This document summarizes research on the application of biofertilizers in major crops in Gujarat state. It discusses various types of biofertilizers including nitrogen fixing (e.g. Rhizobium), phosphate solubilizing (e.g. Bacillus, Pseudomonas), and potassium mobilizing microbes. It describes how these microorganisms fix atmospheric nitrogen, solubilize insoluble phosphates, and increase soil fertility and crop yields. For example, Rhizobium increases nitrogen levels by 40-50 kg/ha and yields by 10-15%, while phosphate solubilizers like Pseudomonas make phosphorus more available to plants. The document reviews the mechanisms, host specificity, and application

1. Research Review on Application of Bio-
fertilizer in Major field crop of Gujarat
State
As the Partial Fulfillment of Subject Agron. 507 (Agronomy of Oilseed, Fiber and Sugar Crops)
Submitted to:- Submitted by:-
Dr. S. N. Shah Jayvirsinh P. Solanki
Associate Professor Reg. no. 04-2917-2016
BACA, Anand Agril. Microbiology

2. Content…
• Introduction
• Classification of bio-fertilizer
• Microorganism which works as bio-fertilizer
• Mode of action
• Review

3. • Bio-Fertilizers: Bio-fertilizers are preparations containing active or
latent cells of efficient strains of certain microbes that can utilise the
atmospheric nitrogen to increase the nitrogen content of soil, and can
dissolve the insoluble phosphate of the soil to release the phosphorus
it contains in the soluble form for increasing crop yield.

4. 4
Acetobacter
Frankia
Azolla
Bacillus spp.
Pseudomonas
Burkholderia
VAM
Aspergillus
Trichoderma
Biofertilizer
Nitrogen fixing
biofertilizer (NBF)
Phosphate
Solubilizing Microbes
Symbiotic Asymbiotic Bacteria Fungi
Rhizobium Azotobacter
Azospirillum
BGA
Potash Mobilizing Biofertilizer
1 2
3

6. • Certain soil microorganisms have an ability to absorb
and convert atmospheric nitrogen to the readily
available form to the plants (e.g., Nitrates).
• Where as certain soil microorganisms solubilise part
of the bound phosphates of the soil and thereby
make them available to the plants.
• Both these attributes make them important to be
used as Bio-fertilizers.

7. • There is an abundance of biopolymers like proteins,
fats, fibers and other carbohydrates in natural soils.
• Microbes in soil digest these large biopolymers to
respective smaller monomers. Proteins are digested
to amino acids, carbohydrates and fiber to sugars and
fats/lipids to fatty acids by the soil bacteria.
• Plants can easily absorb these small molecules or
monomers.
• Additionally, the soil-bacteria help the plant-roots to
absorb Major and minor nutrients present in the soils.
• The soil-bacteria also release biochemicals which
accelerate the plant growth.

8. • Add nutrients (Nitrogen) to the soil / make them available
(Phosphorous) to the crop.
• Secrete certain growth promoting substances.
• Under certain conditions they exhibit anti-fungal activities
and thereby protect the plants from pathogenic fungi.
• Harmless and Eco-friendly low cost agro-input
supplementary to Chemical Fertilizers.
• Improve soil structure (porosity) and water holding
capacity.
• Enhance seed germination.
• Increase soil fertility, Fertilizer Use Efficiency and
ultimately the yield by 15-20 % in general.

9. PGPR
• Certain groups of bacteria like the Pseudomonas fluorescence living in
association with the rhizosphere of most of crop plants (rhizobacteria
promoting plant growth) supply all the essential nutrients required
for the growth of the crop and in addition, protects the plant roots
from the attack by soil-borne pathogens (saprophytic suppression).

