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2. REVIEW OF LITERATURE

Biodynamic farming is one of the most important types of organic farming system

which is gaining increased popularity from past few years due to the increased emphasis

to maintain long-term fertility in soils, increase crop production, and conserve

environment and ecosystems. The increased use of chemical fertilizers and pesticides has

caused considerable environmental pollution through their production and use, and is thus

undesirable (Speth 1992; Ahmed et al. 2012). The concept of sustainable agriculture

denotes the maintenance of crop productivity at levels necessary to meet the requirements

of ever increasing population, without deteriorating the environment and the natural

resources. Experiencing the adverse effects of synthetic input dependent agriculture, the

concept of organic agriculture is gaining momentum. The review of literature pertinent to

the present studies has been presented under the following headings and subheadings:

2.1 Organic farming

2.1.1 Organic Farming: World Scenario

2.1.2 Organic farming in India

2.1.3 Principles of organic farming

2.1.4 Benefits of organic farming

2.2 Biodynamic farming system

2.2.1Biodynamic Preparations

2.3 Cow horn Manure (BD – 500 Preperation)

2.4 Biocomposting and suppressive composts

2.5 Chemical profile of composts

2.6 Bacteria as a biocontrol agent

2.6.1 Competition for substrates and ecological niche

2.6.2 Siderophores production

2.6.3 HCN production

2.6.4 Lytic enzymes production

i Chitinases

ii Proteases

iii Cellulases

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iv Pectinases

2.7 Plant growth promoting attributes

2.7.1 Nitrogen fixation

2.7.2 Phosphate solubilization

2.7.3 Phytohormones production

2.7.4 Zinc solubilization

2.7.5 Potassium solubilization

2.7.6 Ammonia production

2.8 Microbial Diversity

2.1 Organic farming

Organic farming is an ecologically holistic production management system which

promotes and enhances agro-ecosystem health, including biodiversity, biological cycles,

soil biological activity, and avoids the use of synthetic fertilizers, pesticides, herbicides,

growth regulators and livestock feed additives in agriculture (King 2008). Various inputs

are used in organic farming system which mainly include organic manures viz., farmyard

manure, green manure, crop residues, poultry manure, biogas slurry, vermicompost,

vermiwash, biodynamic composts, biofertilizers and biocontrol agents; and management

practices like composting, crop rotation, green manuring, cover crops, mulching,

multicropping and intercropping that restore, maintain, or enhance the physical, chemical

and biological properties of the soil (Rigby and Caceres 2001; Biao et al. 2003; Ramesh

et al. 2005).

According to International Federation of Organic Agriculture Movements

(IFOAM) the major objectives of organic farming are: producing high quality food in

sufficient quantity in harmony with natural systems and cycles, enhancing biological

cycles within the farming system, maintaining long-term soil fertility and genetic

diversity of the production system and its surroundings, promoting healthy use of water

resources and all life therein, creating harmonious balance between crop production and

animal husbandry, and minimizing all forms of pollution (Rigby and Caceres 2001).

2.1.1 Organic Farming: World Scenario

The demand for organic produce has been steadily growing in recent years due to

increased public concerns about health, environment and food security (King 2008).

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According to the latest survey on organic agriculture carried out between July, 2013 and

February, 2014 by the Research Institute of Organic Agriculture FiBL in cooperation

with the IFOAM, organic agriculture is being practiced in 164 countries across the world

with the total area of 37.5 million hectares managed by 1.9 billion producers. There are

31 million hectares of additional non-agricultural areas which include aquaculture,

forests, and grazing areas. Globally 0.9 per cent of total agricultural land of the world is

under organic farming. About one third of the world’s organic agricultural land (10.8

million hectares) and more than 80 percent (1.6 million) of organic producers are in

developing countries (Willer et al. 2014).

During the period from 1999-2012, the area of organic agricultural land increased

from 11 million hectares to 37.5 million hectares. The global demand for organic

products remains robust and is estimated to be 63.8 billion US Dollars according to

Organic Monitor. Consumer’s demand for organic products is mainly concentrated in

North America and Europe. These two regions comprise of 90 per cent of global

revenues. In 2012, the countries with the largest organic markets were the United States,

Germany, and France. Production of organic foods in other regions, especially in Asia,

Latin America and Africa is mainly export-oriented (Petrescu 2013).

2.1.2 Organic farming in India

India has traditionally been a country of organic agriculture but the growth of

modern farming practices in agriculture has lead to the irrational use of chemical inputs

over the past few decades, which have resulted in the loss of natural habitat balance,

environment degradation, reduced food quality and increased cost of crop cultivation.

However, the increasing awareness about the ill-effects of modern agriculture has lead to

the resurgence of interest in organic agriculture (Reddy 2010; Pandey and Singh 2012).

Currently, in India, 0.5 million hectares area is under certified organic farming

along with 4.70 million hectares area under wild harvest collection and forests managed

by 0.6 million producers. India has global market of Rs. 5000 crores for organic food and

non-food commodities which mainly include rice, pulses, cereals, cotton, tea, coffee,

fruits and vegetables. Currently, Uttar Pradesh has maximum area (2.8 million hectares)

under organic farming followed by Himachal Pradesh (0.9 million hectares) (Willer et al.

2014).

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2.1.3 Principles of organic farming

According to IFOAM, the principles of organic farming are the roots of organic

agriculture and express the contribution that organic agriculture can make to the world

with a vision to improve all agriculture in a global context (King 2008). These principles

of organic agriculture are in contrast with conventional farming systems where individual

issues such as nutrition, pests and diseases control are addressed individually rather than

being a part of a system. There are four principles of organic farming. (i) The principle of

health points out organic farming should sustain and enhance the health of every

component of the ecosystem namely soil, plants, livestock, microorganisms and humans.

(ii) The principle of ecology states that production of organic goods should be based on

ecological processes, and adapted to local conditions. The ecological systems should be

self-contained, self-sustained and self-sufficient and should aim to maintain and improve

environmental quality and conserve natural resources. (iii) The principle of fairness states

that fairness is required in systems of production, distribution and trade of the organic

resources and products. (iv) The principle of care states that organic agriculture should be

managed in a precautionary and responsible manner to protect the health and well-being

of all living creatures along with the environment.

2.1.4 Benefits of organic farming

Organic farming benefits the environment through the protection of wild life

habitats, conservation of landscapes, and reduction of environmental pollution. The use

of organic amendments has been associated with the desirable soil physical and chemical

properties (Drinkwater et al. 1995; Doran and Zeiss 2000; Stockdale et al. 2001). Organic

farming has been reported to change the soil characteristics by increasing its soil organic

matter, organic carbon content, size and stability of aggregates, water retention and

decreasing soil bulk density (Herencia et al. 2008). Long-term comparisons between

conventional and organic farms have found that organic methods improve the fertility and

overall health of the soil (Mader et al. 2002; Fliessbach et al. 2007; Birkhofer et al. 2008).

Furthermore, investigations to evaluate the effect of fertilization showed that in

farming systems with regular application of organic manure, higher concentrations of soil

microbial biomass and diversity were observed in comparison to the systems where

mineral fertilizers were used (Lundquist et al. 1999; Mader et al. 2002; Hartmann et al.

2006; Widmer et al. 2006). Microorganisms are key players in biogeochemical cycles of

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various nutrients which are vitally important for plant nutrition (Grayston et al. 1998;

Forge et al. 2003; Bohme et al. 2005) and contribute significantly towards the formation

of soil aggregates as well as the degradation of pesticides (Six et al. 1998). In addition,

many studies have reported a stimulation of microbial activity, diversity and biomass in

organic management systems (Fliessbach and Mader 2000; Gracia-Gil et al. 2000;

Esperschutz et al. 2007).

