Post on 02-Nov-2019
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AGRO-INDUSTRIAL DEVELOPMENT AND ECONOMIC UPLIFTMENT OF WEAKER SECTIONS THROUGH
BIOFERTILIZER MANUFACTURING.
(FINAL REPORT)
SUBMITTED TO
Western Ghats Development Programme
Planning & Economics Affairs Department
Government of Kerala.
DIVISION OF MICROBIOLOGY
TROPICAL BOTANIC GARDEN AND RESEARCH INSTITUTE
PALODE, THIRUVANANTHAPURAM, KERALA- 695 562.
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Title of the project: AGRO-INDUSTRIAL DEVELOPMENT AND
ECONOMIC UPLIFTMENT OF WEAKER
SECTIONS THROUGH BIOFERTILIZER
MANUFACTURING.
Funded by: Western Ghats Development Programme
Planning & Economics Affairs Department,
Govt. of Kerala.
Sanction order No: G.O (MS) No: 31/ 02/ Plg.
Principal investigator Dr. S. SHIBURAJ
Scientist
Tropical Botanic Garden and
Research Institute,
Palode, Pin- 695 562
Thiruvananthapuram
Project Staff Mr. S. Shaju
(Junior Research Fellow)
Project commenced on June 2003
Total Project cost Rs.6.85 lakhs
Amount released Rs. 6.85 lakhs
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PREAMBLE
Azotobacter is one of the free living (non-symbiotic) soil bacteria, which are commonly
used as microbial inoculums in biofertilizers. TBGRI has developed a new Biofertilizer
(Tropbactrin) using a new strain of Azotobacter (Azotobacter chroococcum TBG-1)
which is tolerant to a wide range of pH (4-10). This strain is also thermotolerent and can
survive in soil up to a temperature of 600C. Moisture stresses even as low as 5% will not
inactive this strain. Another important advantage is that it can enhance nodulation in
leguminous plants.
Fertilizers are those substances, which are added to the soil in order to substitute the
deficiency of essential elements required for plant growth and high crop yields. The
principal elements required by plants are Nitrogen, Phosphorus and Potassium. The
nutrient value of a fertilizer is defined in terms of its N P K value.
Uncontrolled and continuous use of chemical fertilizers leads to environmental pollution
and also creates imbalance in soil ecosystem, which in turn leads to the loss of soil vigor.
Biofertilizer will help the soil in restoring the microbial and nutrient balance of the soil
and so is Eco-friendly. Presently the farmers of Kerala are well aware of the importance
of the Biofertilizer and its merit over the chemical ones and so there is a greater demand
for it. But unfortunately the production rate is not up to the demand. The biological soil
fertility management is an ecological approach for sustainable agriculture and is mainly
concerned with the maintenance of yield, closely associated with the desire to conserve
natural resources, including a greater value accorded to the maintenance of bio diversity.
Disadvantages of chemical fertilizers
1. Excessive use leads to imbalance in the soil pH causing soil alkalinity or soil acidity
depending upon the kind of fertilizer used.
2. Imbalance in the soil pH causes imbalance in the availability of native
micronutrients, leading to deficiency syndromes.
3. Imbalanced pH also causes impairment in the population of beneficial
microorganisms and their activity.
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4. Under imbalanced pH macronutrients also get fixed into unavailable form.
5. Use of high analysis fertilizers to supply macronutrients results in plants taking up
native micronutrient in higher quantities leading to impairment of micronutrient
supply system.
6. Most of the chemical fertilizers are based on fossil fuel energy and so are energy
consuming.
7. Chemical fertilizers are found to pollute the environment in various ways. Chemical
factories producing these fertilizers are always a source of pollution.
8. Results of many research studies suggest that the quality and shelf life of the food
products raised exclusively on chemical fertilizers are poor.
Due to these negative aspects of chemical fertilizers, the importance of biological
fertilizers is increasing over the years. Bio fertilizers are carrier based microbial
inoculants of live and latent cells of efficient strains of nitrogen fixing, phosphate
solublizing, cellulolytic or mycorrhizal micro organisms used for the application to soil,
seed or seedlings with the objective of increasing the number of such microorganisms and
accelerate certain microbial processes to augment the extent of availability of nutrients in
a form in which it is easily assimilated by plants. The term may also be used to include all
organic resources for plant growth, which are rendered in an available form for plant
absorption through microorganisms or microorganism-plant associations or interactions.
A wide range of microorganisms including bacteria, fungi and algae beneficially
contribute to the plant development through the supply of nutrients essential for plant
growth. Certain species of these groups of microorganisms may either fix atmospheric
nitrogen or solubilize insoluble phosphorus and make them available to plants. Such
beneficial organisms are domesticated in suitable carriers, which on application to soil
augment crop growth and yield. These carriers and microbes are called bio fertilizers or
appropriately called bio inoculants or microbial inoculants or microbial fertilizers.
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These microorganisms also improve soil fertility and crop productivity due to their
capability to fix atmospheric nitrogen, solubilise insoluble phosphates and decompose
wastes, which results in the release of plant nutrients. The extent of these benefits
depends on their number and efficiency. When the number and activity of specific
microorganisms becomes sub optimal, artificially multiplied cultures of those
microorganisms called microbial inoculants or Bio fertilizers are used to hasten
biological activity to enhance the availability of plant nutrients. Therefore, application of
biofertilizers in the field is a viable alternative for sustainable agricultural activities.
Advantages of Biofertilizers
1. These are excellent in maintaining soil vigour in terms of physical and biological
conditions and favor the growth of beneficial microorganisms.
2. These form the excellent source of micronutrient supply.
3. As these sources are renewable and recyclable these are economical and
sustainable ones for the farmers.
4. They are ecofriendly and can conveniently convert all kinds of organic wastes to
nutrient rich organic manures, which can be the source of pollution.
5. There is no need for strict soil chemical diagnosis before the application of
biofertilizers.
6. Research results suggest that the quality and shelf life of the food products raised
with the use of biofertilizers are very good and the demand for the food raised with
organic nutrients is in the increase.
Importance of Tropbactrin
Recently scientists are engaged in the development of bacterial strains, which are capable
of converting the atmospheric nitrogen into forms usable, by plants. Azotobacter is one of
the free living (non-symbiotic) soil bacteria, which are commonly used as microbial
inoculums in biofertilizers. Azotobacter species such as A. bejherinkii, A. vinelandii and
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A. chroococcum are well known for their high nitrogen fixing ability. The nitrogen fixing
capacity of most of the Azotobacter strains are dependent on limiting factors such as pH,
temperature and moisture content of the soil.
TBGRI has discovered a new strain of Azotobacter tolerant to a wide variety of pH that is
from 4-10. This strain is also thermotolerent and can survive in soil up to a temperature of
600C. Moisture stresses even as low as 5% will not inactive this strain. Another important
advantage is that it can enhance nodulation in leguminous plants. By using this particular
strain, Azotobacter chroococcum TBG-1 TBGRI has already developed a biofertilizer-
‘Tropbactrin’. Tropbactrin is not getting inactivated when applied with chemical
fertilizers. Tropbactrin also protect roots from root pathogen.
Preparation
Mix 1 Kg of the mother culture of tropbactrin with 5 Kg of the cultivator’s soil, mix well
and add sufficient water to make into a paste. Allow it to remain with the same moisture
content for 6-7 days, and then dry and powder it and pack in HDP bags. This biofertilizer
is having a shelf life of more than six months under normal conditions. This process of
multiplication is suggested in order to give better results in the field. This enables heavy
bacterisation in the rhizosphere and root zone as the inoculam gets familiarized with soil
microorganisms and conditions.
Tropbactrin is capable of fixing 40-60 Kg of N/hec/yr. Besides it can mobilize contain
some amount of phosphorus, potassium, Mg, Ca etc. It is suitable for both acidic and
alkaline soil. It can be used as an integrated crop along with various other fertilizers.
Tropbactrin is comparatively a cheap biofertilizer and its method of application is also
very easy. It can be prepared and applied by farmers without much facilities and training.
The organism remains in the soil for a long time and fix atmospheric nitrogen. Besides
tropbactrin can be applied to all kinds of agricultural crops.
Package of Practices of Tropbactrin
1. Paddy & other cereals
Add 100 Kg/ha of the prepared fertilizer, plough well and transplant.
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2. Coconut & other palms
Add 2 Kg of the prepared fertilizer at the base of the plant and mix well with soil.
Boost dose at 1Kg per plant can also be added every year.
3. Pepper
Add 1 Kg of the prepared fertilizer at the base of the plant and mix well with soil.
Application at ½ Kg per plant can be repeated every year.
4. Vegetables
Add 100 Kg/ha of the prepared fertilizer, mix well with soil. Cow dung or compost
can also be added along with this for better results.
5. Tapioca & other tuber crops
Add 1 Kg of the prepared fertilizer at the base of the plant and mix well with soil.