10. • The Rhizobium culture strains are very selective and
require particular host or nodulation.
• The surface antigen on the Rhizobial cells recognizes the
binding sites (specific root exudates) on the roots of the
leguminous plants.
• This characteristic makes them host-specific. Specific
Rhizobial cell can penetrate the roots of the specific
leguminous plants only and form nodules.
• They multiply within the nodule using the carbon source
from the plant and in turn fix part of the atmospheric
nitrogen to the plant.
• Each Rhizobium culture is useful only for the respective
crop. This culture should be applied by seed treatment
only.

11. 11
MORPHOLOGY
• Unicellular,
• Cell size less than 2µ wide.
• Short to medium rod,
plemorphic, motile with
peritricus flagella,
• Gram – negative,
• Accumulate poly B-hydroxy
butyrate granules.
Rhizobium capsule staining

12. 12
1- Recognition and
attachment (Rhicadhesin
+ Host Lectin)
2- Excretion of Nod factors
Steps involved in nodule formation
6.- Nodule
formation
3. Invasion –Rhizobia
penetrate root hair and
multiply within
“Infection thread”
Infection
Thread
Uninfected
root hair4-Bacteria Grow
toward root cell
5.- Formation of bacteroid
state within plant cell

13. • The effective strain used in Rhizobium culture increases
the healthy nodulation and thereby nitrogen fixation
(about 40 to 50 kg/ha.).
• About 10 to 15% increase of crop yield can be achieved
with the use of this culture.
• The residues of pulses (legume crops) left in the soil after
harvesting the crop are also advantageous to the
subsequent crops to be sown.
Dose
• Seed Treatment (for One Acre) : 250 gm / 10 kg seeds for
liquid preparation 10 ml/kg seed.
• Seedling Treatment (for One Acre) : 250 gm.

14. • The cells of Azotobacter remain free in soil or in vicinity of the root
system and fix part of the atmospheric nitrogen.
• Azotobacter is useful for the vegetables and cash crops viz. Brinjal,
Chilli, Okra, Cotton, Cumin, Banana, Sugarcane, Tobacco, Castor,
Vegetables etc., as well as horticultural crops.

15. • The effective strain used in Azotobacter fixes about 15 – 20 kg atmospheric
nitrogen/ ha.
• Certain growth promoting substances released by these bacteria are useful
for increasing the seed germination, plant growth and ultimately the yield
• About 10 to 15 % increase of crop yield can be achieved with the use of
these cultures
• In certain condition they also exhibit anti-fungal activities and thereby
fungal diseases may be controlled indirectly.
Dose
• Seed Treatment (for One Acre) : 250 gm / 10 kg seeds.
• Seedling Treatment (for One Acre) : 250 gm.

16. • The cells of Azospirillum remain in association with the roots and fix
part of the atmospheric nitrogen.
• Azospirillum is useful for the cereals and cash crops viz. Wheat, Paddy,
Bajra, Jowar, Maize, Mustard, Cotton, Cumin, Banana, Sugarcane,
Tobacco, Castor, Vegetables etc., as well as horticultural crops.

17. 17
• Microorganism like Bacteria and fungi viz. Bacillus coagulans, B.
circulans, B.polymaxa, Pseudomonas striata, Aspergillus
awamori, and Penicillium digitatum includes PSM.
• Having ability to solubilize insoluble phosphate present in the
soil by lowering the pH due to secretion of organic acids there
by making unavailable form of P to available form.
• The PSM soiubilize unavailable form to available form by
enzymatic mechanism.
• The P solubilizing bacteria or fungi can be mass multiplied on
Pikovasakys broth and mixed with the carrier material used.
Experiments conducted by scientist have shown the possibility
of saving of 25-50 kg P2O5 mere through application of
recommended PSM culture.

18. 18
Bacillus coagulans,
B. polymaxa
Pseudomonas striata
Aspergillus awamori
Penicillium digitatum

19. 19
Mechanism of action
PSMs secrete organic acids such as formic, acetic, propionic, lactic,
glycolic, citric, fumaric, succinic acids. These acids lower the pH and
bring about the dissolution of bound forms of phosphates
Produce plant growth promoting substances like IAA, IBA ; GA &
members of vitamin B group.