2.2 Biodynamic farming system

Organic farming comprises of several components of which biodynamic farming

system is a very popular area for researchers worldwide. Biodynamic farming is an

important method of organic farming system that treats the farm as a unified self-

nourishing system and emphasis on its holistic development which involves their

interrelationship between all the components of the farm viz. soil, plants, animals,

microbes. The philosophy of biodynamic farming is to feed the farm rather than crops to

maintain soil health and increase crop production (Reganold 1995; Spaccini et al. 2012;

Ponzio et al. 2013). Biodynamic farming system originated in 1924 in Germany in

response to the concerns from farmers about the deteriorating soil health, resulting from

the introduction of the synthetic chemical fertilizers in agriculture at the turn of the

twentieth century. Dr. Rudolf Steiner, the Austrian philosopher from Central Europe was

the founder of this form of farming. In his series of eight lectures known as the

“Agricultural Course” presented at Koberwitz in 1924, Steiner taught that a farm should

be able to create and maintain everything needed to stay healthy and fruitful, and should

not include the use of synthetic chemical fertilizers to enhance the crop productivity

(Droogers and Bouma 1996; Turinek et al. 2009). Since then biodynamic farming has

developed as one of the most sustainable forms of organic agriculture practiced in many

countries across the world (Shiming and Sauerborn 2006).

Biodynamic system is a combination of "biological" practices which include well-

known organic farming techniques that improve soil health, and “dynamic” practices

which involve the influence of cosmic forces to enrich the farm, its inhabitants and

products with energy (Sharma 2012). In Biodynamic farming self-sufficiency in balance

with the entire ecosystem is practiced, as opposed to simply maximizing yields in farms

with mechanical and technological inputs which result in unlimited exploitation of the

earth’s resources (Paull 2011; Ponzio et al. 2013). This contributes holistically towards

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improving soil health, soil fertility, biodiversity, nutrient recycling, food quality and

environmental protection which results in maintaining long-term sustainability and self-

sufficiency in biodynamic farms along with the increased consumer’s acceptance towards

the biodynamically grown foods (Happerly et al. 2006; Turinek et al. 2009).

Biodynamic farming involves all principles and techniques of organic farming

system but is set apart from other organic farming systems on the basis of use of nine

fermented herbal and mineral preparations which are used as compost additives and field

sprays (Reganold et al. 1993; Drinkwater et al. 1998; Zaller and Kopke 2004).

Biodynamic farming practices also follow the astronomical calendar for sowing and

planting of crops and for the application of biodynamic preparations in the fields to

enhance plant and crop productivity (Zaller and Kopke 2004; Ponzio et al. 2013).

2.2.1 Biodynamic Preparations

A distinguishing feature of biodynamic farming is the use of nine BD preparations

for the purpose of enhancing soil quality and stimulating plant growth promotion. These

consist of either mineral, plant extracts or animal manure, usually fermented and applied

in very small proportions to composts, soils, or directly onto plants (Reeve et al. 2011).

The BD 500 preparation also known as cow horn manure is made from cow dung

fermented in a cow horn which is buried in soil for six months during the winter season

and is used as a foliar and soil spray to stimulate plant growth and humus formation

(Reeve et al. 2010; Spacanni et al. 2012) (Table 2.1). The BD 501 preparation known as

horn-silica is made from powdered quartz which is packed inside a cow horn and buried

in the soil for six months during summer season, and is applied as a field spray to

stimulate plant growth in a concentration of 3 g per hectare soil (Bacchus 2010).

The next six biodynamic preparations viz., BD 502-507 are used as compost

additives for preparing the composts. The preparation BD 502 is prepared by stuffing

moistened Yarrow blossoms (Achillea millefolium) into the urinary bladder of red deer

(Cervus elaphus). The bladder is hung in Sun during summer season, then buried in earth

during winter season and retrieved during the spring season. BD 503 is prepared by

stuffing moistened Chamomile blossoms (Matricaria recutita) into the small intestine of

cow and is buried in humus-rich soil in the autumn and withdrawn during the spring

season. BD 504 is prepared by burying stinging nettle (Urtica dioica) plants in soil for 1

year, enclosed in a mantle of peat moss. The preparation aids in the process of

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humification of compost. BD 505 is prepared by placing the scrapings of the outer rind of

oak bark (Quercus robur) in the skull cavity of domesticated animal and is buried in earth

in a place where rain water percolates. BD 506 is prepared by stuffing dried flowers of

dandelion (Taraxacum officinale) into the peritoneum of cow; and buried in soil during

winter and withdrawn during spring season. The preparation BD 507 is prepared by

extracting the juice of Valerian flowers (Valeriana officinalis) and diluting in rain water.

These six preparations are then sprayed over the compost piles to hasten the process of

composting (Carpenter-Boggs et al. 2000; Zaller and Kopke 2004; Turinek et al. 2009;

Reeve et al. 2010; Reeve et al. 2011). Finally, BD 508 preparation made from silica-rich

horsetail plant (Equisetum arvense) is used as foliar spray to suppress fungal diseases in

plants (Reeve et al. 2011). These preparations are used in very low doses and are reported

to improve soil quality, stimulate nutrient cycling, enhance crop productivity and hasten

the process of composting (Carpenter-Boggs et al. 2000; Zaller and Kopke, 2004; Reeve

et al. 2005).

Table 2.1. Preparations used in biodynamic farming and their main ingredients and

agricultural uses (Reeve et al. 2011):

Preparation Main Ingredient Application

BD-500 Cow manure Field spray

BD-501 Silica Field spray

BD-502 Yarrow flowers (Achillea millefolium L.) Compost additive

BD-503 Chamomile flowers (Matricaria recutita L.) Compost additive

BD-504 Stinging nettle shoots (Urtica dioica L.) Compost additive

BD-505 Oak bark (Quercus robur L.) Compost additive

BD-506 Dandelion flowers (Taraxacum officinale Web.) Compost additive

BD-507 Valerian extract (Valeriana officinalis L.) Compost additive

BD-508 Horsetial (Equisetum arvese L.) Field spray

Studies by Carpenter-Boggs et al. (2000) demonstrated that biodynamically

treated composts maintained a significantly higher temperature throughout the

composting period and resulted in faster development of compost. The finished

biodynamic compost contained 65 per cent more nitrate, and higher dehydrogenase

enzyme activity as compared to untreated compost. A narrower C:N ratio, more nitrates

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and greater cation exchange capacity in finished BD composts was also reported by

Heinze and Breda (1978). Lei and Vander-Gheynst (2000) reported that the microbial

inoculants present in biodynamic compost additives speed up the process of composting.

2.3 Cow horn Manure (BD-500 Preparation)

Cow horn manure or BD-500 preparation is the most promising and widely used

biodynamic preparation in organic farming system. The preparation is applied as a field

and foliar spray at a concentration of 150 g dissolved in 15 L water per hectare soil and is

reported to show a significant improvement in soil quality and crop productivity (Mader

et al. 2002; Reeve et al. 2011; Spaccini et al. 2012).

Brinton (1998) prepared the manure in a similar fashion by putting cow dung

inside the artificial horns made of glass and observed that the manure did not undergo

any remarkable change in its appearance after its maturation of 6 months. The final

mature manure appeared more similar to the initial cow dung that was put inside the

horns. In another experiment, horse dung was put inside the cow horns and remarkable

changes in the appearance of the final ripened manure were observed. These changes

were similar to those observed in the case of cow horn manure prepared by putting cow

dung inside the cow horns. Brinton (1998) found in his studies that 6 months mature

samples of cow horn manure retained more than 90 per cent of N. It was also reported

that BD-500 preparation prepared after 6 months of maturation had lower values of pH,

CO2 respiration, C:N ratio, higher nitrate content and reduced losses of organic matter.