Experiments were conducted at various fields with the help of farmers. Then yield, total
cost, net income, and benefit- cost ratios were worked out from the data collected from the
farmers. These results were compared with the results obtained from the use of chemical
fertilizer in their fields.
From the results, it could be concluded that the ‘Tropbactrin’ increased the yield of
various crops and reduced the cultivation cost. Weed growth was lesser with the
application of tropbactrin. So expenditure on weeding and herbicide application was
reduced. It also gives certain resistance to fungal diseases. In banana the maturity period
was reduced at least by a month and the fruits were uniform from top to bottom. Overall
increase in the yield of crops up to 15% was observed due to the application of
Tropbactrin. Application of Tropbactrin decreased environmental pollution and
encouraged the growth of beneficial microorganisms.
Objectives of the Project
Supply of mother culture of Tropbactrin to interest growers in Kerala.
To popularize biofertilizer and its merits.
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Improvement of the strain (Azotobacter chroococcum TBG-1) for better
performance.
Evaluation and comparison of the performance of similar strains.
Development of a molecular marker for the strain Azotobacter chroococcum
TBG-1.
Evaluation of the change in rhizosphere microflora of the Tropbactrin applied
crops.
Achievements of the Project
Molecular phylogeny of the strain was evaluated using RAPD and ARDRA along
with 5 other Azotobacter strains viz, Azotobacter chroococcum, Azotobacter
vinelandii, Azotobacter vinelandii, Azotobacter beijerinkiii, Azotobacter
vinelandii (MTCC 446,MTCC 2459, MTCC 2460, MTCC 2641, MTCC 124)
from Chandigardh and Azotobacter chroococcum (TBG-2) from Calcutta. It was
noticed that the strain Azotobacter chroococcum (TBG-1) is significantly diverse
from the rest of the strains. The 16S portion amplified with specific primers has to
be sequenced for further BLAST analysis.
Two potential phosphate solubilising bacteria were isolated for using as a co-
culture in Tropbactrin. Phosphate solubilising activity of the isolated strains was
checked and compared the activity with two authentic strains (MTCC 490 &
MTCC 428).
Salt tolerance, Temperature tolerance, pH tolerance, Nitrogen fixing ability and
antagonistic properties of Azotobacter chroococcum (TBG-1) were studied and
compared with the strains collected from different sources. It is observed that no
other strains are in par with TBG-1 for different parameters.
Mother culture of Tropbactrin was supplied to various growers for mass
multiplication and field application.
Training was given to 64 persons (selected from rural farmers) in the preparation
and application of Tropbactrin.
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BIOFERTILIZER
TRAINING PROGRAMMES
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Introduction
The green revolution brought impressive gains in food production but with insufficient
concern for sustainability. In India the availability and affordability of fossil fuel based
chemical fertilizers at the farm level have been ensured only through imports and
subsidies. Dependence on chemical fertilizers for future agricultural growth would mean
further loss in soil quality, possibilities of water contamination and unsustainable burden
on the fiscal system. The Government of India has been trying to promote an improved
practice involving use of bio- fertilizers along with fertilizers. These inputs have multiple
beneficial impacts on the soil and can be relatively cheap and convenient for use.
Biofertilizers, more commonly known as microbial inoculants, are artificially multiplied
cultures of certain soil organisms that can improve soil fertility and crop productivity.
Although the beneficial effects of legumes in improving soil fertility was known since
ancient times and their role in biological nitrogen fixation was discovered more than a
century ago, commercial exploitation of such biological processes is of recent interest
and practice. Biofertilizers are defined as 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 seed or to soil. Biofertilizer
from N2 fixing bacteria come in three forms: liquid, solid and lyophilized. For liquid and
lyophilized ones, only solution medium is used, but for solid form, carriers such as peat,
activated charcoal and chicken dung are needed. The first representative of the genus,
Azotobacter chroococcum, was discovered and described in 1901 by the Dutch
microbiologist and botanist Martinus Beijerinck. They are found in neutral and alkaline
soils.
The commercial history of biofertilizers began with the launch of ‘Nitragin’ by Nobbe
and Hiltner, a laboratory culture of Rhizobia in 1895, followed by the discovery of
Azotobacter and then the blue green algae and a host of other micro-organisms.
Azospirillum and Vesicular-Arbuscular Micorrhizae (VAM) are fairly recent discoveries.
In India the first study on legume Rhizobium symbiosis was conducted by N.V.Joshi and
the first commercial production started as early as 1956. However the Ministry of
Agriculture under the Ninth Plan initiated the real effort to popularize and promote the
input with the setting up of the National Project on Development and Use of
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Biofertilizers (NPDB). Commonly explored biofertilizers in India are mentioned below
along with some salient features.
Azotobacter (AZT): This has been found beneficial to a wide array of crops covering
cereals, millets, vegetables, cotton and sugarcane. It is free living and non-symbiotic
nitrogen fixing organism that also produces certain substances good for the growth of
plants and antibodies that suppress many root pathogens. Azotobacter is Gram-negative,
motile, pleomorphic aerobic bacterium which produces catalase, oval or spherical that
form thick-walled cysts and may produce large quantities of capsular slime. Azotobacters
are the most intensively investigated heterotrophic group possessing the highest
respiratory rates. Members of these genera are mesophilic, which require optimum
temperature of about 30ºC. There are some microorganism which establish symbiotic
relationships with different parts of plants and may develop special structures as the site
of nitrogen fixation.
Rhyzobium (RHZ): These inoculants are known for their ability to fix atmospheric
nitrogen in symbiotic association with plants forming nodules in roots (stem nodules in
sesabaniamrostrata). RHZ are however limited by their specificity and only certain
legumes are benefited from this symbiosis.
Azospirillum (AZS): This is also a nitrogen-fixing micro organism beneficial for non-
leguminous plants. Like AZT, the benefits transcend nitrogen enrichment through
production of growth promoting substances.
Blue green Algae (BGA) and Azolla: BGA are photosynthetic nitrogen fixers and are
free living. They are found in abundance in India i. They too add growth-promoting
substances including vitamin B12, improve the soil’s aeration and water holding capacity
and add to bio mass when decomposed after life cycle. Azolla is an aquatic fern found in
small and shallow water bodies and in rice fields. It has symbiotic relation with BGA and
can help rice or other crops through dual cropping or green manuring of soil.
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Phosphate solubilizing (PSB)/Mobilizing biofertilizer: Phosphorus, both native in soil
and applied in inorganic fertilizers becomes mostly unavailable to crops because of its
low levels of mobility and solubility and its tendency to become fixed in soil. The PSB
are life forms that can help in improving phosphate uptake of plants in different ways.
The PSB also has the potential to make utilization of India’s abundant deposits of rock
phosphates possible, much of which is not enriched.
Non nodule forming diazotrophs for example , Azotobacter, Beijerinckia play an
important role in the nitrogen cycle in nature, binding atmospheric nitrogen, which is
inaccessible to plants, and releasing it in the form of ammonium ions into the soil. Apart
from being a model organism, it respire aerobically which uses the organic matter present
in soil to fix nitrogen asymbiotically and receiving energy from redox reactions, using
organic compounds as electron donors. Azotobacter can use a variety of carbohydrates,
alcohols and salts of organic acids as sources of carbon and can fix at least 10
micrograms of nitrogen per gram of glucose consumed so used by humans for the
production of biofertilizers, food additives and some biopolymers.
Azotobacter, a free living microbe, acts as plant growth promoting rhizobacteria (PGPR)
in the rhizosphere of almost all crops. A group of beneficial plant bacteria, as potentially
useful for stimulating plant growth and increasing crop yields has evolved over the past
several years to where today researchers are able to repeatedly use them successfully in
field experiments. Such PGPRs also fix nitrogen for non-legume crops like wheat, rice,
sunflower, sugarcane, cauliflower, cotton, maize and sorghum which helps in saving 20-
40kg chemical nitrogen i.e. 45-90 kg urea per hectare. Yield of several non-legume was
increased by PGPRs symbionts through plant growth promoting substances, it helps in
root expansion, improve uptake of plant nutrients, protects plants from root diseases and
most important improves biomass production of fast growing at wasteland.
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REVIEW OF LITERATURE
Azotobacteraceae
Two genera of bacteria in family Azotobacteraceae that fix nitrogen as free-living
organisms under aerobic conditions: Azotobacter and Azomonas. The basic difference
between these two genera is that Azotobacter produces drought-resistant cysts and
Azomonas does not. Aside from the presence or absence of cysts, these two genera are
very similar. Both are large gram-negative motile rods that may be ovoid or coccoidal in
shape (pleomorphic). Catalase is produced by both genera.
There are six species of Azotobacter and three species of Azomonas (Jan, 2006).
