ABSTRACT
Different strains of phosphate-solubilizing bacteria (PSB) were isolated from the rhizosphere of different plants of different agricultural lands located at Afugiri Ohuhu community Umuahia and Uzuakoli rice plantation. The objective of the study was to explore the capabilities of PSB and evaluate their efficiency to enhance growth of maize plants under various condition. The soil samples were collected from the rhizosphere of plants at a depth of 6-15cm from the top soil. The purified isolates were identified as Bordetella bronchiseptica, Bacillus thuringiensis and Bacillus cereus respectively based on the characteristic morphological, biochemical behavior and 16SrRNA sequencing. The efficiency of different PSB isolates for phosphate solubilization was evaluated from the zones they formed on agar plates of Pickovaskaya growth medium (agar) by solubilizing the tricalcium phosphate of the medium. The results showed that B. cereus with a phosphate solubilization index of 2.3 was the strongest phosphate solubilizer and maize plant simulated with B. cereus inoculant performed better. There was relative increase in plant height, number of leaves, root length and grain yield in plants inoculated with PSM than in uninoculated plants (control). for all the tested parameters. The results of this greenhouse evaluation are encouraging and need to be confirmed under field condition in combination with organic and chemical fertilizers.
TABLE OF CONTENTS
Title Page i
Certification ii
Declaration iii
Acknowledgement iv
Table of Contents v
List of Tables vi
List of Figures vii
Abstract viii
CHAPTER 1: INTRODUCTION
1.1 Problem Statement 4
1.2 Justification of the Research 5
1.3 General Objective 5
1.4 Specific Objectives 5
CHAPTER 2:
LITERATURE REVIEW
2.1 Phosphate Solubilization by Microbes 6
2.2 Biodiversity of Phosphorus Solubilizers 9
2. 3 Mechanism of P-solubilization by PSB 11
2.4 Constraints in using Phosphate Fertilizers 17
2.5 Phosphate Solubilization by Microbes 18
2.6 Mineralization: Enzymatic Degradation
of Complex
Organic
Phosphate Compounds 20
2.7 Role of Exopolysaccharides in P
Solubilization 22
2.8 Role of Exopolysaccharides in P Solubilization 24
2.9 Plant Growth Promoting Attributes of Phosphate Solubilizing
Bacteria 24
2.10
Genetic engineering of PSM 25
CHAPTER 3: MATERIALS AND METHODS
3.1 Sample Collection 27
3.2 Preparation of Medium 27
3.3 Isolation of Phosphate Solubilizing
Bacteria 27
3.4 Morphological Characterization 28
3.5 Analysis of Phosphate Solubilizing
Bacterial Isolates 28
3.6 Optimization of Physiological
Conditions (Temperature and pH) 28
3.7 Gram Staining 29
3.8 Biochemical Characterization 29
3.9 Application of PSB and its Effect on the
Growth Rate of Maize Plant 33
3.10 Molecular characterization 33
CHAPTER 4: RESULTS
AND DISCUSSION
4.1 Morphological and Biochemical Characteristics of Phosphate
Solubilizing
Bacteria from Soil
36
4.2 Phosphate Solubilizing Activities of Selected Isolates 37
4.3 Attributes of the PSB Isolates
48
4.4 Discussion 51
CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusion 54
5.2 Recommendations 54
References 55
LIST OF TABLES
4.1: Morphological and biochemical characteristics
of bacterial isolates from loamy soil. 37
4.2:
Phosphate Solubilizing Activities of selected isolates 38
4.3: Week one performance of maize inoculated
with phosphate solubilizing bacteria 42
4.4: Week three performance of maize inoculated
with phosphate solubilizing bacteria 42
4.5: Week five performance of maize inoculated
with phosphate solubilizing bacteria 43
4.6: Week 7 performance of maize inoculated with
phosphate solubilizing bacteria 44
4.7: Week 10 performance of maize inoculated
with phosphate solubilizing bacteria 45
4.8: Yield Assessment of the Maize Plant With
Phosphate Solubilizing Bacteria. 46
LIST OF FIGURES
1.0
An illustration depicting functional diversity among phosphate
solubilizing
bacteria 21
2.0
Schematic representation of mechanism of soil p solubilization
/mineralization
and mobilization of PSB 23
3.0 Effect of pH on phosphate
solubilization 39
4.0 Effect of temperature on phosphate
solubilization
40
5.0 Agarose electrophoresis of the amplified
16SrRNA gene of the
bacterial isolates 41
6.0 Phylogenetic tree showing the
relationship between the organisms
49
CHAPTER 1
INTRODUCTION
Phosphorus (P) is a major
growth-limiting nutrient, and unlike the case for nitrogen, there is no large
atmospheric source that can be made biologically available for root
development, stalk and stem strength, flower and seed formation, crop maturity
and production, N-fixation in legumes, crop quality, and resistance to plant
diseases are the attributes associated with phosphorus nutrition (Ahmad et al., 2009). However, a greater part
of soil phosphorous, approximately 95-99% is present in the form of insoluble
phosphates and hence cannot be utilized
by the plants Phosphorus plays an indispensable biochemical role in
photosynthesis, respiration, energy storage and transfer, cell division, cell
enlargement and several other processes in the living plant. It helps plants to
survive winter rigors and also contributes to disease resistance in some plants
(Amin et al., 2012). P availability
is low in soils because of its fixation as insoluble phosphates of iron,
aluminum and calcium. Since deficiency of P is the most important chemical
factor restricting plant growth, chemical phosphate fertilizers are widely used
to achieve optimum yields. Soluble forms of P fertilizer used are easily
precipitated as insoluble forms, this leads to excessive and repeated
application of P fertilizer to cropland.