Karthikeyan et al. (2013) conducted the physiochemical analysis of cow horn manure and

reported C (71.2%), N (3.84%), P (0.06%) and C/N ratio (18.5%) in the manure. Matteo

et al. (2013) studied the chemical changes occurring in cow horn manure during its

maturation and found stable C:N ratio in the mature samples of cow horn manure.

Reeve et al. (2005) observed that winegrapes grown in biodynamically managed

plots had significantly higher degree brix, total phenol and total anthocyanin contents

than control plots where no fertilization was done. Jogergensen et al. (2010) reported that

the application of farmyard manure, bio-dynamic manures and bio-organic manures led

to an increased accumulation of bacterial residues in the soil cultivated with wheat as

compared to the inorganic fertilizers. The contents of microbial biomass C, adenylates,

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ATP, glucosamine, muramic acid, and galactosamine were highest in the bio-dynamic

manure treatment and lowest in the treatment with inorganic fertilizers.

Rupela et al. (2003) isolated a total of 17 bacterial isolates from BD 500, BD 502-

506 showing antagonistic activity against the phtopathogenic fungi Rhizoctonia

bataticola, Sclerotium rolfsii, Fusarium oxysporum and Aspergillus flavus and reported

that a high population of antagonists present in biodynamic preparations resulted in

reduced incidence of diseases in plants. Similarly, the study by Valdez and Fernandez

(2008) showed that the application of BD 500, 501 and 508 resulted in the disappearance

of tungro symptom present in the rice crops and the recovery of crop vitality.

Reeve et al. (2011) reported that wheat seedlings receiving 1 per cent BD-treated

compost extract had similar root and shoot biomass as the fertilized seedlings and was

significantly higher than the untreated compost. Similarly, the use of BD sprays 500, 501,

and 508 was correlated with higher yield of lentil (Lens culinaris) per unit plant biomass,

higher nitirate content in soft white spring wheat (Triticum aestivum L.), and greater

ammonia concentration in soil (Carpenter-Boggs et al. 2000).

Valdez and Fernandez (2008) reported higher root length and root biomass in rice

in biodynamic farming system in comparison with the organic and conventional farming

systems. After cropping, available soil P increased by 20 and 18 per cent in biodynamic

and organic farming system, respectively. The available P content in conventional and

control treatments decreases significantly from the initial values after cropping. The high

level of available P in biodynamic farming system was attributed to the higher microbial

activity in this type of farming system. Colmenares and Miguel (1999) found that the BD

preparations, sprayed on permanent grassland in Spain over 3.5 years, increased dry

matter content in the absence of any fertilization.

Tung and Fernandez (2007) conducted experiments on soybeans under organic,

biodynamic and chemical production methods and observed significantly higher increase

in soil organic matter, earthworm population, seed yields, quality and net returns in

organically and biodynamically managed soils than conventionally managed soils.

Sharma et al. (2012) found that the application of BD 500 and BD 501 in

combination with farmyard manure gave significantly higher yield and harvest index in

cumin in comparison with other treatments and control. Similar results have been

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reported by Jayasree and George (2006) who showed that the application of BD 500 and

BD 501 preparations in chilli by adopting biodynamic calendar resulted in better fruit

quality.

Ryan and Ash (1999) reported that plants in the biodynamic soils had a higher

level of colonisation by VAM fungi than the soils managed through conventional

practices.

Droogers and Bouma (1996) compared soils of biodynamic and conventional

farms, where each farming practice has been applied for at least 70 years and found

significant differences in soil organic matter content, water availability, microbial activity

and biomass, better soil structure and lower soil density in favour of biodynamic soils. In

DOK (biodynamic, organic and conventional) trial, the ratio between yield levels and

nitrogen applied turns out to be highest in biodynamic and organic farming systems,

when compared with conventional farming systems, thereby, indicating more efficient

use of the nitrogen supplied in biodynamic and organic farming systems (Mader et al.

2002). Studies have also shown that long term biodynamic cultivation resulted in higher

soil organic matter levels, soil microbial biomass and soil biological activities than other

farming practices (Gracia 1989; Mader et al. 1993; Reganold et al. 1993; Droogers and

Bouma 1996; Scheller and Raupp 2005; Fliessbach et al. 2007).

Studies have found plant growth stimulatory hormones, auxins and cytokinins in

BD preparation 500 (Goldstein and Koepf 1982). Deffune and Scolfield (1995) found

that the humic acid extracted from BD 500, BD 505 and BD 507 caused a positive

growth response in wheat seedlings relative to control.

Spaccini et al. (2012) characterized the molecular composition of BD 500 by solid

state nuclear magnetic resonance and found polysaccharides, alkyl and aromatic

compounds of microbial origin in the manure, thus suggesting the active role of

microorganisms behind the working of the manure in very low doses. It was further

suggested that the presence of active microflora in the manure makes it potentially

conducive for biostimulation of soil microflora and plants.

Matteo et al. (2013) studied the microbiological features and the bioactivity of BD

500 preparations and found the dominance of Gram positive bacteria in the manure. The

manure also exhibited a strong auxin-like effect on plants and exhibited elevated values

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of enzymes viz., alkaline phosphates, chitinase, esterase and β-glucosidase. The presence

of microorganisms in the manure results in its activity as a biostimulant in the soil.

Unlike normal composts which are added in bulk in the soil, cow horn manure is

added at a very low rate in soil. This amount of manure is not sufficient to act as a

nutrient supplement in the soil but is thought to stimulate the processes of nutrient and

energy cycling in soil through microbial activity (Zaller and Kopke 2004). Reeve et al.

(2010) reported that biodynamic preparations are mainly effective microbial inoculants

which stimulate the process of nutrient cycling and nutrient transformation in soil and

thus lead to the greater availability of essential nutrients to the plants. However, the role

of microorganisms in showing plant growth promoting attributes and antagonistic activity

towards the plant pathogens has not yet been studied extensively.

2.4 Biocomposting and suppressive composts

Composting involves the biological decomposition of organic matter by

microorganisms under aerobic conditions to produce the final humus like product.

Residues such as yard waste, manure, sewage-sludge, municipal solid waste or industrial

wastes are recycled through composting. Hence, compost is the final product of an

extensive process of biological decomposition of organic residues with increased

bioavailability of nutrients and reduced pathogen presence. These composts besides

serving as a source of nutrients to plants also have the potential to provide biological

control of several plant diseases (Moreira et al. 2013).

Several microorganisms antagonistic to plant pathogens have also been isolated

from composts, thereby, suggesting that these microorganisms are responsible for the

suppressive activity of the composts (Brandon et al. 2008; Cayuela et al. 2008; Aviles et

al. 2011). The suppressive activity of microorganisms may be attributed to their ability to

degrade polymers through the production of extracellular enzymes. Their presence in the

finished product is important as it reflects the compost quality, and its properties as a

nutrient supplier and pathogen suppressor (Hadar and Papadopoulou 2012). Since cow

horn manure is applied at a very low dose in fields, so its microbial diversity gains

significant importance in having its role in antagonism towards the plant pathogens and

in increasing plant growth (Matteo et al. 2013).

