Extension Bulletin No.

Bio-fertilizers in
Pomegranate Production

NATIONAL RESEARCH CENTRE ON POMEGRANATE

NH-9, Bypass Road, Shelgi, Solapur-413 006 (MS)
Prepared by
Ashis Maity
R.A. Marathe
K. Dhinesh Babu
N.V. Singh

Published by
Dr. V.T. Jadhav
Director
National Research Centre on Pomegranate
Solapur 413 006 (Maharashtra)

Phone : 0217-2374262, Fax : 0217-23745333

E-mail: nrcpomegranate@indiatimes.com;
director@nrcpomegranate.org
Website: http://www.nrcpomegranate.org

Published
Omkar Graphics, Parbhani
Cell : 9422877977
 Bio-fertilizers in Pomegranate Production

Pomegranate (Punica granatum L.), an economically important fruit crop is

gaining popularity in arid and semi-arid regions of India due to its immense
medicinal/ therapeutic value and higher economic return. This region is
characterized by nutrient deficient shallow gravelly soils, high temperature, low
and irregular distribution of rainfall and water scarcity during plant growth period.
Plant growth in this region is often hindered due to insufficient resident micro-flora
which acts as both source and sinks for essential plant nutrients.
The presence of adequate resident micro-flora is a pre-requisite for the
transformation of various nutrients either inherited from soil or applied through
anthropogenic sources. Some of them have the ability to harness vast reservoir of
atmospheric nitrogen and make it available to crops either directly or indirectly.
Solubilization of phosphorus and potassium is also accomplished by many
heterotrophic soil microbes. Besides major nutrients, available micronutrient
status of soil is also directly or indirectly regulated by microorganisms. To harness
maximum benefit from these microbes, the need of artificial inoculation of efficient
microbes was felt and accordingly bio-fertilizer technology was invoked by using
these microbes as source of inocula. Production of fruit crops has undergone
significant changes in recent years due to development of innovative technology
like integrated nutrient management practice in which bio-fertilizer technology is
an essential component for sustainable fruit production.

Bio-fertilizer
The term 'bio' refers 'living' and fertilizer means 'source of nutrition'. Thus,
bio-fertilizers are the preparations containing living cells or latent cells of efficient
strains of microorganisms that help crop plants' uptake of nutrients by their
interactions in the rhizosphere when applied through planting material or soil.
They accelerate certain microbial processes in the soil which augment the extent
of availability of nutrients in a form easily assimilated by plants.
Bio-fertilizers have gained popularity and are being widely used due to the
following reasons.
Cost-effectiveness : Bio-fertilizers are cheaper compared to chemical
fertilizers. They have lower manufacturing costs especially the nitrogenous and

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 Bio-fertilizers in Pomegranate Production

phosphorous bio-fertilizers, hence, easily affordable to marginal and small

farmers.
Soil fertility : Bio-fertilizers help to maintain soil health and enrich soil fertility.
The microbes in the biofertilizers make symbiotic association with plant roots.
They convert complex organic material in to simple compound, so that the
nutrient is readily available to the plant.
Eco-friendly : Bio-fertilizers help in minimizing the environmental pollution.
Organic farming:Bio-fertilizers are ideal input for practicing organic farming.
There are numerous species of soil bacteria which flourish in the
rhizosphere of plants, but which may grow in, on or around plant tissues, stimulate
plant growth. These bacteria are collectively known as plant growth promoting
rhizobacteria (PGPR). Some PGPR appear to promote plant growth by acting as
both bio-fertilizer and bio-pesticides. Mode of PGPR action include N2 fixation,
augmenting the availability of nutrients in the rhizosphere, influencing root growth
and morphology and promoting other beneficial plant-microbe symbiosis. The
search for PGPR and investigation of their mode of actions are increasing at a
rapid pace as efforts are made to exploit them commercially as bio-fertilizers.

Bio-fertilizer for Pomegranates

Several microorganisms and their association with plants are being
exploited in the production of bio-fertilizers. They can be grouped in different ways
based on their nature and function.