Although some rhizobia may fix nitrogen nonsymbiotically, unlike Azotobacter, they can
only do so under reduced oxygen tension. Furthermore, their cells are generally smaller
than Azotobacter cells (A. paspali excepted). Moreover rhizobia need a more complex
medium (supplemented with growth substances, etc.) for growth .Other nonsymbiotic
nitrogen-fixing organisms have a different cell morphology and widely different
physiological and nutritional requirements depending on the taxonomic group of the
prokaryote class to which they belong (Jan, 2006). Differentiation of the six species of
the genus Azotobacter and three species of Azomonas is based primarily on the presence
or absence of motility, the type of water-soluble pigment produced, and carbon source
utilization. Four species of Azotobacter and all three species of Azomonas are motile.
Pigmentation these organisms produce both water-soluble and water-insoluble pigments
(Benson, 2001).
Azotobacter
The first species of the genus Azotobacter, named Azotobacter chroococcum, was
isolated from the soil in Holland in 1901. These nitrogen-fixing bacteria are important for
ecology and agriculture (Mrkovac & Milic, 2001). Free-living, aerobic N2 fixing bacteria
of the genus Azotobacter were discovered at the turn of the century (Beijerinck, 1901)
and their N2 Fixing associations with plants were then soon investigated to improve the
productivity of non-leguminous crops (Hong et al, 2006). Azotobacter is able to fix at
least 10 mg N per gram of carbohydrate (Tejera, et al, 2004). Although the free-living
Azotobacteraceae are beneficial nitrogen-fixers, their contribution to nitrogen enrichment
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of the soil is limited due to the fact that they would rather utilize NH3 in soil than fix
nitrogen. In other words, if ammonia is present in the soil, nitrogen fixation by these
organisms is suppressed (Benson, 2001). Among the free-living nitrogen-fixing bacteria,
those from genus Azotobacter have an important role, being broadly dispersed in many
environments such as soil, water and sediments (Mirjana et al, 2006). Azotobacter sp, are
free-living aerobic bacteria dominantly found in soils, present in alkaline and neutral
soils. They are nonsymbiotic heterotrophic bacteria capable of fixing an average 20kg
N/ha/year. Besides, it also produces growth promoting substances and are shown to be
antagonistic to pathogens. Azotobacter sp. are found in the soil and rhizosphere of many
plants and their population ranges from negligible to 104 g-1 of soil depending upon the
physico-chemical and microbiological (microbial interactions) properties (Ridvan, 2009).
In soils, Azotobacter sp. populations are affected by soil physico-chemical (e.g. organic
matter, pH, temperature, soil depth, soil moisture) and microbiological e.g. microbial
interactions) properties (Ridvan, 2009). The genus Azotobacter includes 6 species, with
A. chroococcum most commonly inhabiting various soils all over the world. The
occurrence of other Azotobacter species is much more restricted in nature, e.g. A. paspali
can be found only in the rhizosphere of a grass. Soil populations of Azotobacter sp. rarely
exceed several thousand cells per gram of neutral or alkaline soils, and in acid (pH < 6.0)
soils these bacteria are generally absent or occur in very low numbers (Martyniuk and
Martyniuk, 2002). Azotobacter sp. is gram negative bacteria, polymorphic i.e. they are of
different sizes and shapes. Old population of bacteria includes encapsulated forms and
have enhanced resistant to heat, desication and adverse conditions. The cyst germinates
under favorable conditions to give vegetative cells. They also produce polysaccharides.
These are free living bacteria which grow well on a nitrogen free medium. These bacteria
utilize atmospheric nitrogen gas for their cell protein synthesis (Khanafari et al, 2006).
The genus Azotobacter comprises large, gram-negative, primarily found in neutral to
alkaline soils, obligately aerobic rods capable of fixing N2 nonsymbiotically. Azotobacter
is also of interest because it has the highest respiratory rate of any living organism. In
addition to its ecological and physiological importance, Azotobacter is of interest because
of its ability to form an unusual resting structure called a cyst. Azotobacter cells are
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rather large for bacteria, many isolates being almost the size of yeast, with diameter of 2-
4 µm or more (Gül, 2003). Besides, nitrogen fixation, Azotobacter also produces,
thiamin, riboflavin, indole acetic acid and gibberellins. When Azotobacter is applied to
seeds, seed germination is improved to a considerable extent, so also it controls plant
diseases due to above substances produced by Azotobacter. The exact mode of action by
which azotobacteria enhances plant growth is not yet fully understood. Three possible
mechanisms have been proposed: N2 fixation; delivering combined nitrogen to the plant;
the production of phytohormone-like substances that alter plant growth and morphology,
and bacterial nitrate reduction, which increases nitrogen accumulation in inoculated
plants (Mrkovac & Milic, 2001).
Effect of External Environmental Factors on the Growth of Azotobacter
PH Effect: The presence of A. chroococcum in soil or water is strongly governed by the
pH value of these substrates. In an environment below pH 6.0, Azotobacter is rare or
absent. The soils above pH 7.5 contained A. chroococcum varying in numbers between
102
and 104 per gram of soil. In nitrogen-free nutrient media, the lower pH limit for
growth of A. chroococcum strains in pure culture is between pH 5.5 and 6.0 (Jan, 2006).
Temperature: In relation to temperature, Azotobacter is a typical mesophilic organism.
Most investigators regard 25-30ºC as the optimum temperature for Azotobacter. The
minimum temperature of growth of Azotobacter evidently lies a little above 0ºC.
Vegetative Azotobacter cells cannot tolerate high temperatures, and if kept at 45-48ºC
they degenerate (Gül, 2003).
Aeration: Owing to the fact that Azotobacter is an aerobe, this organism requires
oxygen. As many investigators have noted, aeration encourages the propagation of
Azotobacter. Effect of different oxygen tensions on the biomass formation of A.
vinelandii was studied and shown that biomass formation was optimum at PO2 2-3% (air
saturation) and decreased with increasing PO2. In another study, both increasing
dissolved oxygen tension and increasing agitation speed increased cell concentration of
Azotobacter when grown diazotrophically. The initiation of growth of nitrogen-fixing
Azotobacter species was prevented by efficient aeration but proceeded normally with
gentle aeration (Gül, 2003).
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Inorganic Salts: Azotobacter needs some basic nutrient to proliferate in nitrogen-free
medium. Beside the carbon source, it needs several salts to fix nitrogen so to propagate.
Iron and molybdenum are the co-factors of the nitrogenase enzyme, responsible for the
nitrogen fixation, so essential for growth. The propagation of Azotobacter is largely
dependent on the presence of phosphorous and potassium compounds in the medium.
Calcium and magnesium play an important role in the metabolism of Azotobacter.
Although manganese is evidently not an essential element for nitrogen fixation, its
favorable action was reported with the highest requirement of A.chrooccocum at the 20-
30 ppm in the medium. According to the information about the action of copper on
Azotobacter is toxic even in very low concentrations (Gül, 2003).
Nitrogen: Although Azotobacters in general are nitrogen fixers, addition of nitrogen in
the medium decreases the lag phase and generation time and thus fermentation time.
When nitrogen is supplied in the NaNO3 form, up to 0.5 g/L concentration, there was an
increase in growth, but further increases in concentration did not altered the growth
pattern. The best results are obtained with NH4Cl form at 0.1 g/L (Gül, 2003).
Azotobacter chroococcum
Taxonomy
Domain Bacteria
Phylum Proteobacteria
Class Gammaproteobacteria
Order Pseudomonadales
Family Pseudomonadaceae/Azotobacteraceae
Genus Azotobacter
Species Azotobacter chrococcum
Characteristic sings of A. chroococcum as follows; Size of cell 3.1 x 2.0 ìm; Forms cyst;
Motile, especially in young culture or if grown in ethanol; The colonies of
A.chroococcum at free nitrogen media were slightly viscous, semi-transparent at first,
later dark-brown. Utilizes starch; In some cases utilizes sodium benzoate; utilizes
mannitol benzoate; utilizes rhamnose benzoate (Martinez et al, 1985, Gül, 2003). Cells of
A. chroococcum are pleomorphic, bluntly rod, oval or coccus-shaped. Mean dimensions
are 3.0.7.0 ìm long × 1.5.2.3ìm wide. The cell shape changes dramatically in time or with
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changes in growth (medium) conditions. Cells are often in pairs show figure 2.1 . Young
cells are motile by peritrichous flagella. Microcysts and capsular slime are formed.
Colonies are moderately slimy, turning black or black-brown on aging, the pigment
produced is not water-diffusible (Jan, 2006).