The system of composting used, composition of raw material and the composting

environment affect the species richness, diversity and therefore, the degree and spectrum

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of suppressive effect (Hoitink et al. 1993). The composition of microorganisms in

composts is affected by the chemistry of the materials from which the compost is

prepared (Castano et al. 2011). The ligno-cellulosic material in the compost affect the

duration of the composting process and the microbial diversity present in the compost

(Hoitink and Fahy 1986). Composts with high lignocellulosic substances are mostly

colonized by Trichoderma spp. In contrast, grape pomace with low cellulosic substances

and high sugars become colonized by Penicillium spp. and Aspergillus spp. (Kuter et al.

1983; Gorodecki and Hadar 1990). In addition, different species of Bacillus being highly

competitive and spore-forming in nature have been reported to be present in many

composts (Chandna et al. 2013; Moreira et al. 2013).

The characteristic succession of microbial communities occurring during the

compost’s life cycle is also directly related to the suppression phenomenon (Aviles et al.

2011). A low concentration of readily available nutrients during the later stages of the

compost maturation increases the competition among the microorganisms for nutrients

and causes them to secrete lytic and degradative enzymes for the lysis of complex organic

material left out during composting (Mandelbaum et al. 1988). These lytic enzymes in

turn check the growth of fungal plant pathogens, thereby reducing the disease incidence

caused by them (Yogev et al. 2006; Danon et al. 2007). Reduction of plant diseases is

also related to the levels of maturity of composts. Hadar and Mandelbaum (1986) showed

that the immature compost could not suppress the damping-off in cucumber seedlings

caused by P.aphanidermatum, while mature compost could. Similar results were obtained

in case of R. solani, where fresh undecomposed organic matter could not exert

suppressive effect on the pathogen, while the growth of the fungal pathogen decreased

significantly with the maturity of the compost (Trillas et al. 2006). A link between the

proliferation of chitin-degrading microorganisms and suppression of fungal pathogens

was reported by Chae et al. (2006).

Chen et al. (1988) suggested that the competitive microorganisms present in

compost when added to the soil compete with the plant pathogens for nutrients and space,

and hinder the growth of the pathogens. The addition of composts to soil also alters the

soil microbiota. Zaccardelli et al. (2013) found that the application of compost prepared

from the municipal solid wastes to the soil showed a substantial increase in the number of

spore forming bacteria in the soil. Approximately 80 per cent of these bacteria were able

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to inhibit the growth of the fungal plant pathogens R. solani, F. oxysporum, F. solani,

Sclerotinia minor and Pyrenochaeta lycopersici. The application of the compost to the

soil can change the composition of soil microbial community, thereby, producing a

decrease in the growth and activity of plant pathogens. Similar results were found by

Alvarez et al. (1995) who also reported the addition of compost to soil the decreased

incidence of rhizobacteria in exhibiting antagonism towards F. oxysporum, P. lycopersici,

P. ultimum and R. solani. These findings suggest that the composts stimulate the

antagonists in the rhizosphere.

2.5 Chemical profile of composts

The chemical profile determines the composition and activity of the microbial

diversity in the compost and its succession upon the maturation of compost. The quality

of compost mainly depends on the level of organic matter stability (Wu et al. 2000). The

application of non-stabilized organic materials in soil could affect both crops and the

environment because of the presence of phytotoxic compounds (Butler et al. 2001).

During composting the most biodegradable organic compounds are broken down and part

of the remaining organic material is converted into humic-like substances (Hsu and Lo

1999). Of many elements required for microbial decomposition, C and N are the most

important. C acts as both energy source and the basic building block for microorganisms,

making up about 50 percent of the mass of microbial cells. N is a crucial component of

the proteins, nucleic acids, amino acids, enzymes and co-enzymes necessary for cell

growth and function. The ideal C:N ratio for composting is generally considered to be

around 30:1. As composting proceeds, the C:N ratio gradually decreases from 30:1 to 10-

20:1 for the finished product (Wu and Ma 2002; Brito et al. 2008).

A pH between 5.5 and 8.5 is optimal for compost microorganisms. As bacteria

and fungi digest organic matter, they release organic acids. In the early stages of

composting, these acids often accumulate. The resulting drop in pH restricts the bacterial

growth and encourages the growth of fungi and the breakdown of lignin and cellulose

(Brandon et al. 2008). Blaker and MacDonald (1983) showed that the majority of

Phytophthora root rot diseases are inhibited by the low pH of the compost. Spencer and

Benson (1981) showed that low pH of pine bark compost reduced sporangium formation,

zoospore release and motility in Phytophthora citricola and Pythium cinnamomi in Acuba

japonica.

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Moisture content of compost is also a very important factor in the proliferation of

the microorganisms and breakdown of the complex nutrients present in the compost

(Arora et al. 2005). Moisture content of the compost is also responsible for inducing

disease suppression. A negative water potential inhibits zoospore release from the

sporangia of several Phytophtora spp. (Wilcox and Mircetich 1985).

Furthermore, the presence of micronutrients such as calcium, magnesium,

manganese, iron, sulphur, copper and zinc in the compost are required for the growth of

microorganisms present in the manure. These micronutrients serve as growth inducers

and cofactors in several enzymatic processes occurring within the microbial cells.

Composts when added to soils serve as micronutrient supplement to the plants and help in

plant growth (Hargreaves et al. 2008).

2.6 Bacteria as a biocontrol agent

Pathogenic microorganisms affecting plant growth are a major and chronic threat

to food production and ecosystem stability worldwide. As agricultural production

intensified over the past few decades, producers became more and more dependent on

agrochemicals. However, increasing use of chemical inputs causes several negative

effects such as soil and water pollution, environmental degradation and harmful effects

on human health. Further, agrochemicals accumulate in the soil, leach-off to the water

bodies and cause environmental pollution and can enter the food chain (Murata and Goh

1997; Shoda 2000). Additionally the growing cost of pesticides, particularly in less-

affluent regions of the world, and the consumer’s demand for pesticide-free food has led

to a search for substitutes for these products (Costa et al. 2006). Worldwide biological

control via microorganisms which have the ability to suppress plant pathogenic fungi and

insect pests are potentially important alternatives to chemical pesticides and are gaining

attention (Macagnan et al. 2008). The widely recognized mechanisms of biocontrol

mediated by microorganisms are competition for an ecological niche or substrates,

competition for ferric ions, production of inhibitory substances and lytic enzymes and

induced systemic resistance (ISR) in host plants (Gomes et al. 2001). Different

mechanisms or combination of mechanisms may be involved in the suppression of

different plant diseases (Whipps 2001). Antagonism towards other microorganisms is the

survival advantage to the bacteria in highly competitive but resource-limited

environments. Several biocontrol agents like Agrobacterium, Alcaligenes, Arthrobacter,

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Bacillus, Burkholderia, Collimonas, Pantoea, Pseudomonas, Serratia, Stenotrophomonas

and Streptomyces have been reported (Raaijmakers and Mazzola 2012).

2.6.1 Competition for substrates and ecological niche

Competition for nutrients and space between the microbial antagonist and the

plant pathogen is considered as a major mode of action by which microbial antagonists

suppress phytopathogens (Sharma et al. 2009). Disease suppression based on competition

is related to the availability and rate of utilization of nutrient and energy sources by the

microorganisms. This nutrient sink restricts the further growth, development and

reproduction of the pathogen (Whipps 2001).

Competition for C source was suggested as mechanism of suppression of P.

aphanidermatum. The fungi could not grow in the presence of microbial antagonists and

consequently the disease could not develop (Mandelbaum and Hadar 1990). Similarly, F.

oxysporum is also susceptible to competition for nutrients, and is controlled by bacteria

competing for nutrients and space present in the composts (Alabouvette et al. 2006).

