Isolation, Purification And Liberation Of Free Phosphate By Indigenous Phosphate Solubilizing Bacteria And Effect On Plant Growth Promotion.

摘要

Solubilization of insoluble organic phosphate has been the focus of many studies as it increases the availability of phosphorus to vegetation and improves plant growth. The aim of this study was to study those bacterial strains which were positive for phosphate solubilization in plate assay as well as in liquid media. The isolates which showed efficient phosphate solubilization activity both in plate and liquid media were examined for the release of free phosphate in liquid media. The amount of free phosphate liberated by the organisms was estimated. The efficient phosphate solubilizing bacteria which also released high amount of free phosphate in the media were scrutinized for auxin production via bioassay. Five bacterial isolates CMG851, CMG854, CMG857, CMG860 and CMG861 found positive for auxin production were checked for the production of indole acetic acid and indole butyric acid via high performance liquid chromatography (HPLC). The amounts of indole acetic acid and indole butyric acid were also estimated. These five efficient phosphate solubilizing and auxin producing bacterial isolates were identified via 16srRNA analysis. They were Acinetobacter lwofii (CMG851), Pseudomonas aeruginosa (CMG860) and Bacillus thuringiensis (CMG854, CMG857, CMG861) showed efficient phosphate solubilization and auxin production abilities. Mung beans were selected to study the effect of plant growth promotion abilities by the three phosphate solubilizing and auxin producing bacteria. The root and shoot length of Rye plants were measured and it was found that the selected bacteria had noticeable effect on them. Introduction The amelioration of phosphate deficiency by the application of costly and environmentally hazardous phosphate fertilizers is not an ideal solution and has generated serious issues about the continued viability of current agriculture practice. This has led to a search for more environmentally friendly and economically feasible strategies to improve crop production in low phosphorus soils. In an ideal manner, such strategies should enable the efficient use of phosphate solubilizing microorganisms. Several scientists have reported the ability of different bacterial species to solubilize insoluble inorganic phosphate compounds, such as tricalcium phosphate and dicalcium phosphate (Gar and Gaind, 1999).According to Statistics, the worldwide transaction amount of fertilizer is roughly US$40 billion. Out of this, 135 million metric tons of chemical fertilizer is applied each year, with sales volume of about US$30 billion. Although there are no clear application statistics for biofertilizer, however, its sales volume is estimated to be as much as US$3 billion. Materials and Method Sample Collection, Media and Chemicals Soil samples were collected from the following locations of Karachi. Nurseries (Glushan-e-Iqbal) Garden soil (Safari park) Staff town (Karachi University) Agriculture fields (Malir and Gaddap) Nutrient broth and Nutrient agar were used for isolation and purification of the bacterial strains. Tris minimal salt medium (Fasim et al., 2002) was used for the initial screening of metal solubilization activity. NBRIP (Nautiyal, 1999), Pikovskya (1948) and MPVK medium (Son et al., 2006), were also used to check solubilization activity of selected bacterial strains. Tris minimal medium (Appendix 7) was employed for solid and liquid culture, containing the carbon source, usually D-glucose10g l-l. All chemicals were either of Oxoid, BDH or Merck.Isolation of Bacterial Strains from Soil Samples by Serial Dilution MethodSoil samples from nurseries, garden soils and agricultural fields (Malir and Gaddap) were brought to the laboratory in sterilized universals. Ten gram soil was aseptically weighed and transferred to Erlenmeyer flasks containing 100 ml nutrient broth and incubated at 37°C for 24 hrs on 100 rpm. After 24 hrs, incubated samples were diluted by serial dilution method. A series of tenfold dilution was made for each s