BIOCHEMICAL CHARACTERISATION OF PHOSPHO BACTERIUM ISOLATED FROM RHIZOSPHERE OF COSTUS SP. WITH SPECIAL REFERENCE TO BACILLUS SPECIES

BIOCHEMICAL CHARACTERISATION OF PHOSPHO BACTERIUM ISOLATED FROM RHIZOSPHERE OF COSTUS SP. WITH SPECIAL REFERENCE TO BACILLUS SPECIES

Sabiha Sultana M.*, C. Maya , Victoria Peter  and Omar Farooq

Department of Botany, Microbiology & Biotechnology, Al-Ameen Arts, Science and Commerce College, Bangalore University , Karnataka, India

Department of Botany, Bangalore University, Karnataka, India

ABSTRACT

Soils generally contain adequate amount of inorganic and organic phosphorous but most of these remain unavailable to plants. The efficiency of water soluble phosphorus alone is usually low and soluble phosphorus when added to soil along with phosphate solubilizing microorganisms indicates utilization. The presence of soil microorganisms capable of transforming insoluble phosphorus to soluble form of phosphorus was studied Different phosphate dissolving bacterial  strains are isolated from the rhizosphere soil of Costus plant and biochemical characterization and P₂O₅ uptake in liquid culture was studied.

INTRODUCTION: Phosphorus is applied to soils in inorganic fertilizers or in organic manures in the form of plant animal residues. Of all forms of phosphorus occurring in soils, the monovalent anion phosphate is the only one available to the growing plants when soluble forms of inorganic phosphorus fertilizers are applied to soils. It is found that high densities of phosphate dissolving bacteria harbor in soil. Fortunately, it is found that high densities of phosphate dissolving bacteria harbor in soils. Bacteria may play an important role in supplying the growing plants with their needs of phosphorus¹. The USSR investigator had isolated an active phosphate dissolving bacteria which enhanced phosphorus uptake ².

Costus pictus D. Don is an antidiabetic herb and its rhizosphere microflora was   assessed. The role of plant growth promoting rhizo-microorganisms in improving plant growth is well documented ³. The rate of metabolic activity of bacterium widely ranges and is based on the breakdown of organic material by the microbial enzyme which includes lyases, oxidoreductase, hydrolyase, amylase, catalase and transferase besides, cellulase. This is assessed by carrying out biochemical analysis of the Bacillus species isolated from rhizosphere soil of Costus plant.

MATERIALS & METHODS:

• Bacteria were isolated from rhizosphere soil by dilution plate technique, and Bacillus species are selected ⁴.

• Three strains of Bacillus species were selected ⁵, and labeled as B1, B2 and B3 strains.

• All the three strain are screened in liquid medium of Pikovskaya broth ⁶ using different phosphate like Tricalcim phosphate, Aluminium phosphate, Rock phosphate and commercial phosphate. Incubated for seven days at 28C in 250ml conical flasks and shaken for five minute on a rotary shaker. The isolates liberate different soluble phosphates (Table 1).

TABLE 1: RELEASE OF PHOSPHATE FROM ‘P’ FERTILIZER BY THE PSB IS LIQUID CULTURE

• Isolated strain B1, B2 and B3 are subjected to various biochemical analysis such as IMViC tests, starch hydrolysis, gelatin liquefaction tests ⁷, Acid and gas production was observed in sugar fermentation tests ⁸.

Observation & Chemical Analysis:

Chemical Analysis:

1. Phosphate solubilization by; Ammonium molybdate phosphoric blue color method ⁹.
2. Biochemical Analysis ^10&11;

• Indole test (I)
• Methyl Red test (MR)
• Voges Proskauer test (VP)
• Citrate utilization test (CU)
• Sugar Fermentation (SF)
  • {Dextrose (D), Mannitol (M) and Sucrose (S)}
• Gelatin liquefaction (GL)
• Starch hydrolysis (SH)

RESULTS & DISCUSSION: The results obtained by this study shows significantly high solubilization of commercial fertilizer than the other inorganic phosphate. All the Bacillus species show significant high solubilization of commercial fertilizer (Table 1). When compared with other phosphate fertilizer Aluminium phosphate shows enhanced results in all the three Bacillus species. The pH of liquid culture of phosphate solubilizing Bacillus strains has  revealed that them they have released acid and hence the liquid broth culture shows p^H more towards the acidic range, Bacillus species-3 has not shown very good acid formation when compared to control and other strain in Aluminium phosphate fertilizer (Table 2).

TABLE 2: CHANGE OF PH VALUE BY THE PSB IN LIQUID CULTURE

The Biochemical characteristic shows that all the three Bacillus species utilize the monosaccharides and produce acid / gas, where as sucrose is negative i.e. disaccharides are not utilized by the Bacillus species (Table 3).

TABLE 3: BIOCHEMICAL CHARACTERISTICS OF THE ISOLATED BACILLUS SPP

Acid soils shows deficiency of phosphorus is plants, under such condition inoculate soil with phosphate dissolving microorganisms along with phosphate fertilizer. Also phosphate solubilizing bacteria are proved beneficial in biological control in root diseases¹².  Phosphate solubilizing microbes has proved to enhance the growth of the antidiabetic plant Costus sp. The Biochemical Analysis reveals that all the Bacillus species were capable of hydrolysing gelatin and starch. The Methyl red shows positive results and Voges Proskauer test show negative results Bacillus species 1 and Bacillus 2 utilized citrate as the sole carbon source whereas Bacillus 3 did not. Indole test showed negative for B1 and B2 species where as it gave a positive result for B3. Catalase test also showed positive for B1 and B2 and negative for B3. The results indicate a decrease in the pH due to the formation of organic acids and support the view that the acidic end products produced as the result of the microbial activity affect the solubilization of phosphate Table 1 & 2. The results of Table 3 correlated with the results of Table 1 & 2, a significantly enhanced production of acid and gases in biochemical analysis was revealed. Soils with pure cultures of indigenous phosphate dissolving bacteria may assist in rendering more phosphorus to the plant, especially in soils which may not have an adequate number of efficient phosphorus solubilizing bacteria. Study of the morphology, cultural characteristics and biochemical properties helped to identify these organisms as Bacillus species. These biochemical tests also revealed the diverse enzymatic activity of the soil bacteria and therefore capable of solubilization of different phosphates.

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