Addition of compost to soil rendered soil suppressive to Fusarium wilt of flax caused by

F. oxysporum (Serra-Wittling et al. 1996).

A non-pathogenic F. oxysporum strain, designated F2, isolated from a suppressive

compost, was reported to reduce Verticillium wilt in eggplant under greenhouse and field

conditions. F2 was shown to colonize the root surface along the intercellular junctions,

excluding V. dahliae from that ecological niche. It was suggested that competition for

space or nutrients on the root surface was the main mechanism of action of F2 against V.

dahliae (Pantelides et al. 2009).

2.6.2 Siderophores production

Iron (Fe) is an essential plant micronutrient and it serves as a cofactor of many

enzymes present in plants. A large portion of Fe is present in soil in the form of highly

insoluble ferric hydroxide. Therefore, Fe acts as a limiting factor for plant growth even in

Fe rich soils. Under Fe-limiting conditions, bacteria secrete siderophores which are low

molecular weight, ferric ion specific chelating compounds (Neilands 1981). Siderophores

can be defined as small peptidic molecules containing side chains and functional groups

that can provide a high-affinity set of ligands to coordinate ferric ions (Crosa and Walsh

2002). Siderophores are secreted to solubilize Fe from their surrounding environments,

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forming a complex ferric-siderophore that can move by diffusion and be returned to the

cell surface (Andrews et al. 2003).

In most of fungi, Fe uptake is essential for viability and under Fe-limiting

conditions, the bacterial antagonists secrete siderophores which sequester the limited

supply of iron available in the rhizosphere, making it unavailable to the pathogenic fungi,

thereby restricting their growth (Haas and Defago 2005). Microbial siderophores enhance

Fe uptake by plants that are able to recognize the bacterial ferric-siderophore complex

(Kloepper et al. 1980; Katiyar and Goel 2004; Dimkpa et al. 2009) and thus plays an

important role in plant growth promotion.

A pseudobactin siderophore produced by P. putida B10 strain was also able to

suppress F. oxysporum in soil deficient in Fe; this suppression was lost when the soil was

replenished with Fe, a condition that represses the production of Fe chelators by

microorganisms (Kloepper et al. 1980). Boopathi and Rao (1999) reported that plant-

beneficial flourescent Pseudomonas isolated from chickpea rhizosphere showed

production of siderophores. The purified compound exhibited siderophore activity for P.

putida and antifungal activity on phytopathogens. Chakraborthy et al. (2006) isolated B.

megaterium from tea rhizosphere and tested its abilities to promote growth and disease

reduction in tea plants. It was concluded that one of the reasons of plant growth

promotion and disease suppression was the production of siderophores.

2.6.3 HCN production

Bacteria produce a wealth of volatile compounds mainly HCN which are

responsible for limiting the fungal growth and contribute towards the antagonism

(Rezzonico et al. 2007). HCN is a volatile, secondary metabolite produced by bacteria

that suppresses the development of microorganisms as it is a powerful inhibitor of many

metal enzymes, especially copper containing cytochrome C oxidases (Siddiqui et

al. 2006). HCN inhibits the electron transport as a result of which energy supply to the

cell is disrupted leading to the death of the organisms. HCN is formed from glycine

through the action of HCN synthetase enzyme, which is associated with the plasma

membrane of certain bacteria (Blumer and Haas 2000). Although cyanide acts as a

general metabolic inhibitor, it is synthesized, excreted and metabolized by hundreds of

organisms, including bacteria, algae, fungi, plants, and insects, as a mean to avoid

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20

predation or competition. HCN is produced by many bacteria and is postulated to have a

role in biological control of pathogens (Defago et al. 1990). Many different bacterial

genera have shown to be capable of producing HCN, including species of Alcaligenes,

Aeromonas, Bacillus, Pseudomonas and Rhizobium (Voisard et al. 1989; Ahmad et

al. 2008).

HCN production is a common trait within the group of Bacillus and

Pseudomonas, with some studies showing that about 50 per cent of pseudomonads

isolated from potato and wheat rhizosphere were able to produce HCN in-vitro (Bakker

and Schippers 1987; Schippers et al. 1990). Suppression of black root rot of tobacco

(Stutz et al. 1986) and take-all of wheat (Defago et al. 1990) by P. fluorescens strain was

attributed to the production of HCN. Ramettee et al. (2003) reported that HCN is a

broadspectrum antimicrobial compound involved in biological control of root disease by

many plant associated fluorescent pseudomonads.

2.6.4 Lytic enzymes production

Lytic enzymes such as proteases, cellulases, chitinases and pectinases are

involved in the biocontrol of plant pathogens (Dunn et al. 1997; Gajera et al. 2013).

Bacteria inhibit the growth of disease-causing fungi by the production of enzymes which

cause lysis of fungal cell wall. Some enzyme producing bacteria are able to destroy

oospores of phytopathogenic fungi (El-Tarabily 2006) and affect the spore germination

and germ-tube elongation of phytopathogenic fungi (Sneh et al. 1984). Hence, the

production of extracellular cell-wall degrading enzymes has been associated with

biocontrol abilities of the producing bacteria (Valois et al. 1996; El-Tarabily 2006). Apart

from having a role in biocontrol of fungal plant pathogens, these lytic enzymes have

tremendous industrial application.

(i) Chitinases

Chitinases are a group of antifungal enzymes that catalyze the hydrolytic cleavage

of the β- 1,4-glycoside bond present in the biopolymers of N- acetyl-D-glucosamine

present in chitin which is a component of fungal cell wall (Cohen and Chet 1998;

Taechowisan et al. 2003). Bacteria produce chitinases to digest chitin primarily to utilize

it as a source of energy and C (de-Boer et al. 2001; Van-Nguyen et al. 2008). Several

bacterial strains such as Aeromonas caviae, Bacillus thuringiensis, Serratia plymuthica,

Enterobacter agglomerans and actinomycetes are well-known for their chitinolytic

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21

activity (Ordentlich et al. 1988; Roberts and Selitrennikoff 1988; Inbar and Chet 1991;

Driss et al. 2005; Das et al. 2010).

A positive relationship was observed between chitinase production and the

antifungal activity of chitinolytic P. fluorescens isolates (Velazhahan et al. 1999). Tn5

mutants of one of the Enterobacter deficient in chitinolytic activity were unable to protect

plants against the fungal disease (Chernin et al. 1995). The chitinoytic enzymes produced

by Bacillus cereus and Pantoea agglomerans were involved in the biocontrol of

Rhizoctonia solani (Chernin et al. 1995; Pleban et al. 1997). Bhushan (2000) found the

chitinase activity of 76 U mL-1

by Bacillus sp.BG-11 in liquid batch isolated from the

alkalophilic environment after 72 h of incubation at 50o C. Agrawal and Kotsthane (2012)

found exochitinase activity of Trichoderma isolate in the range of 0.62 – 32.6 x 10-3

U

mL-1

in colloidal chitin supplemented broth.

(ii) Proteases

Proteolytic enzymes or proteases are the group of enzymes whose catalytic

function is to hydrolyze proteins. Proteases form a large group of enzymes, ubiquitous in

nature and are found in a wide variety of microorganisms. Proteases are the most

important group of enzymes produced commercially and are used in detergent, protein,

brewing, meat, leather and dairy industries (Rao et al. 1998). In recent years, there is

renewed interest in the study of proteolytic enzymes, mainly due to the recognition that

these enzymes play an important role in biocontrol of fungal plant pathogens, since, fungi

consist of proteins in their cell wall besides chitin (Hunsley and Burnett 1970). Proteases

may therefore play a significant role in the fungal cell-wall lysis and are also involved in

the competition for protein substrates with the fungal pathogens (Geremia et al. 1993;

Elad and Kapat 1999).