20. 20

21. • The effective strain of Phosphate Solubilizing Bacteria used, increase the
level of available P2O5 in the soil.
• With the increase in available P2O5 level, overall plant growth can be
increased.
• In certain condition they also exhibit anti-fungal activities and thereby
fungal diseases may be controlled indirectly.
• About 10 to 15% increase of crop yield can be achieved with the use of this
culture.
Dose
• Seed Treatment (for One Acre) : 250 gm / 10 kg seeds.
• Seedling Treatment (for One Acre) : 250 gm.

22. • Another group of free-living nitrogen fixers are the
cyanobacteria commonly called the blue-green algae
(BGA). More than a hundred species of BGA can fix
nitrogen.
• Nitrogen fixation takes place in specialised cells called the
heterocysts (large, thick walled and metabolically inactive
cells) which depend on vegetative cells for energy to fix
nitrogen while the fixed nitrogen is utilised by the
vegetative cells for growth and development.
• BGA are very common in the rice fields (the micro-
aerophilic condition and alkalinity are conducive to the
algal population).

23. 23

24. • Another group of free-living nitrogen fixers are the
cyanobacteria commonly called the blue-green algae
(BGA). More than a hundred species of BGA can fix
nitrogen.
• Nitrogen fixation takes place in specialised cells called the
heterocysts (large, thick walled and metabolically inactive
cells) which depend on vegetative cells for energy to fix
nitrogen while the fixed nitrogen is utilised by the
vegetative cells for growth and development.
• BGA are very common in the rice fields (the micro-
aerophilic condition and alkalinity are conducive to the
algal population).

25. • Azolla is a tiny water fern common in ponds, ditches and rice fields.
• It has been used as a bio-fertiliser for rice in all major rice growing
countries including India, Thailand, Korea, Philippines, Brazil and West
Africa.
• The nitrogen accumulated in the Azolla is made available to the rice crop
when the fern decomposes.
• The nitrogen fixing work is accomplished by the symbiotic relationship
between the fern and a BGA, Anabaena azollae.
• The alga inhabits some of the cells on the underside of the Azolla frond and
fixes atmospheric nitrogen.

26. • It is dependent on the fern for photosynthesis which supply the
energy for nitrogen fixation.
• In addition to nitrogen, the decomposed Azolla also provides K, P, Zn
and Fe to the crop.
• It also controls aquatic weeds which would otherwise compete with
the crop for nutrients.

27. • Frankia is an actinomycete and forms nitrogen fixing nodules in trees
and shrubs.
• The organism invades the cells of a developed lateral root and causes
it to fuse into a nodule.
• Entry into the host changes the structure of the microbe.
• Scientists are hopeful that some day they may be able to make fruit
trees like apple, pear, plum, raspberry, etc. by fixing nitrogen through
the involvement of Frankia.

28. • Some non-pathogenic fungi help in plant growth by
forming associations with the host plant roots called
mycorrhizae (myca- fungi, rhiza -root).
• Some examples of such fungi are Trichoderma,
Gigaspora, Glomus, etc.
• One group of mycorrhizae forms a sheath around the fine
lateral roots and replaces the root hairs by dichotomous
branching of the fungal hyphae.
• They are called ectomycorrhizae because they do not
traverse intracellularly.
• The ectomycorrhizae help the plant by Solubilizing
nutrients near the plant roots and making it easy for the
plants to feed

29. • These fungi increase the surface area of absorption of the
roots and thus help in the absorption of nutrients,
specially those less mobile in soil solution like P.
• They also prevent the roots from being attacked by
nematodes (by entangling them).
• Another group called the endomycorrhizae penetrate the
roots and establish symbiotic relation with the plants.
• The fungi help the roots in obtaining inorganic nutrients
while obtaining essential organic nutrients from the host.
• There is yet another group called ect-endomycorrhiza or
vesiculararbuscular mycorrhiza (VAM fungi) wherein they
are partly outside the host roots and partly intracellular.

