requirement of registration for every manufacturing unit with the
State Fertilizer Controller (who is generally the Commissioner or Director of Agriculture
Department). In every district, some officers of the Agriculture Department have been declared
as Fertilizer Inspectors, who are authorized to inspect production and storage facilities and
draw samples for quality analysis.
National Centre of Organic Farming (NCOF), Ghaziabad and its six Regional Centres
located at Bhubaneswar, Bangalore, Jabalpur, Nagpur, Hissar and Imphal have been declared as
notified testing laboratories. Under the provisions of the act, State Governments can also develop
their own quality control laboratories and notify them under the FCO 1985.
For capacity building of personnel associated with quality control initiative, regular
trainings are being organized by National Centre of Organic Farming and its Regional Centres. A
ten-day training module for laboratory analysts and a five-day training module for field level
officers and fertilizer inspectors have been designed.
Minimum specifications of selected biofertilizers as specified in FCO are given below:
Specifications of Rhizobium biofertilizer
S.No. Parameter Requirements
(i) Base Carrier based* in form of moist/dry powder or granules, or
liquid based
(ii) Viable cell count CFU minimum 5x107 cells/g of powder, granules or carrier
material or 1x108 cells/ml of liquid
(iii) Contamination level No contamination at 105 dilution
(iv) pH 6.5-7.5
(v) Particles size (for carrier All material shall pass through 0.15-0.212mm IS sieve
based)
(vi) Maximum moisture % by 30-40%

80
 wt.(for carrier based)
(vii) Efficiency character Should show effective nodulation on all the species listed
on the packet
*Type of carrier: The carrier materials such as peat, lignite, peat soil, humus, wood charcoal or similar material
favouring growth of organism
Specifications of Azotobacter Biofertilizer
S.No. Parameter Requirements
(i) Base Carrier based* in form of moist/dry powder or granules, or
liquid based
(ii) Viable cell count CFU minimum 5x107 cells/g of powder, granules or carrier
material or 1x108 cells/ml of liquid
(iii) Contamination level No contamination at 105 dilution
(iv) pH 6.5-7.5
(v) Particles size (for carrier All material shall pass through 0.15-0.212mm IS sieve
based)
(vi) Maximum moisture % by 30-40%
wt.(for carrier based)
(vii) Efficiency character The strain should be capable of fixing at least 10 mg of
nitrogen per g of sucrose consumed
*Type of carrier: The carrier material such as peat, lignite, peat soil, humus, wood charcoal or
similar material favouring growth of the organism
Specifications of Phosphate Solubilizing Bacterial Biofertilizer
S.No. Parameter Requirements
(i) Base Carrier based* in form of moist/dry powder or granules, or
liquid based
(ii) Viable cell count CFU minimum 5x107 cells/g of powder, granules or carrier
material or 1x108 cells/ml of liquid
(iii) Contamination level No contamination at 105 dilution
(iv) pH 6.5-7.5 for moist/dry powder, granulated carrier based and
5.0-7.5 for liquid based
(v) Particles size (for carrier All material shall pass through 0.15-0.212mm IS sieve
based)
(vi) Maximum moisture % by 30-40%
wt.(for carrier based)
(vii) Efficiency character The strain should have phosphate solubilizing capacity in
the range of minimum 30%, when tested
spectrophotometrically.
In terms of zone formation, minimum 5mm
solubilization zone in prescribed media having at least
3mm thickness.

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Specifications of Mycorrhizal Biofertilizers
S.No. Parameter Requirements
(i) Form/base Fine powder/tablets/granules/root biomass mixed with
growing substrate
(ii) Particle size for carrier based 90% should pass through 250 micron IS sieve (60 BSS)
powder formulations
(iii) Moisture content, max. 8 -12%
(iv) pH 6.0-7.5
(v) Total viable propagules/g of 100/g of finished product
product, minimum
(vi) Infectivity potential 80 infection points in test roots/g of mycorrhizal
inoculum used