Inactivation of enzymes of pathogens by means of proteases produced by

antagonistic microorganisms has been reported (Rodriguez-Kabana et al. 1978).

Radjacommare et al. (2010) showed that the two strains of T.viridae produced

extracellular proteases and chitinases, and caused disruption in the cell wall and

membrane structure of the fungal plant pathogen. Several Bacillus species have been

reported to be involved in protease production viz., B. cereus, B. sterothermophilus, B.

mojavensis, B. megaterium and B. subtilis (Beg and Gupta 2003; Greze et al. 2005;

Soares et al. 2005). Elad and Kapat (1999) proved that the protease-containing culture

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22

liquid of Trichoderma reduced germination and germ tube length of the pathogen Botrytis

cinerea, thereby, suggesting the involvement of proteases in biocontrol.

(iii) Cellulases

The cell wall of fungi contains considerable portion of cellulosic microfibrils in

their hyphae which make them susceptible to the enzymatic destruction by cellulases

secreted by antagonistic bacterial isolates (Downer et al. 2001). These cellulosic

structural polymers in the cell-wall of fungi maintain rigidity and confer protection to

fungi which is generally achieved through the assistance of other cell- wall components,

most commonly chitin. These cellulosic components of fungal cell wall are susceptible to

the degradation by cellulase enzymes secreted by antagonistic microorganisms which

hydrolyze the β-1-4 glycosidic linkages in cellulose polymers (Pitson et al. 1993). The

production of cellulose-degrading enzymes is a characteristic attributed to a wide variety

of microorganisms, such as aerobic and anaerobic bacteria, and fungi. Besides the role in

biocontrol, cellulases have direct implication in food processing, textile, paper and

pharmaceutical industries. Furthermore, cellulase producing microorganisms are required

for bioethanol production from cellulosic raw materials (Lynd et al. 2002).

The biocontrol of Phytophthora cinnamomi root rot of Banksia grandis was

obtained using a cellulase-producing isolate of Micromonospora carbonacea (El-Tarabily

et al. 1996). The actinomycetes isolate producing β-1,3-, β-1-4- and β-1-6- glucanase

showed the suppression of raspberry root rot caused by Phytophthora fragariae (Valois et

al. 1996). Migheli et al. (1998) showed that the transformants of Trichoderma

longibrachiatum overexpressing the β-1,4-endoglucanase Gene egl1 had enhanced

biocontrol of Pythium ultimum on cucumber. Similarly, bean seed colonization and

protection of bean seedlings against Pythium splendens by Trichoderma koningii were

related to high levels of cellulase activity in the biocontrol agent. Ariffin et al. (2006)

showed the cellullase activity of 0.53 U mL-1

by cellulose degrading isolate of B. pumilis.

(iv) Pectinases

The fungal cell wall contains few pectin compounds incorporated in their cell

walls which makes them susceptible to the action by pectinase enzymes which cleaves

the α -1- 4 linked glycosidic bond of pectic acids or polygalacturonates, producing mono-

, di-, and oligo- galacturonates as final hydrolysis products. When the integrity of the

fungal cell wall is disrupted, the cell lyses and collapses. Hence, the production of

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pectinase enzymes by the antagonistic bacterial isolates may prove to be an important

tool in causing disease suppression in the fungal plant pathogens (Macagnan et al. 2008).

Several microbial strains have been shown to produce pectinase enzymes. Pectin is also

one of the major constituent of the plant cell wall. Fruits and vegetables are rich in pectin

which comprise of the large portion of their biomass (>30%). Hence, pectinases also have

direct application in food industry to obtain value-added products from these pectin-rich

substrates (Marquez et al. 2011).

Roco (2001) studied the biocontrol activity of Trichoderma harzianum against

Alternaria alternata and found the antagonist positive for endo-polygalacturonase

activity. Galal et al. (2011) reported high pectinase activity in the marine sea weed

extract antagonistic against various fungal plant pathogens viz. A. alternata, F.

oxysporum, A. brassicicola and Ulocladium botrytis. Gajera and Vakharia (2012) found

pectinase production along with the production of various other lytic enzymes by the

Trichoderma isolates exhibiting antagonistic activity towards A. niger.

2.7 Plant growth promoting attributes

Bacteria posses several plant growth promoting attributes which directly or

indirectly result in plant growth promotion (Glick 1995). These attributes include N-

fixation, P-solubilization, phytohormone production, siderophores production, Zn-

solubilization, K-solubilization, ammonia production and biocontrol of plant pathogens

(Bhattacharyya and Jha 2012).

2.7.1 Nitrogen fixation

N is one of the most common nutrients required for plant growth and productivity,

as it forms an integral part of proteins, nucleic acids and other essential biomolecules.

More than 78 per cent of N is present in atmosphere in free state, but is unavailable to the

plants. Atmospheric N needs to be converted into ammonia which can be readily taken up

by the plants. The major part of N that is present in soil is due to its fixation by certain

specialized group of microorganisms called N-fixers or diazotrophs (Kim and Rees

1994). Biological N fixation is the process of the conversion of atmospheric di-nitrogen

(N2) to the non-gaseous N compound ammonium (NH4+) by N-fixing bacteria. It fixes

about 60 per cent of the earth’s available N and represents an economically beneficial and

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24

environmentally sound alternative to chemical fertilizers (Ladha et al. 1997). The

reaction involved in biological N fixation is:

N2 + 8 H+ + 8 e

− + 16 ATP → 2 NH3 + H2+ 16 ADP + 16 inorganic P

Biological N-fixation is a complex process that involves a number of functional

and regulatory gene products (Triplett et al. 1989). The actual reduction of N is

performed by the nitrogenase protein complex, which consists of two metalloproteins:

nitrogenase and nitrogenase reductase (Halbleib and Ludden 2000).

A study undertaken by Islam et al. (2010) to determine the free living culturable

diazotrophic bacteria of paddy soils in a long-term fertilizer management experiment. Out

of 165 distinct bacterial morphotypes observed during the isolation process, only 32 were

positive for acetylene reduction assay (ARA) and their nitrogenase activity ranged from

1.8 - 2844.7 nM ethylene h−1

mg protein−1

. Chowdhury et al. (2007) isolated Gram-

negative diazotrophic bacterial isolates from the surface-sterilized roots of Lasiurus

sindicus and found high nitrogenase activity in the isolates by acetylene reduction assay.

Park et al. (2005) isolated free-living N-fixing bacteria from rhizosphere of seven

different plants namely sesame, maize, wheat, soybean, lettuce, pepper and rice grown in

Chungbuk Province, Korea. Five isolates with nitrogenase activity of more than 150 nM

h-1

mg-1

protein were identified by acetylene reduction assay. Similarly, other workers

also used ARA for evaluation of N fixing potential of diazotrophic isolates (Pal et al.

2001; Donate-Correa et al. 2005; Piao et al. 2005; Egamberdiyeva 2007; Mehnaz et al.

2007; Hsu and Buckley 2009).

The nitrogenase complex is very sensitive to oxygen and prokaryotes have

evolved various strategies to deal with this problem (Marchal and Vanderleyden 2000).

They reduce or eliminate nitrogenase production and activity at high oxygen partial

pressures (Kullik et al. 1991; Klein et al. 1996; Marchal and Vanderleyden 2000),

produce heterocysts (Cyanobacteria), slime production (e.g. Derxia), possess high

respiration rates, have leghemoglobin production and alginate capsules (Marchal and

Vanderleyden 2000; Oelze 2000; Sabra et al. 2000). However, many diazotrophs are

adapted to microaerobic or anaerobic niches, thus avoiding or reducing the need for

oxygen protection mechanisms. Li et al. (1992) isolates Bacillus sp. from douglas-fir,

which was capable of showing nitrogenase activity under anaerobic conditions.