30. • VAM - Vesicular Arbuscular mycorrhiza - is the symbiotic association
between plant roots and soil fungus.
• They are zygomycetes fungi belonging to the genera Glomus,
Gigaspora etc..
• VAM plays a great role in inducing plant growth.
• Mycorrhizae increase the resistance to root borne or soil borne
pathogens and Nematodes.
• Enhanced colonization of introduced population of beneficial soil
organisms like Azotobacter, Azospirillum, Rhizobium and Phosphate
Solubilizing Bacteria around mycorrhizal roots thereby, exerting
synergistic effects on plant growth.
• Suitable for: Turmeric, Banana, Rubber, Coffee, Tea, Pepper,
Cardamom, Cocoa, Fruit trees, Tree seedlings and species etc.

31. 31
The fungal network around the root increase
the contact surface area between roots and
particles of soil & absorbs nutrients from
long distance away

32. VAM

33. 33
Root penetration of fungal hyphae
hypha grow
intracellularly & also
penetrate the cell
walls of cortical cells,
causing invagination of
the plasma membrane

35. • VAM is highly versatile and colonizes 85 % of the plant families.
• It penetrates the roots, forms arbuscules and vesicles in the cortical cells of
the roots and hyphae and spores in the soil.
• The mychorrhiza penetrates the roots, mobilizes & supplies phosphorus
and other micronutrients to the plants.
• Solubilize phosphate and transports micronutrients such as zinc,
Manganese, iron, copper, Cobalt, Molybdenum etc. from the surrounding
area to the plant.
• Increases the plant vigor by inducing drought resistance of young seedlings.
• VAM protects the plants from the fungal pathogens.

36. Soil application:
• 200 gms per sq.mt. in seed/nursery bed
• 2 gm per seedling in the Nursery stage
• 5 gms per seedling at the time of planting
• 10 – 50 gms per garden trees and fruit trees respectively
• 100 - 200 gms per plant of grown tree species
• 3 – 5 kgs/acre of Manidharma’s VAM can be applied in 2 - 3 cm depth.

37. • Earthworms are farmer’s friends. They dig up and mix the soil, eat up
decayed plants and convert them to fertiliser thus enriching the soil.
• In recent years a low-tech biotechnology has emerged to restore
earthworms to their natural place in the environment through
vermiculture (since the continuous use of chemicals has caused their
depletion from soil).
• Vermiculture requires mineral inputs in terms of ingredients (leaf
litter, household and agricultural wastes along with a starting
population of earthworms).

38. Table 1. Effect of bio-fertilizer on yield and yield attributing
characters of groundnut (Pooled over 3 years)
Treatment
T1 : Rhi.I
T2 : Rhi.II
T3 : PGPR4
T4 : RDF
T5 : Control
Pod yield
(kg ha-1)
Haulm
yield
(kg ha-1)
Kernel
yield
(kg ha-1)
No. of
pods
plant-1
Shelling
(%)
100
KW(g)
SMK
(%)
Oil
(%)
HI
(%)
2477 2992 1736 15.1 70.1 38.7 78.3 42.1 45.2
2536 3145 1765 15.6 69.6 38.4 79.0 42.6 44.6
2658 3189 1887 16.2 71.0 39.0 79.6 42.0 45.4
2213 2784 1509 13.7 68.2 38.1 77.0 42.8 44.2
1225 1641 773 7.8 63.1 37.8 72.1 43.2 42.7
S.Em.± 56.071 67.123 43.31 0.89 0.21 0.09 0.98 0.03 0.02
C.D. at 5% 143.54 171.83 110.87 2.28 0.54 NS 2.51 NS NS
KW = Kernel weight; SMK = Sound Mature Kernel; HI = Harvest Index
Joshi P. K & Kulkarni, J. H, West Bengal