7.5 Biofertilizer-Storage & shelf life

Biofertilizer packets need to be stored in cool and dry place away from direct sunlight
and heat. The packets may be stored at room temperature or in cold storage conditions in lots in
plastic crates or polythene / gunny bags. The population of inoculant in the carrier inoculant
packet may be determined at 15 days interval. There should be more than 109 cells / g of
inoculant at the time of preparation and107 cells/ g on dry weight basis before expiry date.
Liquid biofertilizer formulation could be considered as one potential strategy for
improving the shelf-life of biofertilizer. Unlike solid carrier based biofertilizers, liquid
formulations allow the manufacturer to include sufficient amount of nutrients, cell protectant,
and inducers responsible for cell/spore/cyst formation to ensure prolonged shelf-life. The shelf-
life of common solid carrier based biofertilizers is around six months; however, it could be as
high as two years for a liquid formulation. Further, solid carrier based biofertilizers are less
thermo-tolerant whereas; liquid formulations can tolerate the temperature as high as 55°C.
Hence, improved shelf-life could be achieved by the application of a liquid biofertilizer
formulation.
However, process cost of liquid biofertilizer is significantly higher than a solid
formulation. Thus, successful commercialization of less expensive liquid biofertilizer is a
challenge and shelf-life of such products is still a concern.
Quality Control of Biofertilizer
The proper quality control mechanism of biofertilizer production and application covers
the whole experimental process: from microorganism isolation, through laboratory screening of
the isolated strains for plant growth; greenhouse screening for plant growth promotion; field
screening of the most effective microbes in cropped soil; readjustment and refining of inoculants;
environmental impact test and, finally, production.

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 The quality specifications of biofertilizers differ from country to country and may contain the
following parameters:
 The microbial strain(s) used; the quality of biofertilizers is usually defined in terms of two
important characteristics: presence of a recommended strain in the required quantity and in
active form.
 Microbial density at the time of manufacture and at the time of expiry: the number of
selected microorganisms in the active form per gram or milliliter of biofertilizer. The
guidelines used are limited to the density of the available microorganisms and their viability
and preservation.
 The permissible contamination; it is important to set control schemes that account for
putative contaminating microorganisms.
 The expiry period;
 The pH, the moisture and the carrier
 Each packet of biofertilizer should have the information eg. name of the product, leguminous
crop for which intended, name and address of the manufacturer, type of carrier, batch or
manufacture no, expiry date.
 Each packet should also be marked with the ISI mark. The biofertilizer should be stored in
the cool place and keep away from direct heat.
Quality has to be controlled at various stages of production as well: during the mother
culture stage, carrier selection, broth culture stage, mixing of broth and culture, packing and
storage. The main quality parameters of biofertilizers are as follows:
 Appearance
 Living target bacteria: Rhizobium sp., PSB, Pseudomonas, Azotobacter etc.
 Multi-strain biofertilizer
 Water content
 Size
 Organic matter
 pH
 Non-target bacteria (contaminants)
 Shelf-life

83
General procedure for quality control of biofertilizers

84
 8. METHODS OF APPLICATION OF BIOFERTILIZERS

There are four ways for application of solid biofertilizer.

(1) The most extensively used method is the Seed treatment. The biofertilizer is applied at the
rate of 200gm per 8Kg of seeds. The per acre application rate is determined from the amount
of seed to be sown in a field. Before the application, biofertilizer is mixed in water (1:2) to
form slurry. The slurry is poured in container with seeds to be sown. The combination is
mixed properly such that each seed is coated by biofertilizer. The seeds are dried under the
shade and then sown. This method is recommended for pulses, oilseeds and fodder crops.
(2) The second method is Seedling treatment. Dose wise diluted formulation is required for
seedling treatment. About 1 part of biofertilizer in 10 parts of water is prepared. The roots of
seedlings to be transplanted in field are dipped in biofertilizer solution for 30 minutes. After
the treatment, the seedlings are immediately planted in field without drying. This method is
recommended for crops like tomato, brinjal, potato, cabbage, onion, paddy and chilly which
are replanted at seedling stage. It is also used for the treatment of ornamental bushes like
rose, jasmine, dahlia, marigold and chrysanthemum.
(3) The third method of choice is the Set treatment. For this treatment the ratio of biofertilizer to
water is 1:50. The explants or cut pieces of planting material are immersed in biofertilizer
mixture for 30 minutes. The treated pieces are dried in shade and then planted in field. The
crops like sugarcane, banana, grapes and strawberries are recommended to be treated by Set
treatment.
(4) Biofertilizers can also used for intermittent application for the standing crop or soil treatment
before plantation or sowing. For such direct soil applications, biofertilizer is mixed with
carriers like soil, compost, farmyard manure, rice husks or lignite (1kg per 25kg of carrier)
and then directly put in the soil. The applied area needs to be irrigated immediately.
(5) The liquid biofertilizers are applied by spraying or by fertigation. Spraying is recommended
for standing citrus plants, vines, mango, guava, custard apple, apple and peach orchards. In
fertigation, biofertilizer is mixed in water and other micronutrients in a tank. It is reached to
individual plant via irrigation sprinklers or sprayers or piping. This method is usually
employed in shade nets and green houses.