Figure 1. Azotobacter chroococcum. Two cells in a pair
Azotobacter chroococcum, a free-living diazotroph has also been reported to produce
beneficial effects on crop yield through a variety of mechanisms including biosynthesis
of biologically active substances, stimulation of rhizospheric microbes, modification of
nutrient uptake and ultimately boosting biological nitrogen fixation. The presence of A.
chroococcum in soil or water is strongly governed by the pH value of these substrates. In
an environment below pH 6.0, Azotobacter is generally rare or totally absent. Soils above
pH 7.5 contained Azotobacter (predominantly A.chroococcum) varying in numbers
between 102 and 104 per gram of soil (Jan, 2006; Qureshi et al, 2009). Due to the role of
A. chroococcum in nitrogen fixation, It is an important (PGPR) producing compounds
needed for plant growth and to their potential biotechnological applications. A.
chroococcum produces gibberelins, auxins, and cytokinins (Mrkovac and Milic, 2001).
Mycorrhizae
In 1885, a German botanist, Frank, coined the term "mycorrhizae" which literally mean
"fungus root" to describe the symbiotic association between fungi and roots of higher
plants. The research work done during latter half of this century has clearly demonstrated
that the nature of this association is symbiotic, in which both plants and associated
fungus derive benefit. These fungi are ubiquitous in occurrence and now recognized as
an important group of soil microorganisms that influence plant growth and development.
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Based on the type of infection caused, two broad groups of mycorrhizae are now
recognized. They are "Ectomycorrhizae” and "Endomycorrhizae". The ectomycorrhizae
are also called "sheathing mycorrhizae" because, the fungus grows around the root
surface forming fungal mantle or sheath. They are characterized by the presence of
fungal hyphae in between cortical cells, which form a distinct network called as
"Hartignet". These fungi belong to Deuteromycetes and Basidiomycetes. Under
Basidiomycetes, these are included in families Agaricaceae and Boletaceae. These fungi
preferentially colonize the roots of woody perennials like pines and conifers.
The group of endomycorrhizae includes arbutoid, ericoid, orchid and arbuscular
mycorrhizal (AM) fungi. Among these except AM fungi others have limited and
specified host range. The AM associations are known to be formed by aseptate fungi,
while the other types of endomycorrhizal associations are formed by septate fungi. AM
fungi are the most predominant among different types of endomycorrizal fungi and are
known to occur in different soil types and varying climatic conditions. They colonize
both intra and inter-cellular regions in cortex and produce distinct storage structures
(vesicles) and nutrient exchange structures (arbuscules). These arbuscules are more or
less equivalent to the houstoria of obligate parasitic fungi but are believed to function in
bidirectional transfer of nutrients. Essentially this transfer involves carbohydrates from
plant to fungus and minerals, especially phosphate, from fungus to plants.
AM fungi are obligate symbionts and can not reproduce without a host. Most
angiosperms and some gymnosperms form these symbiosis (Harley and Harley, 1987).
Worldwide about 200 AM fungi have been described. Of those 50% are Glomus, 25%
Gigaspora and 10% Acaulospora and Sclerocystis respectively. Depending on ecological
niche, AM fungi are selectively chosen by nature on criteria of suitability. Criteria such
as pH, temperature, soil-fertility and sensitivity to heavy metal toxicity determine AM's
sort or ecotype. Jones (1924) described an AM fungus in the roots of some leguminous
plants.
Butler (1939) reported the presence of mycorrhizal infection in the roots of cotton and
tea. Cifferi (1941) made preliminary observations on sugarcane mycorrhizae and studied
their relationship to root diseases. Laylock (1945) reported the occurrence of mycorrhizal
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infection in the roots of Theobroma cocoa. Manjunath and Bagyaraj (1981) reported for
the first time occurrence of VAM in cardamom, betlevine and pepper. Studying the
occurrence of VAM in different cultivars of field bean, they concluded that not only plant
species but also different cultivars of same species vary in the extent of harboring
mycorrhizal fungi in the root system. VAM fungi are considered to be most important in
agriculture, since they are mostly associated with agriculturally important crops like
cotton, soybean, citrus, grape, tomato, maize (Gerdemann, 1964), grasses (Nicolson,
1959) groundnut and other legumes (Butler, 1939; Bagyaraj et al., 1979).
VAM fungi are obligate symbionts and have not been cultured on nutrient media
(Sreenivasa, 1986). Few mycorrhizal researchers are of the opinion that these cannot be
cultured, because they might have lost the ability to synthesize certain enzymes of key
biochemical pathways (Hepper, 1984). In AM fungi symbiosis, both plant and fungus are
benefited. AM fungus Supports the plant growth by facilitating the uptake of nutrients
with hyphal networks, which it extends beyond the nutrient depletion zone. Some of the
benefits conferred by arbuscular mycorrhiza include (a) Improved uptake of macro and
micronutrients (b) Increased tolerance to abiotic stresses and (c) Beneficial alterations of
plant growth regulators (Jarstfer and Sylvia, 1993). All these benefits are resultant of the
complex and dynamic interactions occurring between the fungi and the host.
The plants provide a niche for fungal development and proliferation of AM fungi. AM
fungi supplies Nitrogen (N), Phosphorus (P), Zinc (Zn), Potassium (K), Manganese
(Mn), Copper (Cu), lron (Fe), etc., to the plants. Mosse (1957) observed that mycorrhizal
apple plants grew better and contained more of K, Fe, Cu and less of Mn as compared to
non-mycorrhizal plants. In 1959 she also observed that the mycorrhizal inoculated onion
seedlings grew better in both sterilized and unsterilized soils as compared to non-
mycorrhizal control. Gerdemann (1964) reported that, in phosphate deficient soil
mycorrhizal maize grew much longer than non-mycorrhizal maize and had higher
concentrations of phosphorus. He observed P-deficiency symptoms in non-mycorrhizal
plants and correlated the increased growth to the extent of mycorrhizal infection. Gray
and Gerdemann (1969) studied the uptake and accumulation of P by mycorrhizal and
non-mycorrhizal onion plants, and observed that mycorrhizal onion plants accumulated
significantly higher amount of P in the roots and tops than that of non-mycorrhizal plants.
20
At low rates of P, Zn uptake was increased (Pairunan et al., 1980; Bagyaraj and
Manjunath, 1980) in crops like cowpea, cotton and finger millet when inoculated with
AM fungus Glomus fasciculatum.
Phosphate solubilizing Microorganisms (PSM)
Soils having high pH have the problem of phosphorus availability for plants. In such a
situation, phosphate solubilizing microorganisms (PSM) can be useful to reverse this
process. PSM is a group of heterotrophic microorganisms capable of solubilising
inorganic P from insoluble sources. These include the following bacteria, fungi and yeast.
Bacteria: Bacillus megaterium, B.circulans, B.subtilis, Pseudomonas straita, P.rathonis;
Fungi: Aspergillus awamori, Penicillium digitatum, Trichoderma sp.; Yeast:
Schwanniomyces occidentails.
The solubilisation of P by these microorganisms is attributed to excretion of organic
acids like citric, glutamic, succinic, lactic, oxalic, glyoxalic, maleic, fumaric, tartaric and
Ketobutyric. These microorganisms weather rock phosphate and tricalcium phosphate by
decreasing the particle size, reducing it to nearly amorphous forms. In addition to P
solubilisation, these microorganisms can mineralize organic P into a soluble form. The P-
solubilisers also produce fungistatic and growth-promoting substances, which influence
plant growth. The performance of these microorganisms is affected by availability of a
carbon source, P concentration, particle size of rock phosphate and other factors like
temperature and moisture. PSM will be a boon for the farmers where the soil pH is high.
The earliest report of increasing P uptake and dry weight of plants through inoculation of
phosphate solubilizing organisms was made by Geretsen (1948). He found that
sunflower, oat, mustard and rape pure cultures of rhizosphere bacteria in pots containing
sterilized soil amended with poorly soluble phosphate, resulted increased dry weight of
the plants as well as P uptake. Significant increase in N and P uptake and green matter
was observed during the first stage of vegetation in sunflower plants due to soil
inoculation with phosphate dissolving microorganisms (Stefan and Boti, 1960).
However, the first bacterial inoculant named ‘phosphobacterin’ containing Bacillus
megaterium var. phosphaticum was used on large scale in agriculture production in the
erstwhile U.S.S.R. and other east European countries. Many workers in India and other
21
countries also conducted field trials on the use of phosphate solubilizing biofertilizers
prepared out of several bacteria and fungi on different crop plants with varying
responses. Sundara Rao et al. (1963) found that inoculation with Bacillus megaterium
increased the uptake of phosphate from both soil and fertilizer P sources. In sterile soil
series, the introduced organism (Russian strain) was more effective than the Indian strain
in solubilizing the phosphate.
Taha et al. (1969) reported that inoculation of soil with phosphate solubilizing organisms
like Bacillus megaterium, Micrococcus and Pseudomonos increased dry matter yield and
uptake of P by barley plants and most significant response was obtained by inoculation
with Bacillus megaterium. Sharma and Singh (1971) recorded significant increase in
grain yield as well as N and P uptake of rice due to ‘phosphobacterin’ inoculation along
with the application of bone meal to sandy loam soil in a pot experiment. Banik and Dey
(1981) recorded increased P uptake and dry weight of rice plants due to inoculation of
phosphate solubilizing Bacillus sp.