39. Table 2 : Effect of different composts and biofertilizer on yield
attributes and quality of Kharif green gram
Plant height
Treatments (cm) at
harvest
No. of
branches
plant-1
No. of root
nodules
plant-1
No. of
pods
plant-1
No. of
seeds
pod-1
Pods
length
(cm)
Number of
seeds pod-1
1000 test
weight
(g)
Seed
nitrogen
content (%)
Protein
content
%
T1 52.88 7.95 11.50 17.35 9.25 7.15 9.25 37.77 3.18 19.86
T2 61.35 9.30 13.60 21.00 10.40 7.46 10.40 41.15 3.59 22.43
T3 57.95 8.65 12.55 19.35 9.60 7.35 9.60 38.56 3.33 20.82
T4 59.15 8.70 12.58 18.85 9.65 7.32 9.65 39.34 3.44 21.49
T5 66.98 9.70 15.20 24.60 10.65 7.77 10.65 44.24 4.21 26.29
T6 58.80 8.70 12.83 19.85 9.85 7.34 9.85 39.24 3.37 21.01
T7 57.70 8.75 14.50 19.80 9.90 7.32 9.90 38.76 3.52 22.00
T8 58.35 8.65 14.05 20.10 9.95 7.39 9.95 38.75 3.36 21.01
T9 61.43 9.45 17.88 21.48 10.30 7.50 10.30 41.91 3.79 23.71
T10 58.90 8.75 15.00 19.90 10.05 7.42 10.05 39.32 3.54 22.14
S.E. (±) 2.37 0.31 0.98 1.25 0.29 0.19 0.29 1.28 0.17 1.05
C.D. (P=0.05) 6.88 0.91 2.85 3.63 NS NS NS 3.71 0.49 3.03
NS=Non-significant
Kh. Naveen & K. D. Mevada, Anand

40. Table: 3 Fresh Weight as influenced by KMB inoculation in Potato
Fresh Weight (t/ha)
Tuber Treatment Soil Treatment
Trt. 2010-11 2011-12 2012-13 Pool 2010-11 2011-12 2012-13 Pool
T1 21.50 29.94 26.73 24.05 21.47 22.32 26.64 23.81
T2 20.87 22.33 26.25 23.15 20.87 22.64 25.67 23.06
T3 19.97 21.30 22.7 21.32 20.67 22.27 22.46 21.80
T4 20.55 22.69 25.66 22.97 20.71 22.18 25.16 22.68
T5 20.33 23.17 24.47 22.66 19.74 21.36 24.44 21.86
T6 19.35 21.75 24.86 21.99 20.69 22.56 24.67 22.64
T7 19.98 21.42 22.34 21.24 19.58 21.45 21.88 20.96
T8 18.81 20.26 23.35 20.81 17.89 19.47 23.08 20.15
T9 21.48 21.48 26.64 23.20 22.04 24.17 26.30 24.17
T10 8.72 10.05 13.07 10.61 13.01 14.34 12.87 13.50
S.Em+- 1.09 1.01 1.40 1.18 0.90 1.02 1.68 1.25
CV% 9.9 8.4 10.3 9.6 7.9 8.3 12.5 10.1
YxT NS NS
Dept. of Agril. Micro & Agronomy, 2011, 2012 & 2013

41. Table 4: Effect of Azotobacter on Growth and Yield Attributes of
Maize during 2007-08
T1 150 184 167.0 65 92 78.5 4.36 4.21 4.28 1.03 1.16 1.09
T2 194 213 203.5 85 112 98.5 5.56 5.05 5.30 0.20 0.50 0.35
T3 177 216 196.5 78 115 96.5 4.73 4.43 4.58 0.73 0.88 0.80
T4 184 209 196.5 68 108 88.0 4.13 4.66 4.39 1.35 0.83 1.09
T5 188 211 199.5 90 113 101.5 4.33 4.00 4.16 0.96 1.38 1.17
T6 186 214 200.0 96 111 103.5 5.08 4.60 4.84 0.33 0.93 0.63
T7 206 211 208.5 94 111 102.5 4.86 4.83 4.84 0.41 0.76 0.58
T8 205 216 210.5 100 113 106.5 5.33 5.50 5.41 0.21 0.16 0.18
F-test ** ** * ** * * NS NS
CV% 5.2 2.92 9.5 4.95 14.2 10.3 24.7 28.80
LSD(0.05) 13.6 10.7 14.3 9.51 1.00 0.84
-
Bandhu Raj Baral* and Parbati Adhikari
(Nepal)

