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This work studied the biofertilizer potentials of Rhizobium leguminosarum and Pseudomonas aeruginosa on two common tropical vegetables plant Talinum triangulare (waterleaf) and Telfairia occidentalis (pumpkin). The test microorganisms were isolated from agricultural soils, characterised and identified. Each test organism was inoculated into carrier base material (activated charcoals) at three concentrations of 2.50×10cells/kg, 5.0×10cells/kg, and 7.5×10cells/kg for each test vegetable. Controls of uninoculated sterile activated charcoal were set up for each vegetable as well as N.P.K fertilizer applied at rates of Treatment(2.50×10cells/kg dose of the microbes with the carrier base material)Treatment(5.0×10cells/kg dose of the microbes with the carrier base material)and Treatment(7.5×10cells/kg dose of the microbes with the carrier base material) for each vegetable. Three growth performance indices considered for the evaluation of the biofertilizer potentials were plant biomass, plant height and mean leaf area measured after 28 days of cultivation. Result obtained show that there were significant variations in the impact of the test biofertilizer on the both vegetables. Pseudomonas aeruginosa increased the biomass of the vegetables by 100.54% to 142.87% in waterleaf and by 167.75% to 368.10% in pumpkin while Rhizobium leguminosarum caused increases of 107.77% to 286.78% (waterleaf) and 166.52% to 358.26% (Ugu). These were on the average, higher than the measures obtained with N.P.K fertilizer which ranged from 12.78% to 206.69% (waterleaf) and 52.07% to 376.13% (Ugu). Similarly increases were recorded in the mean leaf areas of the vegetables. In the P.aeruginosa innoculated waterleaf, the increase was from 73.03% to 221.16% and for Ugu, it was 92.34% to 208.06%. The corresponding increases in the Rhizobium innoculated vegetables were 172.17% to207.01% (waterleaf) and 23.29% to 164.27% (Ugu). This also varied from the values recorded from the chemically fertilized vegetables; 8.90% to 222.16% (waterleaf) and 48.27% to 187.57% (Ugu). Conversely, much lower impacts were recorded in the plant height of the biofertilized vegetables with values of 4.04% to 30.58% and Pseudomonas and Rhizobium innoculated waterleaf and Ugu recorded increase with the value of 0% to 20.06% respectively. The values for Rhizobium innoculated plants were 36.35% to55.05% (waterleaf) and 13.77% to 36.42% whereas the chemically fertilized vegetables recorded height increases of 0% to 8.09% in waterleaf and 23.33% to 168.53% in Ugu. It was concluded, on the basis of findings recorded in this work, that the two test bacteria (Pseudomonas aeruginosa and Rhizobium leguminosarum), have great potentials for utilization as fertilizers. Since the success of this research work has been established, there is need for tests on these and possibly other organisms on other vegetable types as well as a determination of optimal environment with respect to field validation of this success recorded at in house trials. This is recommended for future works and for trail field validation of this important research finding.


Title Page                                                                                                                    i

Declaration                                                                                                                  ii

Certification                                                                                                                iii

Dedication                                                                                                                  iv

Acknowledgements                                                                                                    v

Table of Contents                                                                                                       vi

List of Tables                                                                                                              ix

List of Figures                                                                                                             x

Abstract                                                                                                                      xi


CHAPTER 1: INTRODUCTION                                                                          1

1.1       Background of Study                                                                                     1

1.2       Statement of Problem                                                                                     3

1.3       Justification of this Research                                                                          4

1.4       Aims of the Study                                                                                          4

1.5       Objectives of the Study                                                                                  4


CHAPTER 2: LITERATURE REVIEW                                                              6

2.1       Definition of Biofertilizers                                                                             6

2.2       Nitrogen-Fixing Bacteria as Microbial Biofertilizers                                      8

2.3       Symbiotic Nitrogen-Fixing Bacteria                                                               10

2.3.1    Rhizobia                                                                                                          10

2.3.2    Frankia                                                                                                            12

2.3.3    Cyanobacteria                                                                                                 13