Brown (1974) suggested that the increase in plant growth sometimes observed after
inoculation with phosphate solubilizing bacteria were primarily the result of synthesis of
plant growth regulators. Rachewad et al. (1991) Obtained enhanced biomass production,
P content and P uptake in plants when seed inoculation of maize was done with Bacillus
polymyxa. and/or 75 kg P ha-1 was applied as single super phosphate or rock phosphate.
Addition of rock phosphate and inoculation with PSM such as Bacillus megaterium,
Pseudomonas striata, Penicillium.sp and Aspergillus awamori increased yields of
cereals, legumes, potatoes and other field crops. The use of low grade rock phosphate is
recommended for both neutral and alkaline soils if PSM is used as inoculant (Dubey and
Billore, 1992).
Kim et al. (1998) studied the interaction of phosphate solubilizing bacteria and VAM
fungi on tomato growth, soil microbial activity and production of organic acids in non-
sterile soil containing hydroxyapatite and glucose. Glomus etunicatum and Enterobacter
agglomerans were used. They reported that the P-concentration was greatest in all
treatments on day 55 and total N and P uptake in plants were higher in treated ones
compared to control suggesting that there was a synergistic interaction between the two
22
organisms. The effect of inoculating wheat with phosphate-solubilizing organisms
Bacillus circulans and Clodosporium herbarum and the VAM fungus Glomus spp. with
or without Mussoorie rockphosphate amendments in a nutrient deficient natural sandy
soil was studied. The result suggests that combined inoculation of three organisms can
improve crop yields in nutrient-deficit soils (Singh and Kapoor, 1999).
Occurrence and distribution of phosphate solubilizing microorganisms have been found
in almost all soils tested, although their populations vary with different soils, climate and
history (Kucey et al., 1989). Raj (1980) enumerated the PSB in four different soil types
and observed the population ranging from 0.11 x 105 to 5.86 x 103 colony forming units
per g dry weight of soil. Barea et al. (1976) reported the ecological significance of
phosphate solubilizing bacteria, which were isolated from the rhizosphere and their
mode of action when used as inoculants. Bacillus megaterium, B. polymyxa and
Pseudomonas stutzeri were the most efficient P solubilizers in the soils of the Kamarajar
District [Tamil Nadu, India]. Glucose was the best C source for P solubilization
(Rajarathinam et al., 1995)
23
MATERIALS AND METHODS
This chapter deals with the materials and methods (Analytical and Experimental), which
were most common by and repeatedly used throughout this work.
MATERIALS
Glassware and Chemicals
All the glassware used were of Borosil/Corning glass. These were first washed with
detergent, then with tap water and finally rinsed in distilled water. If required, they were
immersed for 24 hours in chromic acid and washed in tap water and rinsed in distilled
water. They were drained and dried properly for future use.
All the chemicals used in the analytical method and media preparation were of analytical
grade with maximum available purity supplied by Hi-media (Bombay), SISCO (Chennai)
and Sigma (USA). RAPD primers were from Operon Technologies, USA and PCR
reagents and Taq polymerase from Finzyme (USA).
Microorganisms
The bacterium used in this experiment was A. chroococcum TBG1 strain. This
microorganism was isolated from Lakshadeep island soil. Other strains of Azotobacter
were obtained from MTCC, Chandigarh.
Methods
I. Identification of Azotobacter chroococcum
1. Cultivation of A. chroococcum
An aliquot (0.1 ml) of the bacterial suspension growing out (burks media) was spread on
the plates of Burk's medium agar. Plates were incubated at 28ºC for 3 days. Bacterial
colonies were subcultured onto sterile Azotobacter agar plates and the plates were
incubated at 28ºC for 3 days. Typical bacterial colonies were observed over the streak.
Well isolated single colony was picked up and re-streaked to fresh Azotobacter agar plate
and incubated similarly.
24
2.Characterization of the Isolated Strain
After 3 days of incubation, different characteristics of colonies such as shape, size,
surface, color, pigmentation were recorded. Morphological characteristics of the colony
of each isolate were examined on Azotobacter agar plates. Production of diffusible and
non-diffusible pigments determined on Burk's solid medium after 5 days of incubation at
30 ºC.
3. Colony Shape
Streak a plate of Burks media agar using isolated colonies from 1-2 old media and
incubate at 30°C for 1-5 days and notice the colony shape and color.
4. Gram Staining
A drop of sterile distilled water was placed in the center of glass slide. A lapful of growth
from young culture was taken, mixed with water, and placed in the center of slide. The
suspension was spread out on slide using the tip of inoculation needle to make a thin
suspension. The smear was dried in air and fixed through mild heating by passing the
lower site of the slide 3 to 4 times over the flame. The smear was then flooded with
crystal violet solution for 1 min and washed gently in flow of tap water. Then the slide
was flooded with iodine solution, immediately drained off, and flooded again with Lugal
iodine solution. After incubation at room temperature for 1 min, iodine solution was
drained out followed by washing with 95% ethanol. After that, it was washed with water
within 15 to 30 s and blot dried carefully. The smear was incubated with safranin
solution for 1 min. The slide was washed gently in flow of tap water and dried in air. The
slide was examined under microscope at 100X power with oil immersion and data were
recorded.
5. Motility Test
Bacteria are introduced into a semisoft agar medium by performing a stab with an
inoculating needle. After incubating the tube, motility is determined by examining
whether or not the bacteria have migrated away from the stab line and throughout the
medium.
25
6. Starch Hydrolysis
Starch agar is used for cultivating microorganisms being tested for starch hydrolysis.
Flood the surface of a 48-hour culture on starch agar with Gram Iodine. Iodine solution
(Gram.s) is an indicator of starch. When iodine comes in contact with a medium
containing starch, it turns blue. If starch is hydrolyzed and starch is no longer present, the
medium will have a clear zone next to the growth.
II. Preparation of Bacterial Suspensions for Seeds Inoculation
1. Inoculam preparation
The bacterial inoculants were prepared where a loopful of the respective A. chroococcum
isolate was transferred to 2 ml of the burks liquid medium and incubated overnight then
transferred into 50 ml burks liquid medium and incubated for 7 days on a rotary shaker.
Turbidity, as bacterial growth indicator, of the cultures was adjusted calorimetrically to
optical density of 1.6 at wavelength of 420 nm, or the bacteria was grown on nitrogen-
free media and incubated at 28˚C for 5 days until early log phase.
2. Pot Experiment
The present investigation was carried out during the season of (2003/2004) at greenhouse
at TBGRI. The experiment consisted of seven treatments of chemical, organic and
biofertilizers arranged in a complete randomized blocks design with thirty replicates for
each treatment and 2 seeds were transplanted in each pot (after germination one of two
seeds is disposed), which mean that each treatment had 60 seeds, the treatments as shown
below:
A = Control (no inoculation).
B = Biofertilizer only (A. chroococcum).
C = Organic only (compost).
D = Chemical fertilizer only.
E = Organic + Biofertilizer (A. chroococcum)..
F = Biofertilizer + 20% Chemical fertilizer.
26
G = Biofertilizer (two doses of A. chroococcum ).
The total number of seeds were 420 seeds. All seeds were sowing in 210 pots (d = 20cm,
h = 30cm), these pots were distributed in completely randomized design. There were five
arrows, each one have the 7 treatment (A,B,C,D,E,F,G) distributed randomly, where each
treatment have 6 pots in each arrow.
So 240 seeds were inoculated with A. chroococcum, 60 seeds as control, 60 seeds with
organic, and 60 seeds with chemical fertilizer.
The soil: The basic properties of the soil used for this pot experiment were as follows:
sand = 58.84%, silt = 1.72%, clay = 29.44%, with pH = 7.3, EC = 540 mg/L.
3. Inoculation of the Seeds
The Cucumber seeds were inoculated immediately before sowing, 240 of cucumber seeds
(biofertilizer, organic + biofertilizer, biofertilizer + 20% chemical fertilizer, biofertilizer
(two doses)) were placed in bacterial suspensions for one hour before sowing under
sterilized conditions and then transferred to unsterilized soil, where the other 180 seeds
(control, compost, chemical) were placed in burks media (without sucrose).
The sowing of seeds were at 17-11-2003 and it continue up to the mid of February of
2004. After the plants were harvested, the following data were recorded at flowering
stages and fruiting stage of cucumber plant.
4. The Growth Parameters
The next parameters, plant height (cm), number of branches, stem wet weight (g), root
wet weight (g), stem dry weight (g), root dry weight (g) were measured. Amount of
nitrogen (%) of shoot and root, were measured by automated kjeldahl method.
27
III. Isolation and Characterization of Phoshate solubilising bacteria
Soil sampling
Random sampling method was employed for the collection of soil. Soil samples were
collected from the forest areas of Western Ghats up to a depth of 10 cm. Four sub samples
were taken from each site.
