Chapter crop production. However farmers do not use

Chapter 1


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Pakistan is suffering from poverty therefore it is necessary
to increase crop production for the reduction of hunger and insecurity of food.
Organic fertilizers and minerals are beneficial for increasing the crop
production. However farmers do not use mineral fertilizer for variety of
reasons. Besides this it is necessary to improve the production of crop in an
eco-friendly manner. This has led to the promotion of commercial, biological
and chemical products that can enhance the organic matter content of soils.
Nutrients such as nitrogen (N), phosphorus (P) and sulphur(S) are released as
result of decomposition of organic matter using soil microorganism. This is the
reason microorganism are important component of soil biodiversity and
integrated nutrient management systems.


Taxila, Punjab Pakistan



Chapter 2

Literature Review


In contrast to chemical fertilizer, biological fertilizer
make use of microbes for improving of crop production and this can be done
either by applying directly to the soil or on the seeds before planting. Microorganism
that help plants in uptake of nutrients through their interaction in the rhizosphere
(Soil that surrounds and is influenced by the roots of plant). They speed up
certain microbial processes in the soil which increase the extent of
availability of nutrients in the form easily utilized by plants for their

Most of the times microorganism cannot be replicated in the
natural environment because of diverse nature of environment (temperature, pH,
etc.) and therefore artificially multiplied culture of microorganism play an
important role to speed up their microbial activity. As use of biofertilizers is
cost effective so it is one of the important component of Integrated Nutrient Management












Microbial Fertilizer

It is defined as the addition or injection of microbial
population into the soil to enhance soil productivity. For example fungi,
nitrogen fixing bacteria, phosphorus solubilizing bacteria etc.

fixing bacteria as microbial fertilizers

Although nitrogen is abundantly present in atmosphere yet it
is the most limiting nutrient to the growth of plants. As plant cannot directly
uptake nitrogen from atmosphere therefore nitrogen fixing bacteria present in
the roots of plants, play role in uptake of nitrogen. These bacteria are
associated with plants in three different ways:

Some nonsymbiotic (free
living) nitrogen fixing bacteria are present in soil

Symbiotic nitrogen fixing
bacteria; others have suggested endophytic association with plant

Others are associated with
rhizosphere and don’t form close endophytic symbioses.

The large amount of nitrogen is fixed by using these three
systems in spite of change in environmental conditions or difference in
combination of plant-microbe. Fixed nitrogen is beneficial for minimizing
leaching and denitrification. There are different organism helpful in easy access
of plants to the nitrogen in atmosphere and use them as biofertilizers and
these are discussed below.

Non symbiotic
nitrogen fixing bacteria

Non symbiotic bacteria such as azotobacter, clostridium also
help in fixing nitrogen present in atmosphere. If environmental conditions are
favourble then Frankia (nodulating bacterial symbionts) can also fix N2 in
non-symbiotic association 1. Frankia can be present in rhizosphere
of non-host plants and help in nitrogen fixation. Record has shown Frankia is
present in rhizosphere of Betula Pendula2 and in soil with no
actinorhizal plants3 . N2 fixation cannot be estimated
by free living bacteria. There are different methods of inoculating bacteria in
green house experiment such as leaf spray, seed soaking and side dressing. The
growth of cucumber and barley plants was stimulated by inoculation of
Beijerinckia mobilis and clostridium sp.4 . However, it remained
unclear about the mechanism of growth. The growth is not only stimulated by
inoculating bacteria but it can also be due to plant growth hormones.

Symbiotic nitrogen-fixing

Rhizobia family (Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium)5
is the best known and most exploited symbiotic nitrogen-fixing bacteria. This
kind of bacteria cause infection in legumes. We should consider rhizobia-host compatibility
while selecting bio fertilizer. For N2 fixing, the capability of
rhizobia varies up to 450 Kg N.ha-1 among strains of bacteria and host plant
species. Rhizobial strains that are considered as practical applications as bio
fertilizers must have some additional characteristics like they must have
fixation rate of Nitrogen and can compete with naturally occurring rhizobia.
Survival and easy production of inoculum is necessary in field application when
target seeds are inoculated. Inoculation is of prime importance when rhizobial
populations of resident soil are absent. For example, acidic and basic soil
contain low population density of alfalfa rhizobial symbiont (Sinorhizonium
meliloti) and low inoculum potential of Brady rhizobium sp. respectively 6.
It is highly advantageous to inoculate compatible rhizobia under such
conditions. Evaluating the need for inoculation and performing cost benefit
analysis is necessary before initiating massive scale inoculation program with
rhizobia. Inoculation is useful for productivity of crops if rhizobial
population density is less than 100 rhizobia/gram of soil 7.
Inoculation would prove cost effective in presence of low population densities
in spite of indigenous rhizobia nitrogen fixation efficiency.