S.No. Category Bio-fertilizer

Nitrogen (N2) fixing bio-fertilizer
1. Free living Azotobacter sp.
2. Associative symbiotic Azospirillum sp.
Phosphorus (P) solubilizing bio-fertilizer
1. Bacteria Bacillus megaterium, Bacillus circulans,
Pseudomonas striata
2. Fungi Penicillium bilaiae, P. radicum, P. italicum,
Aspergillus awamori

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 Bio-fertilizers in Pomegranate Production

Phosphorus (P) mobilizing bio-fertilizer

1. Arbuscular Glomus sp., Gigaspora sp., Acaulospora
mycorrhizal fungi sp., Scutellospora sp.
Potassium (K) solubilizing bio-fertilizer
1. Bacteria Bacillus mucilaginosus
2. Fungi Penicillium pinophylum, Aspergillus
fumigatus
Micronutrient biofertilizer
1. Bacteria Bacillus sp., Pseudomonas fluorescens
2. Ericoid mycorrhizae Oidiodendron maius
Plant growth promoting microorganisms
1. Bacteria Pseudomonas fluorescens, Pink
pigmented facultative methylotrophs
(PPFM)
2. Fungi Trichoderma sp., Penicillium chrysogenum

N2- fixing biofertilizer

Azotobacter
Azotobacter is one of the important members of the free-living diazotrophic club
due to its broad spectrum utility for different crops. Its colonies appear flat, soft,
milky and mucoid. Cells are polymorphic and young cells have peritrichous
flagella. Azotobacter chroococcum is the most prevalent species found in arable
-1
soils and capable of fixing N2 (2-15 mg N2 fixed g of carbon source) in culture
media. But other species reported include A. agilis, A. vinelandi, A. beijerinckii, A.
insiginis, A. macrocytogenes and A. paspali. The numbers of A. chroococcum in
Indian soils rarely exceeds 105 g-1 soil due to lack of organic matter and the
presence of antagonistic microorganisms in soil.

Azospirillum
Azospirillum are important member of associative N2-fixer and are found widely
distributed in loose association with roots of most of the agricultural /horticultural
crops. Azospirillum is a general root colonizer and not plant specific.

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 Bio-fertilizers in Pomegranate Production

There are five species of Azospirillum viz.,

1. Azospirillum brasilense- Motile, free-living, gram-negative bacteria that
occur in the soil. They are aerobic or micro-aerophilic and are capable of
nitrogen fixation
2. Azospirillum lipoferum,
3. Azospirillum amazonense,
4. Azospirillum halopraeferens
5. Azospirillum irakense

The bacteria are established mainly on the root surface but some strains of
A. lipoferum and A. brasilense are capable of colonizing the root interior in the
apoplast and intercellular spaces. This ability could mean a lower vulnerability to
harsh conditions imposed by the soil and/or the environment, which in turn could
imply a more efficient promotion of plant growth. The organism proliferates under
both anaerobic and aerobic conditions but it is preferentially micro-aerophilic in the
presence or absence of combined nitrogen in the medium.

Beneficial effects of Azospirillum to crop plants:

Nitrogen fixation - helps to fix atmospheric nitrogen
Production of natural auxins - Production of Indole-3- acetic acid, a growth
promoting substance
Disease resistance - imparts disease resistance to crop plan
Drought tolerance - improves the drought tolerance

Phosphate solubulizing microorganisms

Phosphorus is an important element and plays a key role in the nutrition of
plant as it promotes development of deeper roots. Being a constituent of ATP, it is
involved in diverse processes such as cell division, energy transduction through
photosynthesis and biological oxidations and nutrient uptake. It occurs in soil as
inorganic and organic phosphate. A large proportion of these two forms is present
in insoluble forms and therefore, not available for plant nutrition. Inorganic P

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 Bio-fertilizers in Pomegranate Production

occurs in soil, mostly in insoluble minerals complexes, some of them appearing

after the application of chemical fertilizers. That is why, most of the soil are
deficient in soluble phosphorus. These precipitated forms need to be converted to
soluble form before they can be taken up by plant. Phosphate solubilizing
microorganisms play an important role in converting insoluble phosphate to
soluble phosphate through the following mechanisms:
(1) Production of organic acids : The organic acids help to chelate metal
of insoluble phosphate compounds, thereby releasing phosphorus.
(2) Production of esterase enzyme : The esterase enzyme liberates P from
organic phosphatic compounds.
Examples:
Phosphate solubulizing bacteria
Bacillus subtilis
Pseudomonas striata
Phosphate solubulizing fungi are more efficient P-solubulizers compared to
bacteria
Aspergillus sp.
Penicillium sp.