Isolation
The four sub samples collected were mixed thoroughly and 10 gm of the composite soil
was taken separately and serial dilutions were conducted for the isolation of
microorganisms. Serial dilution allows isolation of discrete colonies that can be later sub
cultered. 10-4
and 10-5
dilutions were used for the isolation of phosphate bacteria. Pour
plate method was used for culturing phosphate bacteria and the cultures are grown in
Glucose Peptone Agar medium.
Bio-chemical methods
The bacterial colonies appeared in Petri dishes were subjected to various biochemical
methods for the conformation of phosphate solubilising capacity. The strains, which show
positive activity were maintained on Glucose Peptone Agar medium.
Gram- Staining
Among the strains showing high activity on phosphate solubilisation is subjected to gram
staining for differentiating into gram negative or gram positive.
Biochemical tests for identification
Different biochemical tests were carried out as listed in the Bergy’s Manual of
Determinative Bacteriology (Kreig, 1981) for the confirmation of the bacteria.
Screening for phosphate solubilization
Among the isolated strains, two strains with high phosphate solubilizing capacity are
used for the study along with an authentic culture (Bacillus circulans (MTCC490). The
28
pH values of the liquid medium were adjusted at different levels ranging from 4-10. 100
ml lots of the medium containing 0.5g of tricalcium phosphate were sterilized and
inoculated in duplicate at each pH value with 0.5 ml suspension of each of the three
organisms. Un inoculated controls were also maintained in duplicates at each level of pH
along with the inoculated cultures. All flasks were incubated at 30ºC on a rotary shaker.
The water-soluble phosphate was estimated at 5, 10 &15 days of intervals. The liquid
culture was centrifuged at 5000 rpm to remove insoluble phosphate and bacterial cells.
The pH values of the clear liquid were determined on a pH meter. The water-soluble
phosphate was estimated calorimetrically in a clear liquid by vanadomolybdate method
with a photoelectric calorimeter using 420 mµ filter.
DNA isolation
Bacterial culture (3ml) were taken in tubes and centrifuged at 12000 rpm for 2 minutes.
The supernatant was completely drained off. The pellet was washed with 1000 μl of Tris
HCL. Then the pellet was completely dissolved in 800ml saline EDTA by vortex
shaking. 2 μl of Rnase was added and kept for 30 min. at 37°C. To the above solution,
2μl of lysozyme (10mg/ml) was added and kept for incubation in water bath at 60°C for
15 minutes. Equal amount of Phenol: Chloroform: Isoamyl alcohol was added to the
above mixture and vortexed to get a uniform emulsion. This was centrifuged for 10
minutes at 12000 rpm. Supernatant was transferred carefully to a new eppendroff tube
without disturbing the intermediate layer. The Phenol: Chloroform: Isoamyl alcohol step
was repeated two times. To the supernatant, equal amount of Chloroform: isoamyl
alcohol was added and centrifuged for 10 min. at 12000 rpm. The supernatant was taken
to a new tube and DNA was pelleted by adding 2 volumes of ice-cold ethanol. The tubes
were centrifuged for 5 min and DNA pellet was collected. The Pellets were air dried and
dissolved in TE Buffer.
RAPD - PCR Analysis.
RAPD assay was carried out in 25 μl reaction mixture containing 2.5 μl of 10 X
amplification buffer (100 mM Tris HCl pH-8 at 25oC, 15 mM MgCl2, 500 mM KCl and
1.0% Triton X-100) 0.25 μl of dNTP mixture (10 mM each), 0.75 U of Taq DNA
polymerase (Finzyme, Finland), 20 pmoles (1.0 μl) of 10-mer primer (BioGen, USA) and
50 ng of genomic DNA. TECHNE thermal cycler was used for amplification with
29
the following PCR profile: an initial denaturation for 5 min at 95o
C, followed by
40 cycles of 1 min at 95o C, 1min at 36
o C and 1 min at 72
o C and a final extension
at 72o
C for 5 min. The amplified products were resolved in 1.2% Agarose Gel
containing 0.5mg/ml ethidium bromide and were visualized under UV illuminator.
PCR Amplification of 16S rDNA
16S rDNA was amplified using the primer pair 27F (5/-GAG AGT TTG ATC CTG GCT
CAG-3/) & 1495R (5
/-CTA CGG CTA CCT TGT TAC GA-3
/) (Bangalore Genei).
Amplifications were carried out in 25 µl reaction mixture containing 19.5µl sterile water,
2.5µl of 10x PCR buffer,, 0.25µl of 10mM dNTPs, 0.2µl of each primer, 1.0U of Taq
polymerase (Finnzymes) and 2 µl (100 ng) template DNA. TECHNE DNA thermal
cycler was used with the following PCR profile: an initial denaturation for 1 min at 95o
C, followed by 39 cycles (30 sec. at 94 oC, 30 sec. at 61
oC and 2 min at 72
o C) and a final
extension at 72o C for 5 min.
ARDRA
The amplified rDNA region was subjected to restriction digestion using four restriction
enzymes EcoR1, Bam H1, Hha, Hinf1, (Bangalore Genei). Restricted fragments were
resolved in 3% Agarose Gel and visualized under UV illuminator. Restriction profile of 7
Azotobacter species were compared with each other and bands of DNA fragment were
scored as present (1) or absent (0). The data for all the 4 enzymes were used to estimate
the similarity on the basis of the number of shared products (Nei and Li, 1979). A
dendrogram based on similarity coefficient was generated by the unweighted pair group
method arithmatic means (UPGMA) using NTSys Software.
Analysis of RAPD profile
Amplification profile of 7 Azotobacter species were compared with each other and bands
of DNA fragment were scored as present (1) or absent (0). The data for all the 10
primers were used to estimate the similarity on the basis of the number of shared
amplification products (Nei and Li, 1979). A dendrogram based on similarity coefficient
was generated by the unweighted pair group method arithmatic means (UPGMA) using
NTSys Software.
30
RESULTS
1. Characterization of Azotobacter chroococcum TBG1
The isolated bacteria was characterized by morphological and biochemical tests.
Colonies are moderately slimy, turning black or black-brown on aging as in Fig- 2. The
pigment produced is water-undiffusible.
Fig.2: Colony morphoogy at Burks media, A, morphoogy at new culture, B, old culture
with black-brown pigments
The cells of A. chroococcum TBG1 are gram
negative, pleomorphic, bluntly rod, oval, or coccus
shaped. The cell shape changes dramatically in time
or with changes in growth (medium) conditions.
Cells are often in pairs (fig.3). In motility test, the
bacteria have migrated away from the stab line and
throughout the medium and is motile. The strain is
hydrolysing starch, shown as pouring gram’s iodine
over the growth on the medium, there were a clear zone next to the growth.
Fig.3: Gram negative, cells of A.
chroococcum, are often in pairs
31
2. Statistical Analysis
2.1 Lengths of Cucumber
Table 1 and figure 4 show the mean of the final length of shoot. The mean of the final
length of shoot of chemically treated plants is higher than that of all other treatments. The
mean of B is higher than A, where F is higher than E, C, G and B. The mean difference is
statistically significant in the case of chemical fertilizer treatment (p value = 0.001),
compared to control and not significant in all other treatments.
Table 1: Mean and standard deviation for the final length of shoot.
Treatments Number Mean/cm Standard
deviation
A - control 30 106.70 36.06
B - Biofertilizer (one dose) 30 1.1413 27.89
C - Organic 30 105.00 33.33
D - Chemical 30 135.33 27.56
E - Organic + Biofertilizer 30 110.33 27.17
F - 20% Chemical + Biofertilizer 30 120.63 25.52
G - Biofertilizer (two dose) 30 104.20 32.44
Total 210 113.76 31.52
Figure 4: Mean for the final length of shoot
Table 2 shows the mean of the length of root. The mean of the length of root of
biofertilizer treated plants B is higher than that of all other treatments. The mean of B is
32
higher than all treatments. The mean difference is statistically not significant in the case
of all treatments.
Table 2: Mean and standard deviation for the root length.
Treatments Number Mean/cm Standard
deviation
A - control 30 45.00 15.48
B - Biofertilizer (one dose) 30 52.23 11.93
C - Organic 30 44.0 15.44
D - Chemical 30 43.63 13.05
E - Organic + Biofertilizer 30 43.60 15.47
F - 20% Chemical + Biofertilizer 30 50.60 22.41
G - Biofertilizer (two dose) 30 51.57 18.50
Total 210 47.23 16.55
Figure 5: Mean for the final length of root
2.2 Dry Weights of Cucumber
Table 3 and figure 6 show the means of the weight of dry root. The mean of the dry root
weight of chemically treated plants is higher than that of all other treatments. The mean
of B is higher than A ,and equal to C, F, G, E. The mean difference is statistically
significant in the case of chemical fertilizer treatment (p value = 0.001) and B, C, F
compared to control and not significant in E, G (table 3).
