An Efficient Method for Qualitative Screening of Phosphate-Solubilizing Bacteria

Transcription

1 CURRENT MICROBIOLOGY Vol. 43 (2001), pp DOI: /s Current Microbiology An International Journal Springer-Verlag New York Inc An Efficient Method for Qualitative Screening of Phosphate-Solubilizing Bacteria Sangeeta Mehta, Chandra Shekhar Nautiyal Microbiology Group, National Botanical Research Institute, Rana Pratap Marg, P.B. No. 436, Lucknow , India Received: 17 November 2000 / Accepted: 22 December 2000 Abstract. An efficient protocol was developed for qualitative screening of phosphate-solubilizing bacteria, based upon visual observation. Our results indicate that, by using our formulation containing bromophenol blue, it is possible to quickly screen on a qualitative basis the phosphate-solubilizing bacteria. Qualitative analysis of the phosphate solubilized by various groups correlated well with grouping based upon quantitative analysis of bacteria isolated from soil, effect of carbon, nitrogen, salts, and phosphate solubilization-defective transposon mutants. However, unlike quantitative analysis methods that involve time-consuming biochemical procedures, the time for screening phosphate-solubilizing bacteria is significantly reduced by using our simple protocol. Therefore, it is envisaged that usage of this formulation based upon qualitative analysis will be salutary for the quick screening of phosphatesolubilizing bacteria. Our results indicate that the formulation can also be used as a quality control test for expeditiously screening the commercial bioinoculant preparations, based on phosphate solubilizers. Correspondence to: C.S. Nautiyal A large portion of inorganic phosphates applied to soil as fertilizer are rapidly immobilized after application and become unavailable to plants [24]. Thus, the release of insoluble and fixed forms of phosphorus is an important aspect of increasing soil phosphorus availability. Seed or soil inoculation with phosphate-solubilizing bacteria is known to improve solubilization of fixed soil phosphorus and applied phosphates, resulting in higher crop yields [1, 8, 10, 24, 25]. These reactions take place in the rhizosphere, and because phosphate-solubilizing microorganisms render more phosphates into soluble form than is required for their growth and metabolism, the surplus gets absorbed by plants. However, their establishment and performance are severely affected by environmental factors, especially under stress conditions [10, 16]. Bacteria growing in alkaline soils in India during the summer season are subjected to high salt, high ph, and high temperature stress. An understanding of the phosphate solubilization by phosphate-solubilizing bacteria isolated from alkali soils is likely only when the physiology and molecular biology of these organisms have been carefully studied under sub-optimal conditions [7, 15]. Therefore, intensive screening of phosphate-solubilizing bacteria with the genetic potential for increased tolerance to high salt, high ph, and high temperature could enhance production of food and forage in semiarid and arid regions of the world [7, 15]. However, the current methods of screening for efficient phosphate solubilizers are time consuming. Phosphate-solubilizing bacteria are routinely screened by a plate assay method using Pikovskaya (PVK) agar [17]. The test of the relative efficiency of isolated strains is carried out by selecting the microorganisms that are capable of producing a halo/clear zone on a plate owing to the production of organic acids into the surrounding medium [9]. However, the reliability of this halo-based technique is questioned, as many isolates that did not produce