Phosphorus is a plant macronutrient that plays a significant role in
plant metabolism, ultimately reflected on crop yields. It is important for the
functioning of key enzymes that regulate the metabolic pathways. The uptake of phosphorus by the plant is only
a small fraction of what is actually added as phosphate fertilizer. Phosphorus
deficiency is widespread and phosphorus fertilizers are required to maintain crop
production. When it is added to the soil in the form of phosphate fertilizer,
only a small portion is utilized by plants.
Phosphate fertilizers can
also be used to immobilize heavy metals in soil. Insoluble phosphate compounds
can be solubilize by organic acids and phosphatase enzymes produced by plants
and microorganisms. Application of biological fertilizers such as biological
phosphate fertilizers improves soil fertility. Phosphorus can naturally be
found in diverse forms in the soil solution. The roots take up several forms of
phosphorus, out of which the greatest part is absorbed in the forms of H2PO4
and HPO4 2- depending upon soil pH. The degree of
fixation and precipitation of phosphorus in soil is highly dependent upon the
soil conditions such as pH, moisture content, temperature and the minerals
already present in the soil.
Some bacterial species
have mineralization and solubilization potential for organic and inorganic phosphorus,
respectively. Phosphate solubilizing activity is determined by the ability of
microbes to release metabolites such as organic acids, which through their
hydroxyl and carboxyl groups chelate the cation bound to phosphate, the latter
being converted to soluble forms. Phosphate solubilization takes place through
various microbial processes or mechanisms including organic acid production and
proton extrusion. A wide range of microbial P solubilization mechanisms exist
in nature and much of the global cycling of insoluble organic and inorganic
soil phosphates is attributed to bacteria and fungi (Ahmad et al., 2009). Phosphobacteria have been found to produce some
organic acids such as monocarboxylic acid (acetic, formic), monocarboxylic
hydroxy (lactic, glucenic, glycolic), monocarboxylic, ketoglucenic,
decarboxylic (oxalic, succinic), dicarboxylic hydroxy (malic, maleic) and
tricarboxylic hydroxy (citric) acids in order to solubilize inorganic phosphate
compounds.
A diverse group of soil
micro flora was reported to be involved in solubilizing insoluble phosphorous
complexes enabling plants to easily absorb phosphorous. Several fungal and
bacterial species, popularly called as phosphate solubilizing microorganisms, (PSMs)
assist plants in mobilization of insoluble forms of phosphate. PSMs include
different groups of microorganisms, which not only assimilate phosphorus from
insoluble forms of phosphates, but they also cause a large portion of soluble
phosphates to be released in quantities in excess of their requirements.
Species of Aspergillus and Penicillium are among fungal isolates
identified to have phosphate solubilizing capabilities.
Several reports have
examined the ability of different bacterial species to solubilize insoluble
inorganic phosphate compounds, such as tricalcium phosphate, dicalcium
phosphate, hydroxyapatite, and rock phosphate. Among the bacterial genera with
this capacity are Pseudomonas, Bacillus, Rhizobium, Burkholderia,
Achromobacter, Agrobacterium, Microccocus, Aereobacter, Flavobacterium and
Erwinia. There are considerable populations of phosphate solubilizing
bacteria in soil and in plant rhizosphere.