33
Table 3: Mean and standard deviation for the weight of dry root.
Treatments Number Mean/g Standard
deviation
A - control 30 0.60 0.25
B - Biofertilizer (one dose) 30 0.78 0.40
C - Organic 30 0.77 0.36
D - Chemical 30 1.08 0.28
E - Organic + Biofertilizer 30 0.72 0.28
F - 20% Chemical + Biofertilizer 30 0.78 0.24
G - Biofertilizer (two dose) 30 0.78 0.26
Total 210 0.78 0.33
Figure 6 Mean for the weight of dry root
Table 4 and figure 7 show the means of the dry shoot weights. The mean of the dry shoot
weight of chemically treated plants is higher than that of all other treatments. The mean
of B is higher than A, and lower than C, F, E. The mean difference is statistically
significant in the case of chemical fertilizer treatment (p value = 0.001) compared to
control and not significant in all other treatment (table 4).
Table 4 Mean and standard deviation for the weight of dry shoot
Treatments Number Mean/g Standard
deviation
A - control 30 13.4 5.00
B - Biofertilizer (one dose) 30 14.68 6.23
C - Organic 30 16.27 6.23
D - Chemical 30 24.32 5.72
E - Organic + Biofertilizer 30 16.79 5.72
F - 20% Chemical + Biofertilizer 30 16.27 6.55
G - Biofertilizer (two dose) 30 14.99 7.88
Total 210 16.74 6.92
34
Figure 7: Mean for the weight of dry shoot
Table 5 and figure 8 show the means of the dry weights of whole plant. The mean of the
dry weight of whole plant of chemically treated plants is higher than that of all other
treatments . The mean of B is higher than A and G, and the mean of F is higher than B,
C, E, G . The mean difference is statistically significant in the case of chemical fertilizer
treatment (p value = 0.001) compared to control and not significant in all other treatment
(table 5 ).
Table 5: Mean and Standard Deviation for the dry weight of whole plant
Treatments Number Mean/g Standard
deviation
A - control 30 14.69 5.29
B - Biofertilizer (one dose) 30 16.33 6.70
C - Organic 30 17.05 6.36
D - Chemical 30 25.95 5.57
E - Organic + Biofertilizer 30 17.44 5.70
F - 20% Chemical + Biofertilizer 30 18.16 5.93
G - Biofertilizer (two dose) 30 15.63 8.16
Total 210 17.64 7.16
35
Figure 8 Mean for the dry weight of whole plant
2.3 Wet Weights of Cucumber
Table 6 and figure 9 show the means of the wet root weights. The mean of the wet root
weight of chemically treated plants is higher than that of all other treatments. The mean
of B is higher than A and G, E and equal to C, F. The mean difference is statistically
significant in the case of chemical fertilizer treatment (p value = 0.001) compared to
control and not significant in all other treatment (table 6).
Table 6: Mean and standard deviation for the weight of wet root weights
Treatments Number Mean/g Standard
deviation
A - control 30 5.22 1.65
B - Biofertilizer (one dose) 30 6.14 2.55
C - Organic 30 6.21 2.11
D - Chemical 30 8.68 1.86
E - Organic + Biofertilizer 30 5.79 1.98
F - 20% Chemical + Biofertilizer 30 6.05 1.55
G - Biofertilizer (two dose) 30 5.14 2.13
Total 210 6.18 2.26
36
Figure 9 Mean for the weight of wet root
Table 7 and figure 10 show the means of the wet shoot weight. The mean of the wet
shoot weight of chemically treated plants is higher than that of all other treatments . The
mean of B is higher than A, where F is higher than E, C and B. The mean difference is
statistically significant in the case of chemical fertilizer and F treatment (p value = 0.001)
compared to control and not significant in all other treatment (table 7).
Table 7: Mean and standard deviation for the weight of wet shoot.
Treatments Number Mean/g Standard
deviation
A - control 30 111.08 46.40
B - Biofertilizer (one dose) 30 117.07 45.71
C - Organic 30 121.79 48.73
D - Chemical 30 209.15 46.61
E - Organic + Biofertilizer 30 128.07 50.59
F - 20% Chemical + Biofertilizer 30 144.38 48.73
G - Biofertilizer (two dose) 30 110.59 55.01
Total 210 58.67 58.06
37
Figure 10: Mean for the weight of wet shoot
2.4 Different Parameters of Growth of Cucumber.
Through the 2 month of culture, at the first two week the branches are equal in all
treatment, then branches were increased at B, F, E, C, than A, at the end of two month
the higher measurement of branches were at B and D (48 and 46 branches respectively).
Table 8 shows the mean and the standard deviation of number of branches .The mean of
number of branches of chemically treated plants is higher than that of all other
treatments. The mean of B is higher than A (which is the least one), C, F, G and equal to
E. The mean difference is statistically not significant in the case of all treatments.
Table 8: Mean and standard deviation for the number of branches
Treatments Number Mean Standard deviation
A - control 39 18.15 6.95
B - Biofertilizer (one dose) 39 20.08 10.11
C - Organic 39 19.67 10.91
D - Chemical 39 24.26 10.74
E - Organic + Biofertilizer 39 20.00 8.50
F - 20% Chemical + Biofertilizer 39 18.33 8.19
G - Biofertilizer (two dose) 39 19.38 9.29
38
Figure 11: Mean for the number of branches
Table 9 shows the mean of the length of leave. The mean of the length of leave of
chemically treated plants is higher than that of all other treatments. The mean of B, C, E,
F and is higher than A. The mean difference is statistically significant in the case of
chemical fertilizer treatment (p value = 0.001) compared to control and not significant in
all other treatments (table 9).
Table 9: Mean and standard deviation for the length of leaves
Treatments Number Mean Standard deviation
A - control 45 13.62 1.56
B - Biofertilizer (one dose) 45 14.14 1.61
C - Organic 45 14.38 1.51
D - Chemical 45 18.27 3.29
E - Organic + Biofertilizer 45 13.66 2.19
F - 20% Chemical + Biofertilizer 45 13.83 2.02
G - Biofertilizer (two dose) 45 14.40 2.23
Total 315 14.66 2.63
39
Figure 12: Mean for the length of leave.
Table 10 shows the mean of the number of leaves. The mean of the number of leaves of
chemically treated plants is higher than that of all other treatments. The mean of B is
higher than A, F and G, where equal to C. The mean difference is statistically significant
in the case of chemical fertilizer treatment (p value = 0.001), compared to control and not
significant in all other treatments (table 10).
Table 10: Mean and standard deviation for the number of leaves
Treatments Number Mean Standard
deviation
A - control 44 12.39 4.65
B - Biofertilizer (one dose) 44 15.83 6.48
C - Organic 44 15.07 6.40
D - Chemical 44 18.93 6.35
E - Organic + Biofertilizer 44 14.77 6.99
F - 20% Chemical + Biofertilizer 44 13.75 6.05
G - Biofertilizer (two dose) 44 13.63 6.61
Total 308 14.80 6.82
40
Figure 13: Mean for the number of leaves.
2.5 Nitrogen Percentage
Table 11 and figure 14 show means and standard deviations for the shoot nitrogen
percentage. The mean of the shoot nitrogen percentage of 20% chemical and biofertilizer
treated plants is higher than that of all other treatments. The mean of B is higher than A,
C, E, G, where D is higher than B and lower than F. The mean difference is statistically
significant in the case of chemical fertilizer treatment (p value = 0.002), and in the case
of F (p value = 0.001) compared to control and not significant in all other treatments
(table 11).
Table 11: Mean and standard deviation for the shoot nitrogen percentage
Treatments Number Mean Standard deviation
A - control 3 2.00 0,20
B - Biofertilizer (one dose) 3 2.30 0.36
C - Organic 3 2.20 0.20
D - Chemical 3 2.63 0.21
E - Organic + Biofertilizer 3 2.00 0.00
F - 20% Chemical + Biofertilizer 3 2.80 0.10
G - Biofertilizer (two dose) 3 2.06 0.11
Total 21 2.28 0.34
41
Figure 14 Mean for the shoot nitrogen percentage
Table 12 shows mean and standard deviation for the root nitrogen percentage. The mean
of the number of leaves of chemically treated plants is higher than that of all other
treatments. The mean of B is higher than A, C, and equal to E and G, where F is higher
than B. The mean difference is statistically significant in the case of chemical fertilizer
treatment (p value = 0.001) compared to control and not significant in all other treatments
(table 12).