any visible halo/zone on agar plates could solubilize various types of insoluble inorganic phosphates in liquid medium [5, 11]. Therefore, a novel defined microbiological growth medium, National Botanical Research Institute s phosphate growth medium (NBRIP), which is more efficient than PVK, was developed for screening phosphate-solubilizing microorganisms [13]. It was concluded that soil microbes should be screened in NBRIP broth assay for the identification of the most efficient phosphate solubilizers [13]. However, while screening several thousands of bacteria and/or

2 52 CURRENT MICROBIOLOGY Vol. 43 (2001) phosphate solubilization-defective mutants, the time taken to screen the most efficient phosphate solubilizers is crucial. Therefore, in the present investigation an attempt has been made to develop a new protocol based upon visual observation, for a quick and reliable detection of phosphate-solubilizing microorganisms. Materials and Methods Samples (soil and plant rhizosphere) were collected from 35 sites representing varied ecological sites. Different alkaline soil sites located in and around Lucknow were used to prepare a library of diverse bacterial strains. Samples were also collected from Almora, Mukteshwar, and Nainital representing various altitude (600 to 6000 feet), moisture, and temperature levels. Roots were thoroughly washed with tap water for 2 min to remove all the loosely adhering soil particles, followed by washing with sterile 0.85% (wt/vol) saline Milli Q water (MQW). The roots were then macerated in 0.85% saline MQW with a mortar and pestle. Serial dilution of the root homogenate and soil (10% soil in 0.85% saline MQW) samples were then individually plated on Pseudomonas isolation agar, Nutrient agar, Nutrient broth, and Tryptone-Glucose-Yeast extract (TGY) agar (from HI-Media Laboratories Pvt. Ltd., Bombay, India), as described earlier [12]. Bacteria representative of the predominant morphologically distinct colonies present on the plates were selected at random and purified on minimal medium based on AT salts [9]. Two thousand fifteen bacterial strains, representative of different morphological types present on the plates, were selected, purified, and stored on slants. Out of 2015 bacterial strains, based upon the quantitative estimation of phosphate solubilization in broth, ten bacterial strains ASLU13, 12.33, AS7.8, 2661, 3246, 4003, N, P, SN15, and SN16 were selected for further work (data not presented). Bacteria representative of the predominant morphological types present on the plates were selected at random and purified on minimal medium, based on AT salts, which contained the following ingredients (per liter): glucose, 10.0 g; KH 2 PO 4, 10.9 g; (NH 4 ) 2 SO 4, 1.0 g; MgSO 4 7H 2 O, 0.16 g; FeSO 4 7H 2 O, g; CaCl 2 2H 2 O, g; and MnCl 2 4H 2 O, g [13]. National Botanical Research Institute s phosphate growth medium (NBRIP) contained (per liter): glucose, 10 g; Ca 3 (PO 4 ) 2, 5 g; MgCl 2 6H 2 O, 5 g; MgSO 4 7H 2 O, 0.25 g; KCl, 0.2 g, and (NH 4 ) 2 SO 4, 0.1 g [13]. NBRIP medium containing BPB (designated NBRI-BPB) contained (per liter): glucose, 10 g; Ca 3 (PO 4 ) 2, 5 g; MgCl 2 6H 2 O, 5 g; MgSO 4 7H 2 O, 0.25 g; KCl, 0.2 g, (NH 4 ) 2 SO 4, 0.1 g, and BPB, g. Many variations of the NBRI-BPB media were tested, as indicated in the text and Fig. 1 and 3. The ph of the media was adjusted to 7.0 before autoclaving. ASLU13, an efficient phosphate-solubilizing strain resistant to ampicillin (100 g/ml), was used as a parent strain. Transposon5 (Tn5) was introduced into ASLU13 cells by conjugation with Escherichia coli WA803/pGS9 [20], using a modification of the procedure by Rostas and colleagues [19]. Transconjugants were obtained by membrane filter mating on NA medium for 48 h at 30 C and were selected on NA containing ampicillin (100 g/ml) and kanamycin (100 g/ml) as described earlier [14]. Qualitative (qualitative estimation of bromophenol blue) and quantitative analyses (quantitative estimation of phosphate solubilization) in broth were carried out by using 5 ml of NBRI-BPB medium in a 30-ml test tube inoculated in triplicate with the bacterial strain (50 l inoculum with approximately 1 to cfu/ml). Autoclaved, uninoculated medium served as controls. For commercial bioinoculant products, CPB1, CPB2, CPB3, CPB4, and CPB5, autoclaved 10 mg/ml product, served as control. Our ASLU13-based bioinoculant was prepared by growing the culture for 2 days in Nutrient broth. The culture was added to sterile vermiculite (AEl Ltd., Sihor, Rajasthan, India) to 20% saturation. For ASLU13, autoclaved 10 mg/ml product served as control. Unless stated, the test tubes were incubated for 3 days at 30 C on a New Brunswick Scientific, USA, Innova Model 4230 refrigerated incubator shaker at 180 rpm. The cultures were harvested by centrifugation at 10,000 rpm for 10 min, with Sorvall RC 5C centrifuge, Dupont, USA. The culture supernatant thus obtained was used for qualitative and quantitative assays. Absorption spectra of 0.01, 0.025, 0.05, and 0.1 mg/ml bromophenol blue (BPB) in the range of nm revealed absorption maxima at 600 nm by using Shimadzu spectrophotometer, Model UV-1601, Japan (data not presented). Therefore, for qualitative estimation of BPB in NBRIP-BPB, the optical density of the culture supernatant was measured at 600 nm, by using Milton Roy Spectronic 20D, USA. For the quantitative analysis, phosphate in the culture supernatant was estimated by using the Fiske and Subbarow method [3]. Effect of carbon, nitrogen, and salts on qualitative and quantitative analysis by ASLU13 was tested by growing them on NBRIP-BPB, containing various carbon, nitrogen, and salts as indicated. To check the effect of carbon sources (arabinose, glycerol, xylose, and fructose), glucose in the NBRIP-BPB was replaced by the carbon source, as indicated. To check the effect of nitrogen sources [(NH 4 )2Cr 2 O 7,C 4 H 12 N 2 O 6,NH 4 HCO 3, and C 24 H 20 Bi 4 O 28 6NH 3 10H 2 O], (NH 4 ) 2 SO 4 in the NBRIP-BPB was replaced by the nitrogen source as indicated. The effect of salts [MnCl 2 4H 2 O (2.5 mg/ml), NaNO 3 (2.5 mg/ml), CaCl 2 2H 2 O (0.25 mg/ml), and MnSO 4 H 2 O (0.25 mg/ml)] on qualitative and quantitative analysis was tested by growing ASLU13 on NBRIP-BPB containing the salt, as indicated. The data are means of three independent experiments. Results and Discussion Several authors attribute the solubilization of inorganic insoluble phosphate by microorganisms to the production of organic acids and chelating oxo acids from sugars [10, 24]. Therefore, most of the quantitative tests to assay the relative efficiency of the phosphate-solubilizing bacteria are based on the lowering of ph, owing to production of organic acids into the surrounding medium [2, 4, 6, 18, 21]. The initial isolation of phosphate solubilizers is usually made by using a medium suspended with insoluble phosphates such as tri-calcium phosphates [23]. The production of clearing zones around the colonies of the organism is an indication of the presence of phosphate-solubilizing organisms. Such cultures are isolated and the extent of phosphate solubilization is determined quantitatively, by biochemical methods [22, 23]. However, screening a large number of isolates for phosphate solubilization by quantitative methods requires investment of time, labor, and chemicals. Therefore, it was envisaged to modify the NBRIP medium by using BPB, a blue-colored dye, that decolorizes owing to a drop in ph of the medium, as an indicator to quickly evaluate the level of phosphate solubilization based upon visual observations. BPB has been used earlier by Gupta and colleagues [5] in a modified PVK medium for qualitative estimation of phosphate solubilization by bacteria and fungi in plate