The soils that exhibit highest P fixation capacity occupy 1,018 million
hectares (ha) in the tropics (Sanchez and Logan, 1992). It is for this reason
that soil P becomes fixed and available P levels have to be supplemented on
most agricultural soils by adding chemical P fertilizers, which not only
represent a major cost of agricultural production but also impose adverse
environmental impacts on overall soil health and degradation of terrestrial,
freshwater and marine resources . Thus, increased P levels have been identified
as a main factor for eutrophication of surface waters that may lead to algal blooms.
The repeated and injudicious applications of chemical P fertilizers, leads to
the loss of soil fertility by disturbing microbial diversity, and consequently
reducing yield of crops. The long-term effect of different sources of phosphate
fertilizers on microbial activities includes inhibition of substrate-induced
respiration by streptomycin sulphate (fungal activity) and actidione (bacterial
activity) and microbial biomass carbon (C) (Bolan et al., 1996). Similarly, the application of triple superphosphate
(94 kg/ ha) has shown a substantial reduction in microbial respiration and
metabolic quotient (qCO2) (Chandini and Dennis, 2002).
Moreover, the efficiency
of applied P fertilizers in chemical form rarely exceeds 30% due to its
fixation, either in the form of iron/aluminum phosphate in acidic soils or in
the form of calcium phosphate in neutral to alkaline soils. It has been
suggested that the accumulated P in agricultural soils would be sufficient to
sustain maximum crop yields worldwide for about 100 years if it were available
(Khan et al., 2009). A major characteristic
of P biogeochemistry is that only 1% of the total soil P (400–4,000 kg P/ ha in the top 30 cm) is incorporated into
living plant biomass during each growing season (10–30 kg P/ha), reflecting its low availability for plant
uptake (Quiquampoix and Mousain, 2005). Furthermore, P is a finite resource and
based on its current rate of use, it has been estimated that the worlds known
reserves of high-quality rock P may be depleted within the current century
(Cordell et al., 2009). Beyond this
time the production of P based fertilizers will require the processing of lower
grade rock at significantly higher cost (Isherwood, 2000). The realization of
all these potential problems associated with chemical P fertilizers together
with the enormous cost involved in their manufacture, has led to the search for
environmental compatible and economically feasible alternative strategies for
improving crop production in low or P-deficient soils .The use of microbial
inoculants (biofertilizers) possessing P-solubilizing activities in
agricultural soils is considered as an environmental-friendly alternative to
further applications of chemical based P fertilizers.
1.1 PROBLEM STATEMENT
Most agricultural soils
contain large reserves of phosphorus ,a considerable part of which has
accumulated as a consequence of regular application of phosphorus
fertilizers .However, a large portion of soluble inorganic phosphate
applied to soil as chemical fertilizers is rapidly immobilized soon after
application and becomes unavailable to plants there by leading to low crop
yield. Chemical fertilizers are not environmentally friendly and are cost
effective.
This work therefore seeks
to identify those microorganisms in the soil capable of solubilizing the
immobilized insoluble phosphates in the soil thereby making them available for
plants. These microbial inoculants would provide an effective, environmentally
friendly and alternative to further application of chemical based phosphorus
fertilizers.
1.2 JUSTIFICATION OF THE RESEARCH
This research was based on the recognition of the
increasing deficiency of phosphorus in the soil and the urgent need of
providing an eco-friendly alternative to fixing phosphorus in the soil as an
alternative to chemical fertilizers. The diversities of microorganisms in its
activities-provide the needed alternative eco-friendly means of making
phosphorus available for plants’ use. The capacity to properly address the
world wide incidence of soil phosphorus deficiency and increased application of
chemical fertilizers lies in the ability to identify those microorganisms with
the ability to solubilize soluble soil phosphate making them available to
plants.
1.3 GENERAL OBJECTIVE
To develop microbial
inoculants possessing phosphate solubilizing abilities in agricultural soils as an environmentally
friendly alternative to further applications of chemical based phosphorus fertilizer.
1.4 SPECIFIC OBJECTIVES
1. screening, isolation
and characterization of phosphate solubilizing bacteria from the soil.
2. Molecular
characterization of the isolated phosphate solubilizing bacteria using 16S rRNA
3. Optimization of growth
conditions of isolated phosphate solubilizing bacteria
4. Analysis of Phosphate Solubilizing Activity of the isolates
Click “DOWNLOAD NOW” below to get the complete Projects
FOR QUICK HELP CHAT WITH US NOW!
+(234) 0814 780 1594
Login To Comment