Cyanobacteria find its importance in ecology e.g. an aquatic
cyanobacterium (Trichodesmium) contributes 36% of global nitrogen fixation
according to estimate. 8 Nitrogen fixation using cyanobacteria has
proved beneficial in rice cultivation. Another nitrogen source for rice
cultivation in China was Azolla Anabaena until end of 1970s. Cyanobacteria
still have key role in rice fields in various parts of Asia. Furthermore we can
remediate arid soils with cyanobacteria and nitrogen fixation. However the
production and utilization is yet poorly developed 9. For
supporting sustainable agriculture activities in different environments there
is serious need to consider cyanobacteria as biofertilizer.

associated with nitrogen fixing

There are less associated nitrogen fixing bacteria are
present that can be used for improving crop production. For example acetobacter
Diazotrophicus and Herbaspirillum spp. are associated with sorghum, maize and
sugarcane 10. The use of genus Azospirillum to increase the growth
of crops is not bound by host specificity. In addition to wheat and rice it
includes carrot, tomato, sumflower, peeper, eggplant and cotton 11.
The use of Azospirillum cause large increase in crop yield almost in 70 percent
cases according to a general consensus12. This increase in growth
is not due to nitrogen fixation but due to increase growth promoting
substance’s production 13. Sugarcane nitrogen requirement can be
fixed upto 70% by Acetobacter diazotrophics 14. The environmental
conditions and plant genotype specify the amount of nitrogen fixed.

Solubilizing Bacteria

The second most limiting nutrient for plants is phosphorus
(P) 15. Phosphorus is present in large amount in soil but plant
cannot uptake it. There are some bacteria that are able to mobilize phosphorus
so that plants can easily uptake it. Bacillus and Pseudomonas are most leading
phosphorus-solubilizing bacteria (PSB) in this regard 16. Now we
analyse the potential importance of PSB using field experiment. One of the
field experiment was done by Sundara et al. (2002) and he concluded that the
application of PSB increase 12.6% yield of sugarcane. The phosphorus
requirement is reduced to 25% by applying PSB in combination with phosphorus
fertilizer. In addition, cost effective rock phosphate can replace 50% of
expensive superphosphate. It was observed that production of sugar and juice quality
was also improved using PSB 17. It also allows the use of cheap
source of phosphorus.

Mobilizing Biofertilizer

Endosymbionts of the fungi of the genera Glomus, Gigaspora
and Endogone are responsible for the controlled transformation of nutrients
(Phosphorus, zinc & sulphur) from soil to the roots of plants. Fungi
mobilize the phosphorus so that it can be easily uptake by plants. The most
common genus is glomus having many species present in soil. It is beneficial
when experimental conditions are applied however it is difficult to obtain
AM(Arbuscular Mychorrhiza) fungi pure culture when we need production on large

of Plant Hormones by Bacteria

Bacteria can also increase the growth of plants by producing
the plant hormones including nitrogen fixation and solubilizing phosphorus.
High amounts of gibberellins are produced by Bacillus pumilus and B.
licheniformis 18. Some important bacteria that increase the
production of hormones are Paenibacillus polymyxa (nitrogen fixation, phosphorus
solubilizing and production chitinase and other hydraulic enzymes, increase
soil porosity), taxa (N2 fixation and plant hormones production) 19.


The liquid bio fertilizers provide nitrogen and phosphorus
to the plants by fixing nitrogen and

solubilization of phosphorus such as Rhizobium, Azospirilium
and Phosphobacteria. These fertilizers are more effective for sugarcane,
cotton, vegetable, millets and other crops.


This bacteria forms the root nodules for reduction of
nitrogen (Molecular) to ammonia which is used to produce vitamins, proteins and
other nitrogenous compounds.

Properties of liquid Rhizobium

Dull white in colour

No bad smell

No foam formation, pH


These bacteria are fix the large amount of nitrogen 20-40kg
N/ha in non-leguminous plants like cotton, oil seeds, millets etc. By use of Azospirillum
the amount of nitrogen fertilizers can be saved upto 25-30%. Azospirillum is
able to perform the reduction of nitrate to nitrite and denitrification.

Properties of liquid Azospirillium

Colour of liquid (blue or
dull white)

Quality of product can be
confirmed by formation of yellow gummy colour

Acidic pH assure the
absence of Azospirillum bacteria

Improper liquid formulation
is confirmed by bad odour

Azobacter is nitrogen fixing bacteria and work in aerobic conditions.
Azobacter biofertilizer are applied to the non-leguminous plants including rice,
vegetable, cotton etc. Excess amount of Azobacter cell is present in
rhizospheric region. The growth of organic matter is dependent upon the amount
of organic matter.                                                                                                                   Properties of liquid
Azobacter                                                                                                       Azobacter produce pigmentation of
melanin in old culture because of the oxidation process that is carried by copper
containing enzyme tyrosinase.


This bacteria is associated with sweet sorghum, sugarcane
and sweet potato plants. Its nitrogen

Fixation ability is 30kgs/ N/ ha year. It is used on
commercial scale for production of sugarcane crop. The yield of crop is
enhanced upto 10-20t/acre and sugar content almost 10-15%.