2.3.4    Nitrogen-fixing associated bacteria                                                                13

2.3.5    Free-living nitrogen-fixing bacteria                                                    15

2.3.6    Plant-growth-promoting rhizobacteria                                                            16

2.3.7    Phosphorus-solubilizing bacteria                                                                     16

2.3.8    Plant hormone production by bacteria                                                            17


2.4       Types of Biofertilizers                                                                        18

2.4.1    Nitrogen biofertilizers                                                                                     18

2.4.2    Phosphorus biofertilizers                                                                                 22

2.4.3    Compost biofertilizer                                                                              24

2.4.4    Liquid biofertilizers                                                                                        24

2.5       Equipment Required for Biofertilizers Production                                         28

2.6       Mass Production of Biofertilizers                                                                   30

2.7       Application of Biofertilizers                                                                           36

2.8       The Advantages of Biofertilizers Over Chemical Fertilizers                          40

2.9       Economic Importance of Biofertilizers                                                           41

2.9.1    Constraints in biofertilizers technologies                                                        42

2.9.2    Availability and cost efficient of biofertilizers                                               45

2.9.3    Talinum triangulare                                                                                        46

2.9.4    Telfairia occidentalis                                                                                       48


CHAPTER 3: MATERIALS AND METHODS                                                   53

3.1       Source of Material                                                                                          53

3.2       Experimentation Design                                                                                 53

3.3       Isolation of Bacterial Biofertilizer                                                                  55

3.3.1    Isolation of Rhizobium leguminosarum                                                          56        

3.3.2    Isolation of Pseudomonas aeruginosa                                                            57

3.3.3    Preparation of biofertilizers inoculum                                                 57

3.4       Identification of Test Organisms                                                                    58

3.4.1    Colony morphology                                                                                        58

3.4.2    Microscopic characteristics                                                                             58

3.4.3    Biochemical reaction tests                                                                              58

3.4.4    Carbohydrates (sugar) utilization test                                                             59

3.4.5    Identification of isolates                                                                                 59

3.4.6    Molecular identification of isolates                                                                59

3.4.7    Application of the biofertilizers to the plant                                                   60

3.5       Tests for Biofertilizers Potential of Rhizobium leguminosarum and Pseudomonas aeruginosa                                                                               60


3.5.1    Determination of plant height                                                                         61

3.5.2    Determination of plant biomass                                                                      61

3.5.3    Determination of plant leaf area                                                                     62

3.6       Assessment of the Effects of Biofertilizers on Test Plants                            63

3.6.1    Effects of biofertilizer application on the plant biomass                                63

3.6.2    Effect of biofertilizer on the plant height                                                       63

3.6.3    Effect of biofertilizer on the plant area                                                          64

3.7       Statistical Comparisons                                                                                   64


CHAPTER 4:  RESULTS AND DISCUSSION                                                   65

4.1       Results                                                                                                            65

4.2       The Biomass of the Vegetables Grown with Microbial Biofertilizer

            and the Controls.                                                                                            65

4.3       Vegetative Growth (Plant Height) of Vegetables Grown with

            Biofertilizers and the Controls.                                                                       68

4.4       Leaf area Measurement of the Vegetables Grown with Biofertilizers and

            their Controls.                                                                                                 70


4.5       Effect of Biofertilizers, Pseudomonas aeruginosa and Rhizobium leguminosarum, on the Biomass of the Two Test Vegetables Telfairia occidentalis and Talinum triangulare.                                                    73


4.6       Effect of the Biofertilizer on the Plant Height of the Test Vegetables

            Telfaira occidentalis and Talinum triangulare.                                               75


4.7       Discussion                                                                                                       79



5.1       Summary                                                                                                         81

5.2       Conclusion                                                                                                      82

5.3       Recommendation                                                                                            83        References                                                                                                     







2.1:      The different ways biofertilizers are grouped, based on their nature and

            function                                                                                                           9


2.2:      The seed treatment application method                                                          38

3.1:      Shows the experimental design layout representing the block design            54

4.1:      Mean biomass of vegetables grown with biofertilizer                                    67

4.2:      Plant height of vegetables grown with biofertilizers (cm)                              69

4.3:      Leaf areas of vegetable grown with biofertilizers and control (cm2)              72

4.4:      Effect of biofertilizers on biomass of vegetables                                           74

4.5:      Effect of biofertilizers on the plant height of vegetables                               76

4.6:      Effect of biofertilizers on the leaf area of the grown                                     78








2.1       Schematic representation of mass production of bacterial biofertilizers        35

2.2       Mass production of mycorrhizal biofertilizer                                                  35

2.3       Talinum triangulare (waterleaf)                                                                      48

2.4       Telfaria occidentalis (pumpkin)                                                                      52








1.1       BACKGROUND OF STUDY     

Agriculture is known as the most important economic sector in all developing countries. Particularly, 45-60% of the labour force in Nigeria is engaged in agricultural activities and agriculture contributes up to 30-40% of the gross national product (The World bank, 2008). Vegetables are used as food and as raw materials for industries, which also serves for economic interest. One of the major concerns in today's world is the pollution and contamination of soil. The use of chemical fertilizers and pesticides has caused tremendous harm to the environment. An answer to this is the bio-fertilizer, an environmentally friendly fertilizer now used in most countries. Bio-fertilizers are organisms that enrich the nutrient quality of soil. The main sources of bio-fertilizers are bacteria, fungi, and cyanobacteria (blue-green algae). The most striking relationship that these have with plants is symbiosis, in which the partners derive benefits from each other (Youssef et al., 2014). Bio-fertilizer is a substance which contains living microorganisms which, when applied to the seed, plant surfaces or soil colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant. Bio-fertilizers add nutrients through the natural processes of nitrogen fixation, solubilizing phosphorus, and stimulating plant growth through the synthesis of growth-promoting substances. Bio-fertilizers can be expected to reduce the use of chemical fertilizers and pesticides. Bio-fertilizers provide eco-friendly organic agro-input and are more cost-effective than chemical fertilizers. Since a bio-fertilizer is technically living, it can symbiotically associate with plant roots. Involved microorganisms could readily and safely convert complex organic material in simple compounds, so that plants are easily taken up. It maintains the natural habitat of the soil. It increases crop yield by 20-30%, replaces chemical nitrogen and phosphorus by 25% and stimulates plant growth. It can also provide protection against drought and some soil borne diseases. Very often microorganisms are not as efficient in natural surroundings as one would expect them to be and therefore artificially multiplied cultures of efficient selected microorganisms play a vital role in accelerating the microbial processes in soil. Use of biofertilizers is one of the important components of integrated nutrient management, as they are cost effective and renewable source of plant nutrients to supplement the chemical fertilizers for sustainable agriculture. Several microorganisms and their association with crop plants are being exploited in the production of biofertilizers (Hayat et al,.2010). Generally, the use of microorganisms as biofertilizer has wide application in terms of crops covering legumes, cereals, roof and tuber crops (Mohammed et al., 2009, Paudey, 2006).