Phosphate mobilizing microorganisms

Arbuscular mycorrhizal fungi, popularly known as 'Vesicular Arbuscular
Mycorrhizal (VAM) fungi' are ubiquitous soil microorganisms. In nature, the
association of VAM fungi are found in roots of many members of plant kingdom
viz.,
Angiosperms,
Gymnosperms,
Pteridophytes
Thallophytes.
The VAM fungal hyphae proliferate both outside and inside the root, with
the internal hyphae differentiating into arbuscules and vesicles. The arbuscules
are formed only on internal hyphae in response to contact with host cortical cells;

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 Bio-fertilizers in Pomegranate Production

they are the sites of nutrient exchange between the plant and the fungus. Vesicles,
which contain high concentration of triglycerides at maturity, are thought to serve
as storage and survival organs. The improvement of P-nutrition in plants has been
the most recognized beneficial effect of mycorrhizae. The mechanism which is
generally accepted for this mycorrhizal role consists of a wider physical
exploration of soil by mycorrhizal fungi than by roots. Besides, hyphae that extend
beyond the root depletion zone, various subsidiary mechanisms have been
proposed to explain P-uptake by mycorrhizal fungi, such as (1) the kinetics of P-
uptake into hyphae differ from those of roots either through a higher affinity (lower
Km) or a lower threshold concentration at which influx equals efflux (Cmin); (2) root
and hyphae explore microsites differently, especially small patches of organic
matter,(3) plant root and mycorrhizal hyphae affect chemical changes and P
solubility in the rhizosphere differently.

Examples for AM fungi :

Glomus intraradices, G. mosseae, G. clarum, G. etunicatum
Gigaspora ramisporophora and Gigaspora rosea
Acaulospora laevis,
Archaeospora,
Enterophospora,
Gerdemannia,
Geosiphon,
Paraglomus,
Scutellospora.

Mass production of VAM fungi has been achieved with several species
such as Acaulospora laevis, Glomus. clarum, G. etunicatum, G. intraradices, G.
mosseae, Gigaspora ramisporophora and Gigaspora rosea but Glomus
intraradices is the most common inoculum of endomycorrhizae products.

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 Bio-fertilizers in Pomegranate Production

Potassium solubilizing microrganisms

Potassium plays an essential role for enzyme activation, protein synthesis and
photosynthesis. In soil, it is present in water-soluble, exchangeable, non-
exchangeable and structural or mineral forms. Potassium from water-soluble and
exchangeable pools is directly available for plant uptake. At low levels of
exchangeable-K in certain soil types, non-exchangeable-K can also contribute
significantly to the plant uptake. Non-exchangeable-K consists predominantly of
interlayer K of non-expanded clay minerals such as illite and lattice K in K-bearing
minerals such as K-feldspars. More than 98% of potassium in soil exists in the form
of silicate minerals (microcline, muscovite, orthoclase biotite, feldspars etc.).
Some microorganisms in the soil are able to solubilize 'unavailable' forms of K-
bearing minerals, such as micas, illite and orthoclase, by excreting organic acids
which either directly dissolves rock K or chelating silicon ions to bring the K into
solution. These microorganisms are commonly known as potassium solubilizing
microorganisms or silicate dissolving microorganisms.
Example:
I. K solubulizing bacteria
Bacillus mucilaginosus
Bacillus cereus
II. K solubulizing fungus
Aspergillus fumigatus
Penicillium pinophylum

Bio-fertilizers for micronutrients

Micronutrient deficiencies, especially those of zinc (Zn) and iron (Fe), are
major concern in developing countries causing serious health problems among
children, infants and women. Zn and Fe deficiency in human is a consequence of
the limited bio-availability of Zn and Fe in tropical soils and hence in plants.
Vesicular-arbuscular mycorrhizal (VAM) fungi has shown promise as the mobilizer
of the immobile micronutrient particularly Zn and to some extent Fe and Cu in
various fruit crops. Research progress made in enhancing the solubility of

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 Bio-fertilizers in Pomegranate Production

micronutrients also indicated that ericoid mycorrhizal fungi, Oidiodendron maius

isolates produce organic acid like fumarate, malate and citrate that solubilize the
insoluble inorganic Zn compounds and thus increase its bioavailability to the
plants but mass production of this organism as bio-fertilizer has not been
achieved. There are some bacteria which can produce acids and tolerate acidic
conditions that favours the solubulization of micronutrient cations and increase
their bioavailability.
Examples:
Thiobacilus ferrooxidans
Thiobacillus thiooxidans
Thiobacilus acidophilus

The low solubility and hence bioavailability of iron is overcome by

some organisms through the production and excretion of iron-chelating
compounds, termed 'siderophores'. These compounds bind with iron to form a
siderophore-iron complex which is then taken up by the cell and iron is later
liberated internally.

Examples for 'siderophore' producers:

Pseudomonas fluorescens
Pseudomonas aeruginosa
Pseudomonas putida.

