OCCURRENCE OF PHOSPHATE SOLUBILIZING BACTERIA IN SOME SELECTED SOIL SAMPLES

CHAPTER THREE: MATERIALS AND METHODS                                                        20

3.1       Collection of Samples                                                                                                20

CHAPTER FOUR: RESULTS                                                                                              27

4.0       Results                                                                                                                        27

CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATION             35

5.1       Discussion                                                                                                                  35

5.2       Conclusion                                                                                                                 36

5.3       Recommendation                                                                                                       37

REFERENCES                                                                                                                      38

Appendix                                                                                                                                50

LIST OF FIGURE

Figure 4.1 showing Percentage Relative Abundance of Phosphate Solubilizing Bacteria Isolate

LIST OF TABLES

4.1       Sources of soils used in the study                                                                              28

4.2       Morphological identification of phosphate solubilizing bacteria isolate                   29

4.3       Biochemical characteristics of the isolates                                                                30

4.4       Total bacteria isolate from the soil                                                                             31

4.5       Relative abundance of phosphate solubilizing bacteria isolate                                  32

4.6       Colonies of the isolated phosphate solubilizing bacteria                                           34

CHAPTER ONE

INTRODUCTION

1.1       INTRODUCTION

Phosphorus (P) is one of the essential mineral macronutrients, which is required for maximizing crops yield (Griffith, 2009). In soils, Phosphorous may exist in many forms which can be thought of existing in 3 "pools": solution Phosphorous, active Phosphorous and fixed Phosphorous. Phosphorus is one of the major plant nutrients required in optimum amount for proper plant growth. It is known to involve many functions in the plant growth and metabolism. Several important cellular, metabolic and reproductive functions rely on sufficient phosphorus supply. Only about 25 per cent of the phosphorus applied to the soil is available for the crops and the rest become unavailable due  to  chemical  fixation;  aluminum  and  iron  in  acidic soils.  Soils are characterized by poor and medium status with respect to available phosphorus. Phosphorus ranks next to Nitrogen in importance for living plants, however, in comparison with other nutrients; the concentration of phosphorus in the soil solution is generally low. Phosphorus in decomposing litter is subject to the same pattern of immobilization and uptake by micro-organisms as found for Nitrogen (Bargali et al., 2015). In general, the application of phosphate solubilizing microorganism greatly affects the biomass compared to control plants of Dalbergia sissoo.

Seedlings exhibited maximum biomass production when inoculated with Penicillium chrysogenum and Aspergillus sp. There are various types of soil microbes which can solubilize this fixed form of Phosphorus and make it available to plants. Such organisms are called phosphate solubilizers or Phosphate Solubilizing Microorganisms (PSMs). Phosphate solubilizing microorganisms include bacteria, fungi and actinomycetes. Several soil bacteria, particularly those belonging to the genera Pseudomonas and Bacillus possess the ability to convert the insoluble phosphate into soluble form by secreting organic acids resulting in improved phosphate availability to the plants. Most tropical soils are acidic, rich in iron and deficient insoluble forms of phosphorus (P), one of the essential elements in crop production (Khan et al., 2010). In order to increase their fertility, chemical fertilizers containing soluble forms of Phosphorus are applied on large scale. Yet, a great proportion of soluble Phosphorus is rapidly precipitated into forms of low solubility, particularly Fe- Phosphorus and Al- Phosphorus complexes, which can be unavailable to plants (Parasanna et al., 2011).

As an alternative strategy, phosphate bearing minerals, particularly Rock Phosphate (RP) are also used. Rock Phosphate, which usually contains some forms of the mineral apatite, can be applied directly to the soil with varying agronomic efficiencies depending on the type of soil and crop. The use of such a natural resource constitutes an economic, environmentally friendly, and efficient way of fertilizing crops in many tropical and subtropical countries. Many soil microorganisms, particularly those colonizing the rhizosphere of plants, are able to mobilize insoluble inorganic phosphates from their mineral matrix to the soil solution and making them available to plant roots. Microbial inoculants are also found to be useful in enhancing growth of Dalbergia sissoo seedlings grown under stressful conditions (Bisht et al., 2009).