3 S. Mehta and C.S. Nautiyal: Phosphate-Solubilizing Microbiological Growth Medium 53 Fig. 1. Qualitative ( ) and quantitative ( ) analysis of bacterial strains ASLU13 (A, C, E, G) and N3 (B, D, F, G) grown in NBRIP medium, containing 0.01 (A, B); (C, D); 0.05 (E, F) and 0.1 (G, H) mg/ml of bromophhenol blue, at 30 C, ph 7 for 3 days. The data are means of three independent experiments. Fig. 2. Qualitative ( ) and quantitative ( ) analysis of ten bacterial strains, ASLU13 (A), (B), AS7.8 (C), 2661 (D), 3246 (E), 4003 (F),N(G),P(H), SN15 (I), and SN16 (J) grown in NBRI-BPB medium, at 30 C, ph 7 for 3 days. The data are means of three independent experiments. assays. However, it has been reported that many isolates that did not show any clear zone on agar plates solubilized insoluble inorganic phosphates in liquid medium [10, 11]. Thus, the existing plate assay fails where the halo is inconspicuous or absent. Contrary to indirect measurement of phosphate solubilization by plate assay, the direct measurement of phosphate solubilization in broth assay always led to reliable results [13]. It was suggested that microbes from soil may be screened in NBRIP broth assay for the identification of the most efficient phosphate solubilizers [5, 7, 15]. However, in our attempt to reduce the time required to perform a quantitative assay, we modified NBRIP medium by using BPB as an indicator dye for visual observations, to quickly evaluate the level of phosphate solubilization. It was of interest to compare the influence of BPB on qualitative and quantitative analysis, using bacterial strains ASLU13 (an efficient phosphate solubilizer) and N3 (a moderately efficient phosphate solubilizer) grown in NBRIP liquid medium containing 0.01, 0.025, 0.05, and 0.1 mg/ml BPB, for 3 days (Fig. 1). The highest limit of decolorization ( O.D. at 600 nm) in the presence of 0.01 (Fig. 1A) and (Fig. 1C) mg/ml BPB by ASLU13 was achieved by day 3. For N3, the highest limit of decolorization of BPB was achieved for 0.01 mg/ml BPB (Fig. 1B), while it decolorized mg/ml BPB to 0.25 O.D. by day 3 (Fig. 1D). Our data show that it is possible to distinguish among ASLU13 and N3 based on quantitative analysis, by using mg/ml BPB (Fig. 1). It was observed that NBRIP liquid medium containing 0.01, 0.025, 0.05, and 0.1 mg/ml BPB had no appreciable effect on the quantitative analysis of phosphate solubilization by ASLU13 and N3 (Fig. 1). However, on the basis of a qualitative analysis by increasing the concentration of BPB to more than mg/ml, unlike quantitative analysis, it was not possible to distinguish among ASLU13 and N3 (Fig. 1). Therefore, a concentration of mg/ml BPB was used to formulate the new medium. This National Botanical Research Institute s phosphate growth medium containing mg/ml BPB was designated as NBRI-BPB. The potential of NBRI-BPB to evaluate a large number of phosphate solubilizers was tested by initial screening of 2015 bacterial strains. From the quantitative assay, ten bacterial strains ASLU13, 12.33, AS7.8, 2661, 3246, 4003, N, P, SN15, and SN16 were selected for further work (data not presented). Qualitative and quantitative analysis was carried out with ten bacterial strains, ASLU13 (Fig. 2A), (Fig. 2B), AS7.8 (Fig. 2C), 2661 (Fig. 2D), 3246 (Fig. 2E), 4003 (Fig. 2F), N (Fig. 2G), P (Fig. 2H), SN15 (Fig. 21), and SN16 (Fig. 2J) grown on NBRI-BPB for 3 days. The strains selected could be placed into two distinct groups based upon the level of phosphate solubilization. The first group of five