Plant growth promoting microorganisms

Among the microbial inoculants, bacteria from the plant's rhizosphere have
received considerable attention over the past two decades with respect to plant
growth promotion. Collectively such bacteria are called plant growth promoting
rhizobacteria (PGPR). The PGPR inoculants which are currently commercialized,
seem to promote growth through at least one of the mechanisms viz.
1. Suppression of plant disease (termed Bio-protectants)
2. Improved nutrient acquisition (termed Bio-fertilizers)

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 Bio-fertilizers in Pomegranate Production

3. Phytohormone production (termed Bio-stimulants): Pseudomonas

fluorescens, species of Bacillus and pink pigmented facultative
methylotrophs (PPFM) can produce as yet not well characterized
phytohormones or growth regulators that cause crops to have greater
amounts of fine roots which have the effect of increasing the absorptive
surface of plant roots for uptake of water and nutrients. These PGPR are
referred to as Bio-stimulants and the phytohormones they produce
include indole-acetic acid, cytokinins, gibberellins and inhibitors of
ethylene production.

Types of Inoculum
There are varieties of formulation both in liquid and solid. The main types
currently used for bio-fertilizers have been classified into dry products (dusts,
granules and briquettes) and suspensions (oil or water-based and emulsions). A
wider range of formulations with additives are available in market.

Dry inoculum products

These formulations comprise dusts, granules and briquettes (based on
particle or aggregate size) and wettable powders. Dusts based on inert diluents or
carriers, normally with low absorbent capacity have particle size ranging from 5-20
mm. and mainly contain 30% of an organism in suspension by weight.
3 3
Granules are discrete masses 5-10mm in size, pellets are >10mm , and
briquettes are large blocks up to several cubic centimeters. These products are
made up of 20-30% organisms and inert carrier materials for holding the
organisms. Carriers include clay (e.g. Bentonite, disburse quickly to release the
organism) minerals, starch polymers and ground plant residues. Choice of carrier
depends on absorption (more important for formulating slurries of organisms),
hardness, bulk density and product disburse rate in water.
Wettable powder formulations are predominant among all commercial
products and comprise charcoal, lignite, vermiculite powders blended with 3%
gum to make them stable during storage on the shelf and readily stick with planting
materials.

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 Bio-fertilizers in Pomegranate Production

Liquid Bio-fertilizers
Liquid bio-fertilizers are special liquid formulation containing not only the
desired microorganisms and their nutrients, but also special cell protectants or
substances that encourage formation of resting spores or cysts for longer shelf life
and tolerance to adverse conditions.
The Liquid bio-fertilizers are available in two forms such as,
Dormant aqueous suspension
Dormant oil suspension.

Dormant aqueous suspension

Several current commercial products are available in market following the
dormant technology. Generally, various growth suppressants, contaminant
suppressant like sodium azide, sodium benzoate, butanol, acetone, fungicides,
and insecticides etc. are used in the formulation for the long term viability. These
formulations are particularly useful for long duration fruit crops as the organisms in
the formulation requires reactivation time to revive from dormant stage.

Dormant oil suspension

Microorganisms can be suspended in oil at high concentration in various
degrees of dehydration and remain viable. This formulation delivers organisms in
a physiologically dormant state and does not encourage the growth of
contaminants during storage. Oil suspension also has the limitation in reactivating
the bacteria / fungus. It generally takes 10 to 20 days to regenerate and the initial
requirement of the plant is not fulfilled by them.
There are four basic characteristics which need to be taken care of while
formulating liquid bio-fertilizers:-
(a) To stabilize the organism during production, distribution and storage.
(b) To easily deliver to the field in the most appropriate manner.
(c) To protect the microorganism from harmful environmental factors at the target
site (field), thereby increasing persistence.
(d) To enhance activity of the organism at the target site by increasing its activity,
reproduction, contact and interaction with the target crops.

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 Bio-fertilizers in Pomegranate Production

Mass Production of Mycorrhizal Bio-fertilizers

The large scale utilization of mycorrhizal fungi has been limited due to its
obligate symbiotic nature and hence is difficult in culturing on laboratory media.
Production of AM inoculum has evolved from the use of original infested field soils
to the current practice of using pot culture inoculum derived from the surface
disinfected spores of single AM fungus on a host plant grown in sterilized culture
medium. Advancement in research has explored different methods of AM fungal
inoculum production both as soil based culture as well as carrier based inoculum.
As carrier based inoculum, pot culture is widely adopted method for production of
AM fungal inoculum. Such inocula comprise of infected roots or spores and
hyphae trapped in soil or peat/ clay carrier. As sterilization process is
cumbersome, perlite, vermiculite have been found to be good alternative to soil
sterilization for inoculum production. The method of inoculum production is
outlined as follows:
A trench (1m x 1m x 0.3m) is formed and lined with black polythene sheet to be
used as a plant growth tub.
Mixed 50 kg of vermiculite and 5 kg of sterilized soil and packed in the trench
up to a height of 20 cm
Spread 1 kg of AM inoculum (mother culture) 2-5 cm below the surface of
vermiculite
Maize seeds surface sterilized with 5% sodium hypochlorite for 2 minutes are
sown
Apply 2 g urea, 2 g super phosphate and 1 g muriate of potash for each trench
at the time of sowing seeds. Further 10 g of urea is applied twice on 30 and 45
days after sowing for each trench
Quality test on AM colonization in root samples is carried out on 30th and 45th
day
Stock plants are grown for 60 days (8 weeks). The inoculum is obtained by
cutting all the roots of stock plants. The inoculum produced consists of a
mixture of vermiculite, spores, pieces of hyphae and infected root pieces.
Thus within 60 days 55 kg of AM inoculum could be produced from 1 sq meter
2
area. This inoculum will be sufficient to treat 550 m nursery area.