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25

Minamisawa et al. (2004) isolated endophytic Clostridium spp. from graminaceous plants

and found nitrogenase activity in the range of 16-24 nM ethylene produced h-1

for these

isolates.

2.7.2 Phosphate solubilization

P is one of the major essential macronutrients for plants. P is an important

constituent of nucleic acids, phospholipids, phytins, co-enzymes, phosphorylated sugars

and nucleotides, and is involved in many essential processes like transfer of energy, cell

division, sugar breakdown, photosynthesis, flower and seed formation, stalk and stem

strength, root development, nodule formation in legumes, crop maturity, resistance in

plant diseases, and nutrient uptake (Sanyal and De Datta 1991).

P deficiency in soil is a major constraint to the crop production. The average

content of P in soil is about 0.05 per cent (w/w); of which only 0.1 per cent is available

for plants in the form of monobasic H2PO41-

and dibasic HPO42-

ions, which are

necessary to support maximum plant growth. The bioavailability of P in soil remains low

due to the chemical transformations of P into insoluble forms (Rodríguez and Fraga

1999). The majority of P that is applied to the soil is fixed rapidly through precipitation

reaction with highly reactive Al3+

and Fe3+

ions in acidic, and Ca2+

ions in calcareous and

normal soils. In these forms, phosphate is highly insoluble and is poorly available to the

plants. Therefore, the release of insoluble and fixed forms of P is an important aspect of

increasing soil P availability and plant productivity (Alexander 1977).

Phosphate-solubilizing bacteria (PSB) have been considered as one of the possible

alternatives for mediating inorganic phosphate solubilization and increasing its

availability to the plants. PSB enhance the P availability to plants by mineralizing organic

P and solubilizing precipitated phosphates present in the soil (Rodriguez et al. 2006). The

production of low molecular weight organic acids such as acetate, lactate, oxalate,

tartarate, succinate, citrate, gluconate, ketogluconate, glycolate during phosphate

solubilization leads to drop in pH, which is the main driving force for mobilization of

mineral phosphates (Illmer et al. 1995; Rodriguez and Fraga 1999). The production of

organic acids results in the acidification of the microbial cell surroundings, which results

in the substitution of Ca2+

, Al3+

and Fe3+

ions bound to phosphates by a proton. This

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26

results in the conversion of insoluble forms of P into the soluble monobasic H2PO41-

and

dibasic HPO42-

ionic forms, which could be readily taken up by the plants. The

mineralization of organic P in soil is also carried out by the means of extracellular

enzymes released by PSB, such as acid and alkaline phosphatases, and phytases which

hydrolyze the phosphoester or phosphoanhydride bonds present in organic phosphate

compounds (Goldstein 1986; Kim et al. 1998).

Dugar et al. (2013) isolated P-solubilizers from the rhizosphere of a widely

growing weed, Parthenium hysterophorus, on Pikovskaya’s medium and these were

further assayed for multipe plant growth promoting properties. Two potential isolates, P1

and P2, were employed and resulted into increase in seed germination in red gram

and green gram as compared to control. Ranjan et al. (2013) isolated PSBs from different

soil samples and selected 12 efficient PSB isolates on their ability to form clear zone on

Pikovskaya's agar medium. The isolated PSB released high amount of phosphorus from

calcium phosphate. Hassimi et al. (2013) isolated Bacillus under anaerobic conditions

from thermo-anaerobic grassland waste biodegradation plant and found P-solubilization

in the range of 7-15.8 per cent.

2.7.3 Phytohormones production

One of the direct mechanisms by which bacteria promote plant growth is by

production of plant growth regulators or phytohormones. One of the most common and

naturally occurring auxin with broad physiological effects is indole-3-acetic acid (IAA).

Bacterial biosynthesis of phytohormone IAA is known in many bacteria. IAA is known to

be involved in root initiation, cell division, cell enlargement, differentiation of phloem

and xylem, and delaying of leaf senescence (Glick 1995).

Tryptophan is the main precursor for IAA synthesis and thus plays a role in

modulating the level of IAA biosynthesis (Spaepen et al. 2007). Application of

exogenous tryptophan increases IAA production in various bacteria. However, the

tryptophan-independent pathway is also found in microorganisms but the mechanisms

involved are largely unknown (Costacurta and Vanderleyden 1995; Sergeeva et al. 2002).

The production of auxins in the presence of tryptophan have been reported for

several bacteria, including Acinetobacter, Acetobacter, Alcaligenes, Azospirillum,

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27

Azotobacter, Bacillus, Bradyrhizobium, Pantoea, Pseudomonas, Rhizobium and

Xanthomonas (Patten and Glick 1996; Bent et al. 2001; Kang et al. 2006; Tsavkelova et

al. 2007; Jha et al. 2009).

Tien et al. (1979) demonstrated that production of IAA by Azospirillum

brasilense increased with increasing concentrations of tryptophan from 1 to 100 µg mL-1

.

Synthesis of IAA by Rhizobium spp. in presence and absence of tryptophan has also been

demonstrated (Kittel et al. 1989). Spaepen et al. (2007) found that tryptophan is the main

precursor for IAA synthesis and thus plays a role in modulating the level of IAA

biosynthesis and it plays an important role in increasing production of IAA in Bacillus

amyloliquefaciens FZB42. Karnwal (2009) tested two strains of fluorescent Pseudomonas

isolates for the production of indole acetic acid and found that production of IAA

increased as the tryptophan concentration increased.

2.7.4 Zinc solubilization

Zn is one of the essential micronutrients required for optimum plant growth and

plays a vital role in cell metabolism. It is also the co-factor and metal activator of many

enzymes (Desai et al. 2012). Zn is one of the eight essential micronutrients required for

the normal healthy growth and reproduction of crop plants and is required in relatively

small concentrations in plant tissues (5–100 mg Kg-1

).

Zn deficiency is well reported in the soils of much of the world. The

major reason for the widespread occurrence of Zn deficiency problems in crop plants is

the low solubility of Zn in soils rather than a low total amount of Zn (Cakmak 2008). Zn

in soils associated mainly with hydrous Fe3+

and Al3+

oxides and with clay minerals.

Thus, the release of insoluble and fixed forms of Zn is an important aspect of increasing

soil Zn availability by Zn solubilizing microorganisms that are able to solubilize

insoluble Zn salts through the production and excretion of organic acids

(Venkatakrishnan et al. 2003; Bhupinder et al. 2005).

Simine et al. (1998) reported a Zn solubilizing strain of P. fluorescens from forest

soil. Fasim et al. (2002) isolated a strain of P. aeruginosa from air environment of

tannery and found zinc solubilization of 18 mM by the isolate in a medium containing

ZnO. A fall in pH of the medium from 6.8-3.9 was observed during zinc solubization.

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28

Saravanan et al. (2003) assessed the Zn solubilizing ability of Bacillus sp. and

Pseudomonas sp. and found the Zn solubilization of 13.60 mg Kg-1

and 16.40 mg Kg-1

,

respectively, along with the significant decline in the pH of the medium. Desai et al.

(2012) observed zinc solubilization in the range of 5.6 - 13.12 ppm by the 13 bacteria

(out of 130) belonging to different species of Azotobacter, Azospirillum, Bacillus and

Pseudomonas in the minimal growth medium amended with ZnO, with the highest

solubilization recorded in case of Bacillus sp.