Table 12: Mean and standard deviation for the root nitrogen percentage
Treatments Number Mean Standard deviation
A - control 3 1.2 0.05
B - Biofertilizer (one dose) 3 1.5 0.11
C - Organic 3 1.2 0.20
D - Chemical 3 2.0 0.26
E - Organic + Biofertilizer 3 1.4 0.10
F - 20% Chemical + Biofertilizer 3 1.5 0.17
G - Biofertilizer (two dose) 3 1.4 0.10
Total 21 1.5 0.27
42
Figure 15 Mean for the root nitrogen percentage
2.6 Growth of Cucumber
2.6.1 The Number and Weight of the Last three Collections
As shown in (13), control is the least number and weight, then G which were lower than
the other treatments, where B is higher than A and G, nearly equal E, F, and lower than
C, D, where D is the highest.
Table 13: The number and weight of the last three collections of cucumber
Treatments Number Weight of Cucumber Mean
A - control 90 5000g 55.55
B - Biofertilizer (one dose) 112 6545g 58.43
C - Organic 115 7150g 62.17
D - Chemical 162 10400g 64.20
E - Organic + Biofertilizer 124 7245g 58.42
F - 20% Chemical + Biofertilizer 122 7164g 58.72
G - Biofertilizer (two dose) 106 5834g 55.10
43
Figure 16: The number and weight of the last three collections
2.7 Comparison of the Different Parameters
The next table14 show the different between the means of control and the means of
biofertilizer for different parameters: as showed all means of biofertilizer, 20% chemical
+ biofertilizer mean and compost + biofertilizer mean are higher than the means of
control which show the activity of Azotobacter chroococcum as biofertilizer. As shown
the nitrogen percentage at shoot is the highest at F (20% chem. + bio) where nitrogen
percentage at root at B,F, and E is higher than A. It's clear that the treatments B, E, F, in
most measurements are nearly equal.
Table 14: Comparison of the different parameters means for different
experiments
44
Table 15: Comparison of the different parameters percentage for different
experiments
III. Phosphate Solubilizing Bacteria- Isolation and Characterization
Soil samples were collected from Western Ghats, cultivar lands etc and selectively
isolated phosphate solubilizing bacteria. The two isolates (PSB1 and PSB2) produced a
clear hallow zone around the growth on sperber’s medium which indicated its phosphate
solubilization capacity. These strains were showing promising activity. On gram staining
the two bacterial isolates were appeared as Gram positive and were catalase positive as it
produced gas bubbles on treatment with 3% H2O2. These showed a positive result on
starch hydrolysis by producing a clear zone around the colony. The two isolates produced
Acetyl- carbinol on Voges-Proskaeur broth and changed the color of the medium to dark
red and are scored as positive for Voges-Proskaeur reaction.
On evaluation of effect of pH, maximum decrease in pH was noticed in a pH range of 8-
10 in the 15 days of inoculation. The pH of the medium decreased as a result of the
growth of the organism and thus the maximum growth of the isolates were observed on
the 15 day on a pH range of 8-10.
On phosphate solubilization screening it was observed that the pH of the medium
decreased as a result of inoculation with the organisms. The phosphate solubilization leads
45
to increased acidity. The fall in pH is found to be proportional to increase in the amount of
phosphorus solubilized. In the case of the two bacterial isolates maximum phosphate
solubilization was noticed in the 15th day of inoculation at a pH range of 8-10 and the
authentic culture (Bacillus circulans MTCC490) showed maximum growth in the 15th
day
at a pH of 8.
Figure 17. Effect of phosphate solubilization
PSB1 PSB2
The amount of Soluble P released after the inoculation of the strains at different pH
Soluble 'P' released after inoculation of
the organism at pH 4
0
10
20
30
40
50
60
5th Day 10th Day 15th Day
Days of inoculation
Solu
ble
P (m
g)
PSB 1
PSB 2
MTCC 490
46
Amount of soluble 'P' released after
inoculation of the organism at pH 8
0
10
20
30
40
50
60
70
80
5th Day 10th Day 15th Day
Days of inoculation
Solu
ble
P re
leas
ed (m
g)
PSB 1
PSB 2
MTCC 490
Amount of Soluble 'P' released after
inoculation of the organism at pH 10
0
10
20
30
40
50
60
70
80
5th Day 10th Day 15th Day
Days of inoculation
Solu
ble
P re
leas
ed (m
g)
PSB 1
PSB 2
MTCC 490
47
RAPD Analysis of Azotobacter spp.
10 primers were used for the RAPD analysis and were amplified a total of 52 markers.
Out of them 70% were found to be polymorphic. The levels of polymorphism and the
number of amplicons produced were different with different primers among these
isolates. BGA2, BGA7, BGA8, produced maximum numbers of amplicons and rests of
them were produced comparatively less number of bands. The number of amplified
products from each species varies significantly for most of the primers.
The similarity matrix obtained based on Nei and Lei’s method shows the coefficient of
similarity value ranging from 0.56 to 0.70 with a mean value of 0.635. The observed
value signifies the extent of genetic variation in these isolates. Cluster analysis based on
UPGMA reveals 2 major clusters. Cluster A comprises of 4 isolates (1,6,7,4) and cluster
B comprises of 3 isolates (2,3,5).
Fig18: Dendrogram showing genetic similarity of RAPD data following UPGMA
48
49
ARDRA
The primers used for the 16S rDNA amplification are 27F and 1495R, and the amplified
product was around 1400 bp in length .Four restriction enzymes were used for the RFLP
analysis which produced different banding patterns for different isolates when resolved in
3% Agarose Gel (Plate 2b,c,d,e). Digestion with EcoR1 produced two fragments for the
first four samples (1,2,3,4) and did not produced any fragments for the rest of the three
samples (5,6,7). Digestion with Hinf1 produced different bands with molecular size
ranging from 50 bp to 1100 bp, which clearly shows the difference between the isolates.
HaeIII also produced the similar kind of polymorphic bands as produced by the Hinf1.
restriction digestion with BamH1 produced an undigested pattern with all the seven
isolates.
Fig 18: Dendrogram showing the genetic similarity of ARDRA
50
51
The similarity matrix obtained based on Nei and Lei’s method using the software Ntsys
shows the coefficient of similarity value ranging from 0.44 to 1.00 with a mean value of
0.72. Cluster analysis based on UPGMA reveals 2 major clusters. Cluster A comprises of
4 isolates (1,2,3,4) and cluster B comprises of 3 isolates (5,6,7). Isolates in the cluster B
shows cent percent similarity. Molecular diversity of the strain was evaluated using
RAPD and ARDRA along with 5 other Azotobacter strains viz, A. chroococcum (MTCC
446), A. vinelandii (MTCC 2459), A. vinelandii (MTCC 2460), A. beijerinkiii (MTCC
2641), A. vinelandii MTCC 124) from Microbial Type Culture Collection, (MTCC)
Chandigardh and A. chroococcum (TBG-2) from Vivekanada Institute, Calcutta. It was
noticed that the strain Azotobacter chroococcum (TBG-1) is significantly diverse from
the rest of the strains.
From the results observed it can be concluded that one of the two bacterial isolates have a
high phosphate solubilizing capacity as compared to the authentic culture (MTCC490
Bacillus circulans). Variations were observed among these organisms in solubilizing
insoluble phosphorus. The first isolate (PSB1) was observed to be superior over the
second one in phosphate solubilization. The activities of the organisms, as observed by
the amounts of phosphate solubilization were found to be different at different pH levels.
But the fall in pH is found to be proportional to the increase in the amount of phosphorus
solubilized and each of the organisms examined has different optimum pH value for
maximum solubilization of Phosphate. This indicates that the solubilization depended on
the type of acid produced rather than total acidity. These results indicate that both the
isolated strains are potential in phosphate solubilization and can be utilized for enhancing
the growth of plants.
Molecular diversity of the strain was evaluated using RAPD and ARDRA along with 5
other Azotobacter strains viz, A. chroococcum (MTCC 446), A. vinelandii (MTCC 2459),
A. vinelandii (MTCC 2460), A. beijerinkiii (MTCC 2641), A. vinelandii MTCC 124)
from Microbial Type Culture Collection, (MTCC) Chandigardh and A. chroococcum
(TBG-2) from Vivekanada Institute, Calcutta. It was noticed that the strain Azotobacter
chroococcum (TBG-1) is significantly diverse from the rest of the strains. The 16s rDNA
portion amplified with specific primers has to be sequenced for further BLAST analysis.
52
Publications
S. Shaju, C.N. VishnuPrasad, S. Shiburaj, N.S.Pradeep and T.K. Abraham- “Preliminary
physiological and Molecular studies of a diazotroph–Azotobacter chroococcum TBG-1”
on a National Seminar on’ Microbial diversity, A source of innovation in biotechnology’
organized by Tropical Botanic Garden and Research Institute, Palode,
Thiruvananthapuram, Kerala on 27th –29th May 2004.
S.Shaju, S. Shiburaj and K.Vijayakumar- Effect of pH on Phosphate solubilisation of
selected bacteria from western ghat soil - in the International Conference on
Biotechnology, Biosciences and Biodiversity analysis held at Pune, on 15th-17th
Oct.2005, organized by Modern College of Arts, Science and Commerce.
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