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 Bio-fertilizers in Pomegranate Production

Application of Bio-fertilizers
Pomegranate plant is propagated mainly through air-layering. So, the bio-
fertilizer can be introduced to the plant at two stages i.e. (1) nursery stage, (2)
standing crop in the orchards.
Introduction of bio-fertilizers at nursery stage is reported to have better
microbial survival and activity in the main field after plantation. Growth promoting
microorganisms like Azospirillum sp., Pseudomonas fluorescens and pink
pigmented facultative methylotrophs (PPFM) are advocated to be introduced at
initial rooting stage. For introduction of these microorganisms, a solution has to be
prepared by mixing one part of carrier based bio-fertilizer containing said
microorganisms in ten parts of water. And for liquid bio-fertilizers, similar solution is
made by mixing liquid bio-fertilizer @ 2-3 ml/airlayer in required amount of water.
The air-layers after detaching from the mother plants have to be dipped in the
above mentioned solution containing bio-fertilizers for minimum half an hour
before planting them into polythene bag in the nursery. By the time, substrate (soil:
farmyard manure: sand, 3:1:1) in the polythene bag have to be made ready by
mixing each ten ml of cell suspension (liquid bio-fertilizer) having 109 cell ml-1 of
Azotobacter sp., Azospirillum sp., phosphate solubilizing bio-fertilizer (PSB) and
potassium solubilizing bio-fertilizer followed by spreading of 10 g of AM fungal
inoculum containing about 8-10 viable AM fungal propagules per gram soil as thin
layer at 2 cm below soil surface. Then treated air layers have to be planted in to the
polythene bag. Light watering needs to be given immediately after planting.
For intermittent application of bio-fertilizers in standing crop, solid bio-
fertilizers have to be mixed with carrier like soil, compost, farmyard manure (@ 1
kg bio-fertilizer per 25 kg of carrier), kept overnight with sufficient moisture content
and then applied in the rhizosphere by making trench around the plant. The
application of bio-fertilizer should preferably be made in the evening hour and the
applied area needs to be irrigated immediately. Similarly, liquid bio-fertilizers of
phosphate and potassium solubilizing microorganisms @ 400 ml/acre have to be
mixed with 400 to 600 kg farmyard manure along with half bag of rock phosphate, if
available. The mixtures have to be kept under shed for over-night with moisture

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 Bio-fertilizers in Pomegranate Production

maintaining at 50%. Then this mixture can be used for application in the standing
crop. The liquid bio-fertilizers can also be applied by spraying or through
fertigation. The efficacy of spraying liquid bio-fertilizers particularly of growth
promoting microorganisms is greatly affected by fluctuation of microclimate. In
fertigation, bio-fertilizers are mixed in water @ 400 ml/acre and other
micronutrients in the tank and is delivered to individual plant via piping.

Crop Response
Bio-fertilizers comprising of microbial inocula or assemblages of living
microorganisms improve crop yield because of increased availability or uptake or
absorption of nutrients, stimulation of plant growth by hormone action or antibiosis
and by decomposition of organic residues. Pomegranate is not an exception.
Many positive impacts of bio-fertilizers on pomegranate have been reported.
Inoculation of cuttings with different microorganisms such as Trichoderma
harzianum, Azospirillum lipoferum and Azotobacter sp. was reported to increase
rooting and survival per cent. The sprouting occurred in 7-10 days in biofertilizer
inoculated cuttings while it took 8-10 days longer in non-inoculated cuttings. Soil
inoculation with pure cell suspensions of Azospirillum brasilense or Azotobacter
chroococcum increased plant height and dry weight in rooted cuttings of
pomegranate cv. Jalore seedless which was correlated with a marked increase in
N uptake. Pink pigmented facultative methylotrophs (PPFM), an growth promoting
microorganism was found to increase shoot and root biomass by 46% and 49%
respectively and also uptake of nutrients while Pseudomonas fluorescens was
found efficient in enhancing photosynthetic rate of plant. Dual inoculation with N2-
fixing Azospirillum brasilense and P-solubilizing Pseudomonas striata was found
very effective in promoting plant growth and nutrient uptake through beneficial
interaction
Mycorrhizal (Glomus epigaeum, Glomus mosseae and Gigaspora
calospora strains) inoculation increases root colonization, N and P uptake which
in-turn improves plant growth parameters such as height, root length, number of
leaves, root and shoot dry weight. The effect of mixed inoculation with number of