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 9. FACTORS INFLUENCING THE EFFICACY OF
BIOFERTILIZER

The critical factors which are responsible for the effectiveness of a particular biofertilizer
are as follows:
1. Suitability of the species to the target crop.
2. Suitability of the strain: There are specific strains of Rhizobium for different leguminous
species like Cowpea, Red gram, Soybean, Alfalfa etc. Biofertilizer specific culture should be
used for specific crop.
3. Identification of strains as suited to the agro eco system, particularly the soil pH, moisture
conditions.
4. Rhizo-competence of the microbes for that crop.
5. The aseptic conditions of manufacturing, the cell count of living organism present the carrier
material, purity and level of contamination.
6. The conditions of carrier material in which the culture is packed and the quality of packing
material, which determine the shelf life.
7. The conditions, in which the packed materials are stored, distributed and kept with farmers
before it is applied.
8. Soil conditions particularly pH, organic matter content, moisture level and agronomic
practices, soil salinity/sodicity/acidity etc.
Marketing constraints for wide scale use of biofertilizers
1. Retail fertilizer dealers do not keep biofertilizers mainly because of the short shelf life, limited
demand and lack of storage facilities.
2. These are major constraints in the availability of biofertilizers in the market. Shelf life of
carrier based inoculants, which are currently being produced, is usually not more than six
months.
3. Further due to poor awareness among framers as well as development staff (Extension Staff)
demand of biofertilizers is not increasing.
4. Instability of the inputs and outputs markets
5. Lack of developed marketing channels and infrastructure
6. Initiatives for promotion of biofertilizer business sector
General constraints for wide scale adoption of biofertilizers
 Unawareness regarding the biofertilizers‟ utility, short shelf-life, lack of ready availability in
time and in the desired quality, inconsistency in results with their application.
 Different methods of inoculation application.
 No visual difference in the crop growth immediately after biofertilizer application is
observed in comparison with that of inorganic fertilizers.
 Socio-psychological constraints that lead to unawareness of biofertilizer technology: lack of
motivation form extension agencies; low credibility of source of biofertilizers; farmers‟

86
 belief that chemical fertilizers are more effective than biofertilizers; lack of use of
biofertilizers by fellow farmers or their application being not permitted in farmers‟ culture.
 Lack of awareness of biofertilizers is a major challenge for farmers.
 Insufficient understanding of the technology of agro-dealers, extension services and policy
makers.
Can we use biofertilizers with chemical fertilizers?
There is a huge difference in the application amount and the actual availability of chemical
fertilizers to the plants. Biofertilizers have been reported to enhance the availability of these
inorganic inputs to the plants. Thus Biofertilizers can be used along with chemical fertilizers but
the care should be taken to avoid direct contact of chemical based inputs with Biofertilizers
which is likely to reduce the microbial population of Biofertilizers.
Disadvantages of biofertilizers
 Biofertilizers require special care for long-term storage because they are alive.
 Must be used before their expiry date.
 If other microorganisms contaminate the carrier medium or if growers use the wrong strain,
they are not as effective.
 Biofertilizers lose their effectiveness if the soil is too hot or dry.

87