42. Table 5: Lint, Seed cotton and stalk yield of cotton as influenced by nitrogen, FYM and
biofertilizer
Lint yield
(q/ha-1)
Seed
yield(q/ha-1)
Seed cotton
yield(q/ha-1)
Stalk
yield(q/ha-1)
Treatments 2001 2002 2001 2002 2001 2002 2001 2002
Control 5.10 5.89 9.6 11.0 14.6 16.8 47.9 49.4
30 Kg N ha -1 6.15 6.97 11.6 13.2 17.7 20.2 51.9 54.2
60 Kg N ha -1 6.95 7.89 13.3 15.1 20.2 22.9 55.8 58.8
Azotobacter (Az.) M4 5.40 6.33 10.1 11.4 15.5 17.8 48.9 50.6
Azotobacter (Az.) M5 5.34 6.29 9.9 11.2 15.3 17.5 48.7 50.4
FYM @ 12 t ha -1 6.54 7.34 12.2 14.1 18.7 21.5 52.8 55.7
30 Kg N ha -1 Az.M 4 6.49 7.09 12.1 14.1 18.6 21.2 52.5 55.4
Division of Agronomy, IARI, New Delhi
2005
Mahaagrozyme 45.6 49.4 96.2 97.0 145.3 151.8 170.6 181.6
Sem ± 1.1 1.4 3.3 3.5 3.9 4.4 5.3 4.8
CD (P= 0.05) 3.3 4.0 9.8 10.2 11.6 12.8 15.6 14.1

43. Table 6: The effect of mineral fertilizers alone or in combination with biofertilizers on
yield and its components of wheat at 2013/2014.
Treatments No. of spikes No. of spikelets Spike length 1000 grains Grain yield
Plant -1 Spike -1 (cm) Weight (gm) (t ha -1)
2013/2014 Season
100% NPK 5.03 15.00 8.33 39.21 1.11
75% NPK + BI*
Inoculated
6.91 15.00 9.00 41.89 1.21
75% NPK +
Bu** foliar
7.13 15.67 9.00 43.25 1.31
50% NPK + Bi
inoculated
4.01 14.00 7.33 38.11 1.05
50% NPK + Bu
foliar
4.89 14.67 7.33 36.93 1.01
LSD (5%) 1.83 1.85 0.97 3.23 0.17
Ragab S. Taha, Ayman H.A. Mahdi and
Hamdy A. Abd El-Rahman, Egypt

44. Table 7 : Effect of chemical fertilizer and biofertilizer on yield attributes
and economics of plant
Treatment Weight
(1000 g)
Panicle
weight (g)
Filled grain (Per
panicle)
Grain yield (qt
ha-1)
Straw yield
(qt ha-1)
B:C
ratio
100 kg N + 40
kg P + 20 kg K
21.46 2.43 124 58 83 3.31
150 kg N + 60
kg P + 40 kg K +
Azotobacter@
4 kg/ha
23.59 3.16 138 63 87 3.35
150 kg N + 60
kg P + 40 kg K +
Azotobacter+
PSB @ 5 kg/ ha
24.50 3.58 140 65 88 3.41
C.D. (P=0.05) 0.85 0.23 6.8 2.1 2.5
RAMA KANT SINGH, PANKAJ KUMAR, BIRENDRA
PRASAD AND S. B. SINGH, Katihar, Bihar 2015