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 Bio-fertilizers in Pomegranate Production

strains is superior in promoting plant growth as compared to their individual

counterpart. The pre-inoculation of nursery pomegranate saplings with N2-fixing
bacteria, Azotobacter chroococcum and AM fungi, Glomus mosseae enhancs the
rhizosphere microbial activity, concentration of various metabolites and nutrient
particularly N and P. These bio-inoculants help in better establishment of
pomegranate plants under field condition. A significant improvement in the plant
height, plant canopy and fruit yield (Figure 8) is possible in 5 year old pomegranate plants.
The strategy of introducing beneficial microorganisms in pomegranate soil is of
recent adoption and it needs more study to improve the process of application for getting
higher benefits and such microorganisms in pomegranate fertilization programme will
make its production sustainable.

To Achieve Maximum Benefit

Bio-fertilizer product must contain good effective strain in appropriate population and
should be free from contaminating microorganisms.
Select right combination of bio-fertilizers and use before expiry date.
Use suggested method of application and apply at appropriate time as per the
information provided on the label.
Bio-fertilizer packets/containers need to be stored in cool and dry place away from
direct sunlight and heat.
Other chemicals should not be mixed with the bio-fertilizers.
Both nitrogenous and phosphatic bio-fertilizers are to be used together to get the best
results.
Bureau of Indian Standard (BIS) has developed quality standards for Azotobacter,
Azospirillum and phosphate solubilizing bacteria and regular testing is also done by
National Bio-fertilizer Development Centre (NBDC), Ghaziabad/ Regional Bio-fertilizer
Development Centres (Hissar, Jabalpur, Bangalore, Bhubaneshwar, Nagpur and Imphal)
and State Government laboratories. But there is a need to bring other types of bio-fertilizers
under its ambit.

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 Bio-fertilizers in Pomegranate Production

Bureau of Indian Standard for Bio-fertilizers

Azotobacter Standard
Base : Carrier based or liquid base
.
Viablecell count (CFU) : Minimum 107 cells g -1 of carrier material or

Contamination level : No contamination of 10 5 dilution.

pH : 6.5-7.5
Particle size in case of carrier based

Moisture per cent by weight, maximum : 30-40% (carrier based material).

Efficiency character : The strain should be capable of fixing at

Azospirillum Standard
Base : Carrier based or liquid base
.
Viable cell count (CFU) : Minimum 107 cells g -1 of carrier material or

Contamination level : No contamination of 10 5 dilution.

pH : 6.5-7.5
Particle size in case of carrier based

Moisture per cent by weight, maximum: 30-40% (carrier based material).

Efficiency character : Formation of white pellicle in semi-solid

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 Bio-fertilizers in Pomegranate Production

Phosphate Solubili zing Biofertilizers (PSB) Standard

Base : Carrier based or liquid base.
Viable cell count (CFU) : Minimum 107 cells g -1 of carrier material or

Contamination level : No contamination of 10 5 dilution.

pH : 6.5-7.5
Particle size in case of carrier based

Moisture per cent by weight, maximum : 30-40% (carrier based material).

Efficiency character : The strain should have minimum 30%

* CFU-Colony forming unit

Limitations and Constraints

The major limiting factors include:
Narrow genetic base of mother cultures and lack of efficient and virulent strains
suitable to various agro-environments.
Unsatisfactory carrier material with uniform and consistent good quality
comparable to imported peat material.
Contamination in broth mixing and packing stages, not using completely
closed system of production.
Unsatisfactory packing material which reduces shelf life.
Unsatisfactory storing conditions, particularly during the distribution period.
Exposure to high temperatures and sunlight destroy the microbial culture.
They should be preferably kept in cold storage conditions.

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 Bio-fertilizers in Pomegranate Production

Not employing properly trained microbiologist.

Lack of quality controls and certification procedures.

Despite having certain limitations, biofertilizers are potential component in

integrated nutrient management programme and also alternative to chemical
fertilizers in organic fruit production system for improvement of soil fertility and
sustainable crop productivity in cost effective and eco-friendly manner.