Phosphate solubilizing microorganisms are found in all soils but their number varies with soil climate as well as history. Calcium phosphate dissolving microorganisms were found in larger number than microorganisms that dissolve other mineral phosphate compounds. Pikovskaya (1948) made a pioneering attempt  in isolating an organism capable of actively solubilizing tricalcium phosphate and coined the name "Bacterium Phosphate". In 1999, Reyes et al. formulated the basal medium used for the isolation and enumeration of phosphate solubilizing microorganisms (Reyes et al.,1999)

Phosphorus (P) is one of the three major essential nutrients for plant growth, the other two being nitrogen (N) and potassium (K). Phosphorus plays many vital roles in crop yield. Compared with the other major nutrients, phosphorus is by far the least mobile and unavailable to plants in most soil conditions. To circumvent phosphorus deficiency, phosphate-solubilizing microorganisms (PSM) could play an important role in a more ecofriendly and environmentally sustainable manner Pseudomonads are known for their plant growth promoting property and they are the most studied phosphate-solubilizers (Jyothi et al., 2011). Phosphorus compounds in Indian soils are predominantly inorganic that are chiefly locked as Ca3 (PO4)2 (Tricalcium Phosphate). The group of phosphate solubilizing microorganisms dissolving Ca3 (PO4)2 appears to have an implication in agriculture. The major microbiological means by which insoluble phosphorus compounds mobilized is by the production of organic acids.

Phosphorus is a vital plant nutrient, available to plant roots only in soluble forms that are often in short supply in the soil. In fact, Phosphorous can be tightly bound with soil cations, particularly calcium, iron, or aluminum, leading to precipitation of Phosphorous (P) in the soil.

It has a critical role in plant metabolism and other activities such as cell division, development, photosynthesis, breakdown of sugar, nutrient uptake, nuclear transport within the cell, plant disease resistance and regulation of metabolic pathways (Gupta et al., 2012). Therefore, despite Phosphorous being widely and abundantly distributed in the soil in both its inorganic and organic forms, it is not easily accessible for plant growth. Thus, phosphate solubilizing bacteria (PSB) play an important role in reducing Phosphorous deficiency in soil through transforming insoluble phosphate to available, soluble phosphate (Antoun et al., 2012).

Genera with the ability to solublize phosphorus include Pseudomonas, Bacillus, Rhizobium, Burkholderia, Achromobacter, Agrobacterium, Microccocus, Flavobacterium, Erwinia, Serratia, Ralstonia, and Pantoea, Ewingella, Enterobacter and Photorhabdus. Although several mechanisms may be involved, the main one is through the production of organic acids. It is assumed that these organic acids solubilize insoluble forms of phosphate to usable forms which increase the potential availability of phosphate for plants. Microorganisms isolated from rhizospheric soil may be better adapted to crop plants and provide better growth and disease control than organisms isolated from other sources such as composts or harsh environments as the formers have been already closely associated with the plant system and adapted to the local environment as well (Bakhshandeh et al., 2014). Moreover, some of these microorganisms can induce resistance in plants against some pathogenic bacteria, fungi and viruses, a phenomenon termed induced systemic resistance (ISR). In recent years, beneficial rhizospheric microorganisms have gained special attention due to their potential to enhance plant growth by a variety of mechanisms such as phosphate solubilization (Andrade et al., 2014). Phosphorous is such an important macronutrient which is very often present in the soil in unavailable form. Many soil bacteria particularly those belonging to the genera Bacillus and Pseudomonas possess the ability to bring insoluble phosphates in the soluble forms by secreting organic acids. These acids lower the pH and bring about the dissolution of bound forms of phosphorous. These bacteria are commonly known as phosphobacteria. They can be applied either through seed or soil application. Phosphorus, both native in soil and applied to inorganic fertilizers becomes mostly unavailable to crops because of its low level of mobility and solubility and its tendency to become fixed in soil.

1.2       AIM AND OBJECTIVES OF STUDY

The main aim of this research paper is to isolate and identify Phosphate solubilizing bacteria from rhizosphere soil in Umudike and Umuahia.

The specific objectives are:

1    To  isolate  phosphate  solubilizing  bacteria  (PSB)  from  the  soil  samples  collected  in

Umudike and Umuahia using Pikovskaya media.

2    To identify Phosphate Solubilizing Bacteria (PSB) from the soil on the basis of cultural appearances of organism, colony morphology, differential and selective media and also biochemical test.

3    To assess the natural population of Phosphate Solubilizing Bacteria (PSB) associated with soil samples collected in Umudike and Umuahia.