4 54 CURRENT MICROBIOLOGY Vol. 43 (2001) Fig. 3. Qualitative ( ) and quantitative ( ) analysis of the effect of various carbon, nitrogen, salts, and phosphate solubilization defective transposon5 (Tn5) mutants. To check the effect of carbon sources, glucose (A) in the NBRIP-BPB was replaced by the carbon source, arabinose (B), glycerol (C), xylose (D), fructose (E), as indicated. To check the effect of nitrogen sources, (NH 4 ) 2 SO 4 (A) in the NBRIP-BPB was replaced by the nitrogen source, (NH 4 )2Cr 2 O 7 (F), C 4 H 12 N 2 O 6 (G), NH 4 HCO 3 (H), C 24 H 20 Bi 4 O 28.6NH 3 10H 2 O(I), as indicated. To check the effect of salts, MnCl 2 4H 2 O; 2.5 mg/ml (J), NaNO 3 ; 2.5 mg/ml (K), CaCl 2 2H 2 O; 0.25 mg/ml (L), MnSO 4 H 2 O; 0.25 mg/ml (M) was added in the NBRIP-BPB, as indicated. Tn5 mutants, ASLU13.T035 (N), ASLU13.T168 (O), ASLU13.T268 (P), and ASLU13.T483 (Q) were grown in NBRI-BPB medium, at 30 C, ph 7 for 3 days. The data are means of three independent experiments. strains ASLU13 (Fig. 2A), (Fig. 2B), AS7.8 (Fig. 2C), 2661 (Fig. 2D), and 3246 (Fig. 2E) solubilized phosphate and was at least three-fold more efficient than the second group of five strains 4003 (Fig. 2F), N (Fig. 2G), P (Fig. 2H), SN15 (Fig. 2I), and SN16 (Fig. 2J). Comparative studies on the strains both in qualitative and quantitative assay gave similar results, as the strains could easily be divided into two groups. These findings indicate that there is a correlation between a qualitative and a quantitative assay. However, in a qualitative assay with NBRI-BPB, it was possible to quickly distinguish the two groups of bacteria without any need for timeconsuming biochemical methods usually involved in the quantitative assay of phosphate solubilizers. The results suggest that NBRI-BPB should serve as an excellent formulation for the initial screening of a large number of phosphate solubilizers. An investigation was carried out to assess the probability of an early detection of the effect of various physiological parameters on qualitative and quantitative analysis, by using NBRI-BPB. Phosphate solubilization activity of ASLU13 was monitored in the presence of various carbon, nitrogen, and salts. ASLU13 as compared with control NBRI-BPB (Fig. 3A), demonstrated diverse level of phosphate solubilization activity in the presence of various carbon [arabinose (Fig. 3B), glycerol (Fig. 3C), xylose (Fig. 3D), fructose (Fig. 3E)]; nitrogen [(NH 4 )2Cr 2 O 7 (Fig. 3F), C 4 H 12 N 2 O 6 (Fig. 3G), NH 4 HCO 3 (Fig. 3H), C 24 H 20 Bi 4 O 28 6NH 3 10H 2 O (Fig. 3I)]; and salts [MnCl 2 4H 2 O (Fig. 3J), NaNO 3 (Fig. 3K), CaCl 2 2H 2 O (Fig. 3L), MnSO 4 H 2 O (Fig. 3M)]. The pattern of phosphate solubilization by ASLU13 in qualitative assay with NBRI-BPB correlated well with the quantitative assay. This observation further augurs well for the use of NBRI-BPB for qualitative analysis to detect the effect of various physiological factors on phosphate solubilizers, based upon visual observation. Pure culture evaluation by using NBRI-BPB may be a useful tool in the search for phosphate-solubilizing strains better suited for soil environments where physiological factors may constitute a limitation for phosphate solubilization. An experiment was conducted to screen 500 phosphate solubilization-defective Tn5 mutants of ASLU13. Based upon visual observation, owing to their incapability to decolorize BPB efficiently, as compared with ASLU13 (Fig. 3A), four mutants ASLU13.T035 (Fig. 3N), ASLU13.T168 (Fig. 3O), ASLU13.T268 (Fig. 3P),