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Annexure 1
List of Biofertilizer Production Units in India
States Biofertilizer Production Unit
Andhra Pradesh Sri Aurbindo Institute of Rural Development (SAIRD),
Nalgonda
Bacterial Culture Production Lab., Hyderabad
Biofertilizer Unit-Vijayawada, Madras Fertilizers Limited,
Chennai
Acharya N.G. Ranga Agriculture University, Agriculture
Research Station, Amaravathi
Godavari Fertilizers and Chemicals Limited, Kakinada
Assam Hindustan Fertilizer Corporation Ltd. (FP & ARD),
Parbatpur
North East Green Tech Pvt. Ltd., Guwahati
Microbiology Laboratory, Assam Agro Industries
Development Corporation Limited, Guwahati
Assam Agricultural University, Jorhat
Bihar Hindustan Fertilizer Corporation Limited, Begusarai
M/s Pyrites, Phosphates & Chemicals Limited, Rohtas
Gujarat National Agricultural Research Project Biofertilizer
Project Gujarat Agriculture University, Anand
Gujarat State Fertilizers and Chemicals Limited,
Vadodara
Gujarat State Co-operative Marketing Federation Ltd.,
Ahmedabad
Haryana Regional Biofertilizer Development Centre, Hissar
Ganpati Bio Organic Limited, Safidon
Himachal Pradesh Central Laboratory, Shimla
Jharkhand Hindustan Fertilizer Corporation Ltd., Sindri
Karnataka West Coast Herbochem Ltd., Bangalore
Regional Biofertilizer Development Centre, Bangalore
Biofertilizer Unit-Bangalore, Madras Fertilizer Limited,
Chennai
Karnataka Biofertilizers, Bijapur

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 Bio-fertilizers in Pomegranate Production

Kadur Agro, Bangalore

Samrath Bio-Tech Ltd., Hubli
Shakthi Bio-Tech (P) Ltd., Vijaywada
Karnataka Agro Industries Corporation Limited, Bangalore
University of Agricultural Sciences, Bangalore
Karnataka Compost Development Corporation Limited,
Bangalore
Rhizobium Culture Production Laboratory, Bangalore
University of Agricultural Sciences, Dharwad
Kerala Biofertilizer Production Unit, Ramanathapuram
The Fertilizers and Chemicals Travancore Ltd., Kochi
Soil Testing Laboratory, Pattambi, Palakkad
Agro Bio-Tech Research Centre Ltd., Kottayam
Madhya Pradesh Regional Biofertilizer Development Centre, Jabalpur
Samridhi Bioculture Pvt. Ltd., Indore
Bio-Tech Research Centre, M.P. State Co-operative
Oil-seed Growers’ Federation Ltd., Dhar
Hindustan Fertilizer Corporation Ltd., Bhopal
Trupti Bio-Tech Laboratory, Balaghat
The M.P. State Agro Industries Development
Corporation, Biofertilizer Plant, Bhopal
Narmada Bio-Tech Limited, Barwaha
Nafed Biofertilizer, Indore
National Fertilizers Limited, Indore
Maharashtra Regional Biofertilizer Development Centre, Nagpur
Sainath Agro Vet Industries Pvt. Ltd., Ahmed Nagar
BAIF Development Research Foundation, Pune
Samruddhi Agrotech, Pune
Maharashtra Research & Development Centre, Solapur
Kumar Krishi Mitra Bio Products (I) Pvt. Ltd., Pune
Vasantdada Sugar Institute, Pune
Biofertilizer Production Unit Sahakar Maharshi Shankarrao
Mohite-Patil Sahakari Sakhar Karkhana Ltd, Solapur

19
Bio-fertilizers in Pomegranate Production

Institute of Natural Organic Agriculture (INORA), Pune

K-Ferts Lab, Nanded
Nirmal Seeds Pvt. Ltd., Jalgaon
Rashtriya Chemicals & Fertilizers Limited, Mumbai
Nomain Agri-Bio Pvt Ltd., Pune
Nilayam Bio-Fertilizer Production Unit, Wardha
Kisan Agro Chem, Naded
Nav Maharashrta Chakan Oil Mill Ltd., Sangli
Environmental Protection Research Foundation, Sangli
Arun Biofertilizers, Kolhapur
Mahatama Phule Krishi Vidyapeeth, Pune
Mauli Biotech, Pune
Bio Agro Fertilizers, Pune
M/s Niku Bio-Research Lab, Pune
Manipur Regional Biofertilizer Development Centre, Imphal
Mizoram Biofertilizer Production Unit, Neihbawi, Department of
Agriculture, Aizawl
New Delhi Indian Agriculture Research Institutre (IARI), New Delhi
Indian Farmers Fertilizer Cooperative Limited, New Delhi
Orrisa Regional Biofertilizer Development Centre, Bhubneshwar
Orrisa Agro Industries Corporation Ltd., Bhubneshwar
Deputy Director of Agriculture (PP), Bhubneshwar
Hindustan Fertilizer Corporations Limited, Bhubneshwar
State Bio Chemist, Bhubneshwar
Pondicherry Pondicherry Agro Service and Industries Corporation
Limited (PASIC), Pondicherry
Punjab Biofertilizer Production Unit, Ludhiana
Rajasthan Nafed Biofertilizer, Bharatpur
Rhizobia Scheme Agriculture Department, Jaipur
Tamilnadu Department of Agricultural Microbiology, Agriculture
College and Research Institute,Tamil Nadu Agricultural
University, Madurai
Biofertilizer Production Unit, Department of Agriculture,
Govt. of Tamil Nadu, Cuddalore