The use of microbial biofertilizer has been described as an important component of integrated nutrient management which is not only cost effective but represents a renewable nutrient source which supplements the use of chemical fertilizers. Essentially, biofertilizers help plant to take up nutrients through microbial activities at the rhizosphere which include interactions which accelerate microbial processes in the soil to augment the extent of nutrients availability in easily assimilated form to plants.

Presently, there is growing interest in the use of microorganisms as biofertilizer owing to the relative advantages over chemical fertilizers. Biofertilizers restore soil’s natural nutrient cycle and build up organic matter thus representing an eco-friendly organic agricultural input. They generally promote plant growth and at the same time enhance sustainability and health of soil. It has been observed, that some important plant nutrient like phosphate is rarely available in quantity that meet plants need and this is attributed to phosphate immobilization by some mineral element like iron, aluminum, calcium, etc (Pandey, 2006). Findings also show that less than 20% of added phosphate as chemical fertilizer, is absorbed by plants for the same reasons while the rest is either immobilized or leached out and therefore unavailable to plant thereby resulting in wastage and ecological pollutions. Mohammed et al. (2009) cited the above as one of the factors which make it imperative to resort to the use of phosphate solubulizing microorganisms as biofertilizer in perference to chemical phosphate fertilizers. 

Some scholars (Sullivan, 2001) have described biofertilizers as microbial inoculants which represent agricultural soil amendment that uses beneficial microorganisms to promote plant health as the microbes form symbiotic relationship with the target crop and both benefit mutually. Besides phosphate solubulization and immobilization, biofertilizer organisms improve the soil by increasing the availability of other primary nutrients. The Azotobacter and Rhizobium species fix nitrogen in the soil. While Rhizobium is seen by many as the most efficient biofertilizer due to its symbiotic fixation of atmospheric nitrogen, the Azotobacters are known to in addition, improve soil aggregation by their ability to produce slime.

In view of the successes recorded here with microbial biofertilizers, and in consideration of their numerous agricultural advantages, this project is designed to evaluate the use of some bacterial species as biofertilizer and their impact on growth pattern of two common vegetable plants Talinum trangulare,and Telferia occidentalis.


Crop production needs to be increased substantially to reduce hunger and food insecurity in West Africa. Since most soils in the region are inherently poor, external inputs are necessary to boost crop production. In addition, there is a need to improve crop productivity in an eco-friendly manner and this has led to the promotion of commercial biological products called biofertilizer intended to restore or enhance the fertility and organic matter content of soils.


With the ongoing adverse effects of using Chemical fertilizers to both the plants, human and animal life, it has become imperative to seek an alternative to this menace and the urgent need of providing an eco-friendly alternative to chemical fertilizers, An answer to this is biofertilizer, an environmentally friendly fertilizer now used in most countries. Soil microorganisms play significant role in organic matter decomposition and release of plant nutrients such as nitrogen (N), phosphorus (P) and sulfur (S). Therefore, microorganisms are important component of integrated nutrient management systems and soil biodiversity. Biofertilizer has gained advantage over chemical fertilizers and its usage is cheaper and better.  Therefore, a success in this work will help solve and bridge the gap caused by chemical fertilizer and make biofertilizers readily cheap and available for farmers.


The aim of the project work is to evaluate the use of some soil bacteria (Rhizobium leguminosarum and Pseudomonas aeruginosa) as biofertilizer and their impact on growth performance of two common vegetable plants, Talinium triangulare and Telferia occidentalis.


       I.            To isolate and identify of Rhizobium and Pseudomonas species.

    II.            To do molecular characterization of the isolates

 III.            To test the biofertilizer potential of the two isolates using the two plant seedlings.

 IV.            To assess the growth performances of the two biofertilized vegetables based on plant

height, biomass, and leaf area; after a growth period of eight weeks, relative to that of controls (Non biofertilizer plants).

    V.            To statistically compare the growth performance of the biofertilized plants relative to the controls, as well as between the test organisms.


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