5 S. Mehta and C.S. Nautiyal: Phosphate-Solubilizing Microbiological Growth Medium 55 Fig. 4. Qualitative ( ) and quantitative ( ) analysis of the ASLU13 (A, B, C) and various commercial bioinoculants, based on phosphate solubilzers, CPB1 (D, E, F), CPB2 (G, H, I), CPB3 (J, K, L), CPB4 (M, N, O), and CPB5 (P, Q, R), grown in NBRI-BPB medium, at 30 C, ph 7 for upto 1 day (A, D, G, J, M, P), 2 day (B, E, H, K, N, Q), and 3 day (C, F, I, L, O, R). The data are means of three independent experiments. and ASLU13.T483 (Fig. 3Q) were easily distinguishable by day 3. Quantitative analysis further confirmed the diverse levels of phosphate solubilization ability of the mutants (Fig. 3). These findings further demonstrate that there is a correlation between the pattern of phosphate solubilization by the mutants in qualitative assay and quantitative assay. The data thus show that, with our simple protocol, it is indeed possible to screen a large number of phosphate-solubilizing defective mutants. Phosphate solubilizing bacteria have been used in the commercial preparation of phosphate-dissolving cultures to improve the growth of plants [22, 23]. Based on the observations obtained as above, a study was conducted to evaluate the possibility of using NBRI-BPB for quickly assessing the quality of commercial bioinoculant preparations, based on phosphate solubilizers. In a comparative study, our ASLU13-based bioinoculant preparation (4A, 4B, 4C), along with five commercial products CPB1 (4D, 4E, 4 F), CPB2 (4G, 4H, 4I), CPB3 (4J, 4K, 4L), CPB4 (4M, 4N, 4O), and CPB5 (4P, 4Q, 4R) grown in NBRI-BPB medium, at 30 C, ph 7 for up to 1 day (4A, 4D, 4G, 4J, 4M, 4P), 2 days (4B, 4E, 4H, 4K, 4N, 4Q), and 3 days (4C, 4F, 4I, 4L, 4O, 4R), was subjected to testing by the new formulation (Fig. 4). Decolorization of BPB with our ASLU13-based bioinoculant preparation was achieved by day 2 (Fig. 4B). Among the five products tested, based upon visual observation, the highest limit of decolorization of BPB in products CPB1 (Fig. 4F) and CPB3 (Fig. 4L) was achieved by day 3, while decolorization of BPB in CPB2 (Fig. 4B), CPB4 (Fig. 4O), and CPB5 (Fig. 4R) was under by day 3. Thus, the commercial bioinoculant product CPB1 was easily distinguishable from other products in its ability to solubilize phosphate. Furthermore, the present work indicates that the formulation can also be used as a quality control test for expeditious screening of the commercial bioinoculant preparations, based on phosphate solubilizers. The results suggest that, by using NBRI-BPB based upon visual observations, it is indeed possible to classify the phosphate solubilizers on a qualitative basis, among various groups. Qualitative analysis of the phosphate solubilized by various groups also correlated well with our grouping based upon quantitative analysis of bacteria isolated from soil, the effect of various carbon, nitrogen, and salts, and phosphate solubilization-defective transposon mutants. However, unlike quantitative analysis, the time for screening phosphate solubilizers based upon visual observation is significantly reduced by using our simple protocol. Therefore, it is envisaged that use of this protocol based upon qualitative analysis will be salutary for the quick screening and grouping of phosphate-solubilizing bacteria. ACKNOWLEDGMENTS We are grateful to P. Puspangadan, Director, National Botanical Research Institute, for his valuable encouragement and providing necessary facilities. Sincere thanks are due to K.V.B.R. Tilak, Head, Divi-