20
 Bio-fertilizers in Pomegranate Production

Biofertilizer Production Unit, Department of Agriculture,

Govt. of Tamil Nadu, Thantavur
Biofertilizer Production Unit, Department of Agriculture,
Govt. of Tamil Nadu, Trichy
KRIBHCO, Chennai
Regional Research Station, Tamil Nadu Agricultural
University, Piyur
Monarch Bio-Fertilizers and Research Centre, Chennai
Lakshmi Bio-Tech, Cuddalore
Marygreen Afrotech (P) Ltd., Chennai
Tamil Nadu Agricultural University, Coimbatore
T Stanes & Company Limited, Coimbatore
Esvin Advanced Technologies Limited, Chennai
Southern Petrochemical Industries Corporation Limited,
Chennai
Biofertilizer Unit-Manali, Madras Fertilizers Limited,
Chennai
Biofertilizer Production Unit, Salem
Biofertilizer Production Unit, Kudumiamalai
Main Biocontrol Research Laboratory, Chengalpattu
The SIMA Cotton Development and Research
Association, Coimbatore
Uttar Pradesh National Biofertilizer Development Centre, Ghaziabad
Myodelphia Chemicals Company (Pvt.) Ltd., Ghaziabad
M/s Kanhaiya Agrotech (Pvt.) Ltd., Mathura
Motilal Nehru Farmers Training Institute, Allahabad
Krishak Bharati Cooperative Limited, Noida
West Bengal Amit Biotech, Kolkata
Process Development and Analytical Control Research
Laboratory, Kolkata
Nitrofix Laboratories, Kolkata
Hindustan Fertilizer Cooperation Ltd., Kolkata
Nodule Research Laboratory, Nadia
Green Tech Farms & Agro Pvt. Ltd, Kolkata
Nimpit Ashram, South 24-Parganas

21
 Bio-fertilizers in Pomegranate Production

Excel Bio-Tech Pvt. Ltd., Kolkatta

Micro Bac India, North 24-Parganas
Eastern Enterprises, Kolkata
West Bengal Forest Development Corporation Ltd.,
Kolkata

Source: National Biofertilizer Development Centre, Govt. of India, Ministry of

Agriculture (http://dacnet.nic.in/ncof/productionunit.htm)

22
 (a) (b)
Figure 1. (a) Colonies of Azotobacter on Waksman medium No. 77
(N-free Mannital agar medium) and (b) microscopic view of Azotobacter cell

(a) (b)
Figure 2. (a) Colonies of Azospirillum on congo-red agar media and
(b) electronic micrograph of Azospirillum cell

(a)
Figure 3. Formation of solubilization zone by phosphate solubilizing bacteria on
Pikovskaya medium supplemented with insoluble P source.
 Figure 4. Vesicles (v) and arbuscules (a) of arbuscular mycorrhizal
fungi inside plant root.

(a) (b) (c)

Figure 5. (a) Colonies of Bacillus mucilaginosus on Aleksandrov medium (b)
photomicrograph (1200x) of cells and (c ) electron micrograph of cells.

Figure 6. Electron micrograph of Pseudomonas fluorescens cell.

 Tank for mass Sprinkling of water in Making of furrows to sow
multiplication of AM tank with vermiculite maize seeds

Vermiculite contained raised AM Sowing the seeds in

infected maize plants furrows

View of the maize sown AM pit

Figure 7. Mass production of mycorrhizal bio-fertilizers

 Figure 4. Effect of inoculation with various bio-fertilizers on fruit yield of
pomegranate grown in field condition (C: Control; Ab: Azospirillum brasilense;
Ac: Azotobacter chroococcum; Gf: Glomus fasciculatum; Gm: Glomus mosseae)

(Data source: Aseri et al., 2008 )