title of the project: agro-industrial development and...

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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


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


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 biofertilizers 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


C - Organic 30 44.0 15.44

D - Chemical 30 43.63 13.05




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


C - Organic 30 0.77 0.36

D - Chemical 30 1.08 0.28




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


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


deviation

A - control 30 13.4 5.00


C - Organic 30 16.27 6.23

D - Chemical 30 24.32 5.72




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


deviation

A - control 30 14.69 5.29


C - Organic 30 17.05 6.36

D - Chemical 30 25.95 5.57




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


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


deviation

A - control 30 5.22 1.65


C - Organic 30 6.21 2.11

D - Chemical 30 8.68 1.86




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.


deviation

A - control 30 111.08 46.40


C - Organic 30 121.79 48.73

D - Chemical 30 209.15 46.61




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


C - Organic 39 19.67 10.91

D - Chemical 39 24.26 10.74




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


A - control 45 13.62 1.56


C - Organic 45 14.38 1.51

D - Chemical 45 18.27 3.29




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


C - Organic 44 15.07 6.40

D - Chemical 44 18.93 6.35




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


A - control 3 2.00 0,20


C - Organic 3 2.20 0.20

D - Chemical 3 2.63 0.21




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


A - control 3 1.2 0.05


C - Organic 3 1.2 0.20

D - Chemical 3 2.0 0.26




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


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


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

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

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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