2.7.5 Potassium solubilization

Potassium (K) is an essential macronutrient for plant growth and plays significant

roles in activation of several metabolic processes including protein synthesis,

photosynthesis, as well as in resistance to diseases. Most agricultural soils contain large

reserves of K, a considerable part of which has accumulated as a consequence of regular

applications of K fertilizers. Although, K constitutes about 2.5 per cent of the lithosphere

but actual soil concentrations of this nutrient vary widely ranging from 0.04 to 3.0 per

cent (Parmar and Sindhu 2013). A large portion of K applied to soil as chemical fertilizer

is rapidly immobilized soon after application and becomes unavailable to plants. Hence,

K is one of the major plant nutrients limiting plant growth (Groudev 1987; Rogers et al.

1998; Prajapati and Modi 2012). K solubilizing microorganisms are able to solubilize

insoluble rock K minerals through the production and excretion of organic acids or by

chelation of silicon ions to bring K into the solution (Friedrich et al. 1991; Ullman et al.

1996; Bennett et al. 1998; Sheng et al. 2003).

A wide range of bacteria namely Pseudomonas, Burkholderia, Acidothiobacillus,

Bacillus and Paenibacillus have been reported to release K into accessible form from K-

bearing minerals in soils (Lian et al. 2002; Sheng 2005; Li et al. 2006). Inoculation with

K solubilizing bacteria has been reported to exert beneficial effects on growth of cotton

(Sheng 2005), pepper and cucumber (Han et al. 2006), sorghum (Badr et al. 2006), wheat

(Sheng and He 2006) and Sudan grass (Basak and Biswas 2008). Similarly inoculation of

maize and wheat plants with Bacillus mucilaginosus, Azotobacter chroococcum and

Rhizobium resulted in significant higher mobilization of K from waste mica, which in

turn acted as a source of K for plant growth (Singh et al. 2010).

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29

2.7.6 Ammonia production

The increasing cost of petroleum products required for N fertilizer production has

focused attention on biological system for N fixation. Narula and Gupta (1986) found that

inoculation of wheat and barley with ammonia excreting strains caused increased dry

weight and enzyme activity. Whitehead and Cotta (2004) isolated of 40 bacterial cultures,

which were capable of producing at least 40 mM ammonia in peptone-amino acid

medium, concentrations similar to those produced by hyper-ammonia producing (HAP)

bacteria isolated from the lumen of cattle.

Joseph et al. (2007) isolated a total of 150 bacterial isolates belonging to Bacillus,

Pseudomonas, Azotobacter and Rhizobium from different rhizospheric soil of chick pea

in the vicinity of Allahabad. Ammonia production was detected in 95 per cent of isolates

of Bacillus followed by Pseudomonas (94.2%), Rhizobium (74.2%) and Azotobacter

(45.0%). Selvakumar et al. (2008) reported that two isolates viz. KR-3 and KR-4 were

able to grow in N-free medium and excreted extracellular ammonia which is indicative of

their ability to fix atmospheric N. Karthikeyan and Sakthivel (2012) reported that

ammonia production was detected in 96 per cent of isolates of Bacillus followed by

Pseudomonas (92%), Azospirillum (65%) and Azotobacter (50%) isolated from different

plant rhizospheres.

The production of ammonia is also reported to be involved in antagonistic

interactions that result in disease control (Saraf et al. 2008). Howell et al. (1988) reported

that volatile compounds such as ammonia produced by E. cloacae were involved in the

suppression of P. ultimum-induced damping-off of cotton.

2.8 Microbial Diversity

Microbial diversity includes the number of different microbial species and their

relative abundance in a particular habitat. Microorganisms represent by far the richest

repertoire of molecular and chemical diversity in nature (Joseph et al. 2003). They

underlie basic ecosystem processes such as the biogeochemical cycles and food chains, as

well as maintain vital relationships between themselves and higher organisms. Microbes

provide the fundamental underpinning of all ecosystems. Exploration, evaluation and

exploitation of microbial diversity is essential for scientific, industrial and social

development (Borneman and Triplett 1997).

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30

Microbial diversity is measured by various techniques such as traditional plate

counting and direct counts as well as the newer molecular-based procedures and fatty

acid analysis. Traditionally, the analysis of soil microbial communities has relied on

culturing techniques using a variety of culture media designed to maximize the recovery

of different microbial species and identification of these microorganisms by classical

techniques, including analysis of morphology, physiological characteristics and

biochemical properties (Janssen 2006). It is now recognized that only 1 per cent of

microbial diversity present in various environments is culturable and the remaining 99

per cent of the microbial diversity remains unculturable (Amann et al. 1995).

Molecular approaches for the detection and characterization of microbes have

resulted in dramatic change in our understanding of microbial diversity. Several DNA-

based typing methods are known which provide information for delineating bacteria into

genera and species with the potential to resolve differences among strains. Phylogenetic

comparison based on conserved part of genome, has been shown to be much stable than

classification solely based on phenotypic traits and other features (Dubnau et al. 1965;

Woese 1987; Clarridge 2004). Hence the use of rRNA molecules was advocated for

making phylogenetic comparisons (Woese et al. 1985; Woese 1987). The rRNA approach

opened the window to the enormous diversity of natural microbial communities. All the

three kinds of rRNA molecules i.e., 5S, 16S, 23S and spacers between these can be used

for phylogenetic analyses, but the small and large size of 5S rRNA (120 bp) and 23S

rRNA (3300 bp) have restricted their use. 16S rRNA gene (1550 bp) is the most

commonly used marker that has revolutionized the field of microbial systematic (Woese

et al. 1985; Amann et al. 1995; Mora and Amann 2001). 16S rRNA gene has universal

distribution, highly conserved nature, fundamental role of ribosome in protein synthesis,

no horizontal gene transfer and its slow rate of evolution which represents an appropriate

level of variation between organisms. Thus this gene is used for inferring the

phylogenetic relationship among bacteria (Stackebrandt and Goebel 1994; Mora and

Amann 2001; Clarridge 2004). The 16S rRNA molecule comprises of variable and

conserved regions, and universal primers for amplification of full 16S rRNA gene are

usually chosen from conserved region while the variable region is used for comparative

taxonomy. The differences in the variable region of the 16S rRNA gene sequences

provide the basis for a phylogenetic taxonomy and enable quantification of evolutionary

differences between different groups.

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31

Igual et al. (2001) used various molecular techniques including 16S rRNA

sequencing for the characterization of phosphate solubilizing bacteria. They used

bacterial universal primers i.e. fD1 and rD1 for amplification of 16S rRNA gene, which

yield 1.5 kb band. Similarly, other workers used bacterial universal primers for 16S

rRNA gene for identification and phylogenic studies of bacteria (Yang et al. 2001; Han et

al. 2009; Islam et al. 2010). A continuously increasing number of studies have sought to

explore the diversity of soil microbial communities with the help of molecular methods,

most prominently using the rRNA genes as genetic markers (Rotthauwe et al. 1997).

Chandna et al. (2013) assessed the bacterial diversity during the composting of

lignocellulosic agricultural byproducts using 16S rRNA gene sequencing and found the

dominance of bacteria belonging to firmicutes, actinobacteria, β-proteobacteria and γ-

proteobacteria.

As sequencing of rRNA genes has become a common tool in microbial

investigations, a considerable volume of sequence data of rRNA genes from diverse

bacteria has been deposited in public data bank (NCBI) enabling the phylogenetic

affiliation of bacteria. This line of work has considerably extended knowledge of

microbial diversity, community structure and community response to environmental

conditions.