6 56 CURRENT MICROBIOLOGY Vol. 43 (2001) sion of Microbiology, Indian Agricultural Research Institute, New Delhi, for useful discussions. Financial assistance in part was provided by Super Special Grant from Director General, Council of Scientific & Industrial Research, New Delhi and Department of Biotechnology, Ministry of Science & Technology, New Delhi, Grant No. BT/ PRO322/R&D/12/9/96 awarded to C. S. Nautiyal. Literature Cited 1. Abd-Alla MH (1994) Phosphatases and the utilization of organic phosphorus by Rhizobium leguminosarum biovar viceae Lett Appl Microbiol 18: Bajpai PD, Sundara Rao WVB (1971) Phosphate solubilizing bacteria II. Extracellular production of organic acids by selected bacteria solubilizing insoluble phosphates. Soil Sci Plant Nutr 17: Fiske CH, Subbarow Y (1925) A colorimetric determination of phosphorus. J Biol Chem 66: Gaind S, Gaur AC (1989) Effect of ph on phosphate solubilization by microbes. Curr Sci 58: Gupta R, Singal R, Shankar A, Kuhad RC, Saxena RK (1994) A modified plate assay for screening phosphate solubilizing microorganisms. J Gen Appl Microbiol 40: Johnston HW (1952) The solubilization of phosphate : the action of various organic compounds on dicalcium and tricalcium phosphate. NZ J Sci Technol 33: Johri JK, Surange S, Nautiyal CS (1999) Occurrence of salt, ph and temperature-tolerant, phosphate-solubilizing bacteria in alkaline soils. Curr Microbiol 39: Jones DL, Darrah PR (1994) Role of root derived organic acids in the mobilization of nutrients from the rhizosphere. Plant Soil 166: Katznelson H, Peterson, E. Rouatt JW (1962) Phosphate dissolving microorganisms on seed and in the root zone of plants. Can J Bot 40: Leyval C, Barthelin J (1989) Interactions between Laccaria laccata, Agrobacterium radiobacter and beech roots: influence on P, K, Mg and Fe mobilization from mineral and plant growth. Plant Soil 17: Louw HA, Webley DM (1959) A study of soil bacteria dissolving certain phosphate fertilizers and related compounds. J Appl Bacteriol 22: Nautiyal CS (1997) A method for selection and characterization of rhizosphere-competent bacteria of chickpea. Curr Microbiol 34: Nautiyal CS (1999) An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 170: Nautiyal CS, van Berkum P, Sadowasky M, Keister DL (1989) Cytochrome mutants of Bradyrhizobium induced by transposon Tn5. Plant Physiol 90: Nautiyal CS, Bhadauria S, Kumar P, Lal H, Mondal R, Verma D (2000) Stress induced phosphate solubilization in bacteria isolated from alkaline soils. FEMS Microbiol Lett 182: Pal SS (1998) Interactions of an acid tolerant strain of phosphate solubilizing bacteria with a few acid tolerant crops. Plant Soil 198: Pikovskaya RI (1948) Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17: Rose RE (1957) Techniques of determining the effect of microorganisms on insoluble inorganic phosphates. NZ J Sci Technol 38: Rostas K, Sista PR, Stanley J, Verma DPS (1984) Transposon mutagenesis of Rhizobium japonicum. Mol Gen Genet 197: Salvaraj G, Iyer VN (1983) Suicide plasmid vehicles for insertion mutagenesis in Rhizobium spp. J Bacteriol 156: Sethi RP, Subba Rao NS (1968) Solubilization of tricalcium phosphate and calcium phytase by soil fungi. J Gen Appl Microbiol 14: Subba Rao NS (1993) Biofertilizers in agriculture and forestry. Oxford, New Delhi: Oxford Press and IBH Publishing Co. 23. Tilak KVBR (1993) Bacterial fertilizers. New Delhi, India: Indian Council of Agricultural Research 24. Yadav KS, Dadarwal KR (1997) Phosphate solubilization and mobilization through soil microorganisms. In: Dadarwal KR (ed): Biotechnological approaches in soil microorganisms for sustainable crop production. Jodhpur, India: Scientific Publishers, pp Yahya Al, Al-Azawi SK (1989) Occurrence of phosphate-solubilizing bacteria in some Iraqi soils. Plant Soil 117: