Stress induced phosphate solubilization in bacteria isolated from alkaline soils

FEMS Microbiology Letters 182 (2000) 291^296

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Stress induced phosphate solubilization in bacteria isolated from alkaline soils C. Shekhar Nautiyal *, Shipra Bhadauria, Pradeep Kumar, Hind Lal, Rajesh Mondal, Dinesh Verma Microbiology Group, National Botanical Research Institute, Rana Pratap Marg, P.O. Box 436, Lucknow 226 001, India Received 27 January 1999 ; received in revised form 1 September 1999; accepted 28 October 1999

Abstract Phosphate solubilizing bacteria NBRI0603, NBRI2601, NBRI3246 and NBRI4003 were isolated from the rhizosphere of chickpea and alkaline soils. All four strains demonstrated diverse levels of phosphate solubilization activity under in vitro conditions in the presence of various carbon and nitrogen sources. Acid production may have contributed to phosphate solubilization, but was not the only reason for phosphate release into the medium. Among the four strains, NBRI2601 was the most efficient strain in terms of its capability to solubilize phosphorus in the presence of 10% salt, pH 12, or 45³C. The strains showed varied levels of phosphate solubilization when the effects of different sources of nitrogen were examined during growth. The presence of low levels of Ca2‡ and EDTA in the medium enhanced phosphate solubilization. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Phosphate solubilizing bacteria ; Phosphate mineralization; Alkaline soil; Stress

1. Introduction Improving soil fertility is one of the most common tactics to increase agricultural and forest production. Maintaining high levels of available nitrogen (N) and phosphorus (P), the two most limiting nutrients in soils, remains a major challenge to ecologists and land managers. The availability of soil P is largely controlled by biologically mediated processes such as gross mineralization and immobilization rates [1]. Thus, even if the total P is high and also if P fertilizers are applied regularly, the P is rapidly ¢xed to unavailable forms and accounts for low P use e¤ciency. Were it not for P-releasing mechanisms at work in the soil, biological immobilization and chemical precipitation would soon deplete the available P supply, leaving very little available for plants [2]. Therefore, the pH dependent release of insoluble and ¢xed forms of P is an important aspect of increasing soil P availability. Many soil microorganisms are able to solubilize `unavailable' forms of calcium-bound P through their metabolic activities, by excreting organic acids which either directly dis-

* Corresponding author. Tel. : +91 (522) 271031-35; Fax: +91 (522) 282849/282881.

solve rock phosphate or chelate calcium ions to bring P into solution. The production of microbial metabolites results in a decrease in soil pH, which probably plays a major role in solubilization. Besides changes in pH, chelations by organic acids which bind phosphate anions also bring about phosphate in soil solution [2^4]. Soil inoculation with phosphate solubilizing bacteria (PSB) has been shown to improve solubilization of ¢xed soil P and applied phosphates resulting in higher crop yields. PSB are found in the majority of soils [5^7]. However, their performance is severely in£uenced by environmental factors especially under stress conditions [8,9]. Bacteria growing in alkaline soils in India during the summer season are subjected to high salt, high pH and high temperature stress. In the alkaline soils of the tropics, salt concentrations and pH may be as high as 2% and 10.5, respectively, and temperatures may range between 35^45³C [10]. These conditions may result in poor growth and survival of PSB. The available P in these soils is poor, and the most appropriate solution to this situation is to use PSB as bioinoculants. However, detailed studies have not been made on PSB isolated from alkali soils. To understand phosphate solubilization by PSB isolated from alkali soils, an understanding of the physiology of these organisms, under similarly stressed conditions is re-

0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 6 0 5 - 9

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quired. PSB, 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. Therefore, the objective of this study was to isolate and characterize PSB strains from alkali soils which could solubilize phosphates at high salt, high pH and high temperature. 2. Materials and methods 2.1. Isolation of phosphate solubilizing bacteria from the rhizosphere and soil Bacterial strains were isolated from four di¡erent sites using root-free soil, soil surrounding the roots of chickpea (rhizosphere) growing in alkaline soil (exposed to high salt, pH and temperature stress) in the range of pH 8.5 to 11.0 from Banthara Research Station, Lucknow, India. The soil temperature of the sites from where these bacteria were isolated during the summer varies from 48 to 52³C at Banthra village, Lucknow, India as described earlier [11]. To isolate rhizosphere bacteria, the adhering soil on the root was gently shaken to collect the rhizosphere soil. Roots were thoroughly washed with tap water for two min to remove all the loosely adhering soil particles, followed by washing with sterile 0.85% (w/v) saline Milli Q water (MQW), to isolate rhizoplane bacteria. 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 individually plated on Pseudomonas isolation agar, nutrient agar and tryptone-glucose-yeast extract (TGY) agar (from HI-medium Laboratories Pvt. Ltd., Bombay, India), as described earlier [11]. 2.2. Medium and growth conditions Bacteria representative of the predominant morphological types present on the plates were selected at random and puri¢ed on minimal medium based on AT salts which contained the following ingredients per liter: glucose, 10.0 g; KH2 PO4 , 10.9 g; (NH4 )SO4 , 1.0 g; MgSO4 W7H2 O, 0.16 g; FeSO4 W7H2 O, 0.005 g; CaCl2 W 2H2 O, 0.011 g; and MnCl2 W4H2 O, 0.002 g [11]. Unless otherwise stated, National Botanical Research Institute's phosphate growth medium (NBRIP) contained per liter: glucose, 10 g; Ca3 (PO4 )2 , tricalcium phosphate (TCP), 5 g; MgCl2 W6H2 O, 5 g; MgSO4 W7H2 O, 0.25 g; KCl, 0.2 g; and (NH4 )2 SO4 , 0.1 g [12]. Quantitative estimation of phosphate solubilization in broth was carried out using Erlenmeyer £asks (150 ml) containing 10 ml of medium inoculated in triplicate with the bacterial strain (100 Wl inoculum with approximately 1 to 2U109 cfu ml31 ). The absolute value of the control refers to the amount of P solubilized (Wg ml31 ) by each

strain (NBRI0603, NBRI2601, NBRI3246 or NBRI4003), when individually grown for 3 days in NBRIP at 30³C in the presence of 0% salt (NaCl) at pH 7. The e¡ect of carbon and nitrogen on phosphate solubilization by the strains was tested by growing them on NBRIP as indicated. To check the e¡ect of the carbon source, glucose in the NBRIP was replaced by the carbon source as indicated. To check the e¡ect of the nitrogen source, (NH4 )2 SO4 in the NBRIP was replaced by the nitrogen source as indicated. The e¡ect of salt (NaCl), pH, temperature, calcium, and disodium salt of ethylenediamine tetraacetic acid (EDTA) on the solubilization of phosphate was tested by growing the strains on NBRIP containing various amounts of NaCl, pH, temperature, calcium and EDTA as indicated. Autoclaved, uninoculated batch cultures served as negative controls. Unless stated, the £asks were incubated for 3 days at 30³C on a New Brunswick Scienti¢c, USA, Innova Model 4230 refrigerated incubator shaker at 180 rpm. The strains were harvested by centrifugation at 19 950Ug for 10 min, using a Sorvall RC 5C centrifuge, Dupont, USA. The ¢nal pH of the growth medium was measured using a Mettler-Toledo Inc., USA, pH meter Model MP-220, and the coe¤cient of variation was within 2% for triplicate samples. The concentration of phosphate in culture supernatant was estimated using the Fiske and Subbarow method [13]. Values are given as means þ S.D. for triplicate samples. Data were analyzed by analysis of variance or by regression analysis. Di¡erences were considered to be signi¢cant at the P 6 0.05 level. 3. Results and discussion Tolerance to high salt, high pH and high temperature stresses may be important in the survival, multiplication and spread of bacterial strains in alkaline soils. Stress-tolerant bacteria are likely to be found in environments affected by osmotic, pH and temperature stresses. Recently we have formulated a de¢ned medium for screening PSB and established a procedure for the identi¢cation of most of the e¤cient phosphate solubilizers from soil [12]. Using this method, we isolated four bacterial isolates NBRI0603, NBRI2601, NBRI3246 and NBRI4003. Strain NBRI0603 was isolated from the rhizosphere of chickpea growing in alkaline soil and the other three isolates were isolated from di¡erent alkaline soils. 3.1. Solubilization of phosphate by bacteria from alkaline soils The phosphate solubilization by the four isolates was monitored up to 5 days in NBRIP medium at 30³C in the presence of 0% salt (NaCl) at pH 7. The level of solubilized P by NBRI0603, NBRI2601, NBRI3246, and NBRI4003 increased linearly, until the second, third, sec-

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Table 1 E¡ect of various carbon and nitrogen sources on TCP solubilization by NBRI0603, NBRI2601, NBRI3246 and NBRI4003 Ingredient(s)

Phosphate solubilization compared to control (%) NBRI0603

NBRI2601

NBRI3246

NBRI4003

Controla Absolute valueb Carbon sourcec Fructose Galactose Sorbitol Mannitol Xylose Sucrose Maltose Lactose Nitrogen sourced (NH4 )2 NO3 KNO3 CaNO3 NaNO3

100 250 þ 11.5

100 450 þ 21.7

100 290 þ 13.5

100 200 þ 9.8

105 54 2 95 131 5 1 0

91 50 76 102 130 25 60 150

55 58 2 40 132 5 10 51

11 32 5 28 5 7 26 17

80 57 63 83

110 125 109 101

105 120 94 115

93 60 113 56

a

Control strains were grown at 30³C for 3 days in National Botanical Research Institute's phosphate growth medium (NBRIP) which contained per liter: glucose, 10 g; Ca3 (PO4 )2 , 5 g; MgCl2 W6H2 O, 5 g; MgSO4 W7H2 O, 0.25 g; KCl, 0.2 g; and (NH4 )2 SO4 , 0.1 g at pH 7. Autoclaved, uninoculated batch cultures served as negative controls. b The absolute value of phosphate solubilization (Wg ml31 ) of control corresponding to 100%. Values are means þ S.D. for triplicate samples. c glucose in the NBRIP was replaced by the carbon source as indicated. d (NH4 )2 SO4 in the NBRIP was replaced by the nitrogen source as indicated.

ond and third day, respectively. Maximum solubilization of phosphate was achieved after 3 days of incubation. Further incubation up to 5 days did not improve the extent of solubilization (data not presented). Therefore, the batch tests were analyzed after 3 days for all four strains. Under these conditions, NBRI2601 was the most e¤cient strain in solubilizing phosphate followed by NBRI3246, NBRI0603 and NBRI4003 in decreasing order of e¤ciency (Table 1). At the end of each day, the ¢nal pH was determined to ¢nd out whether solubilization of phosphate was accompanied by the production of acid in the growth medium. All four strains produced acid and lowered the pH of growth medium from neutral to 3^3.5 after one day. Thereafter, the pH of the medium remained stable for up to 5 days (data not presented). The decrease in pH clearly indicates the production of acids, which is considered to be responsible for P solubilization. It has been suggested that microorganisms which decrease the medium pH during growth are e¤cient P solubilizers [14,15]. 3.2. E¡ect of carbon and nitrogen sources on phosphate solubilization Phosphate solubilization activity of NBRI0603, NBRI2601, NBRI3246 and NBRI4003 was evaluated in the presence of nine carbon and four nitrogen sources, by replacing glucose and (NH4 )2 SO4 , respectively (Table 1). All four strains demonstrated diverse levels of phosphate solubilization activity in the presence of various carbon and nitrogen sources. Among the four strains, NBRI2601 proved to be the most e¤cient strain consider-

ing its capability to solubilize P utilizing a wide range of carbon and nitrogen sources (Table 1). Xylose, lactose, xylose and glucose were the best carbon sources for the phosphate solubilization by strains NBRI0603, NBRI2601, NBRI3246 and NBRI4003, respectively (Table 1). NBRI0603 could not solubilize phosphate e¤ciently with sucrose, sorbitol, maltose and lactose as the sole carbon source. Sucrose and sorbitol were identi¢ed as poor carbon source for phosphate solubilization for NBRI3246. Sucrose, sorbitol and xylose were identi¢ed as poor carbon source for phosphate solubilization for NBRI4003 (Table 1). All nitrogen sources were utilized for phosphate solubilization by the four strains (Table 1). Contrary to previous reports [15,16] we observed ammonium and nitrate source to be equally e¡ective for phosphate solubilization. Previous reports on PSB [14,15] have attributed the di¡erences in phosphate solubilization when ammonium and nitrate were used to the use of di¡erent mechanisms for the generation of acidity in the culture [17^19]. The presence of ammonium in the growth medium of Penicillium cyclopium was reported to result in the development of inorganic acid by a proton exchange mechanism [17]. However, a second mechanism did not require the presence of ammonium and probably involved the excretion of organic acid metabolites by phosphate solubilizing fungi [18,19]. Our results revealed that phosphate solubilization by NBRI0603, NBRI2601, NBRI3246 and NBRI4003 varied when ammonium and di¡erent sources of nitrates were used as nitrogen source (Table 1). Our study suggests that, contrary to the previous reports on PSB [15,16],

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more versatile mechanism(s) for phosphate solubilization may be adopted by PSB. Thus the two mechanisms suggested for fungi for the generation of acid may not be the only mechanisms for phosphate solubilizers [17^19]. 3.3. E¡ect of high salt, high pH and high temperature on phosphate solubilization To study the e¡ect of high salt, high pH and high temperature stresses on the phosphate solubilization ability of NBRI0603, NBRI2601, NBRI3246 and NBRI4003, the strains were grown in the presence of high salt (2.5, 5, 7.5 and 10% NaCl), high pH (8, 9, 10, 11 and 12) and high temperature (37³C and 45³C) (Table 2). The strains seemed generally well adapted to the environments from which they had been isolated (i.e. hot, dry or salt a¡ected ecosystems). All four strains demonstrated diverse levels of phosphate solubilization in the presence of high salt, high pH or high temperature. The phosphate solubilization abilities of the four strains were higher than the controls in the presence of 2.5% salt and pH 8 (Table 2). It seemed, therefore, that the strains isolated from alkaline soils have the potential to solubilize phosphates at high salt, high pH and high temperature. Among the four strains, NBRI2601 proved to be the most e¤cient strain in solubilizing P individually in the presence of 10% salt, pH 12, or 45³C (Table 2). To our knowledge, this is the ¢rst report of a PSB demonstrating phosphate solubilization ability in the presence of such extreme conditions. In general, phosphate solubilization was accompanied by a decrease in the pH of the medium. All four strains demonstrated a signi¢cant

decline in phosphate solubilization when the ¢nal pH of the medium was about 8.0, and above (Table 2). However, decrease in the medium pH was not always correlated with phosphate solubilization activity. For example, the ¢nal pH of the NBRI2601 and NBRI4003 in the presence of 0^ 10% salt were approximately comparable, contrary to disparate level of phosphate solubilization (Table 2). The overall results of the study that acid production was not the only reason for phosphate release into the medium were in agreement with data obtained earlier by AbdAlla [16]. 3.4. E¡ect of calcium supplement on phosphate solubilization To determine the e¡ect of increasing amounts of various calcium salts (CaCl2 , CaSO4 , CaCO3 and Ca(OH)2 ) supplements on phosphate solubilization, the cells were cultivated in NBRIP medium containing 0, 0.25, 0.5, 1.0, 2.5 and 5.0 mg ml31 as Ca of calcium salts. Data presented in Table 3 show diverse levels of calcium salt induced phosphate solubilization ability by the four strains. An increase up to 122% in the phosphate solubilization ability of the strains was observed in the presence of calcium salts (Table 3). The trait of enhanced phosphate solubilization in the presence of certain calcium salts might be of some signi¢cance for the survival of PSB in alkaline soils. The decrease in the phosphate solubilization with increasing CaCl2 and CaSO4 concentrations was not associated with the ¢nal pH of media. This indicates a role of calcium activity in the phosphate solubilization. Studies of Wilson

Table 2 E¡ect of salt, pH and temperature on TCP solubilization by NBRI0603, NBRI2601, NBRI3246 and NBRI4003 Treatments a

Control Absolute valueb Salt (NaCl, w/v)c 2.5 5 7.5 10 pH 8 9 10 11 12 Temperature (³C) 37 45

Phosphate solubilization compared to control (%) NBRI0603

NBRI2601

NBRI3246

NBRI4003

100 (3.5) 250 þ 11.5

100 (3) 450 þ 21.7

100 (3.5) 290 þ 13.5

100 (3.2) 200 þ 9.8

119 (3.5) 88 (3.7) 8 (4.8) 0 (5.4)

115 (3) 80 (3) 33 (3.2) 18 (3.5)

110 (3.5) 93 3.8) 9 (5) 2 (5.5)

110 (3.2) 80 (3.2) 65 (3.4) 3 (3.8)

112 (3.5) 114 (3.6) 4 (8) 0 (8.3) 0 (9)

113 (3) 95 (3) 37 (4.8) 16 (5.2) 8 (8)

110 (3.5) 124 (3.6) 9 (8) 3 (8.2) 0 (9)

115 (3.2) 10 (3.4) 5 (7.8) 0 (8.4) 0 (9)

56 (3.8) 8 (4.5)

87 (3.8) 73 (4.2)

58 (3.6) 20 (4.4)

55 (3.7) 2 (5)

a

Control strains were grown for 3 days in National Botanical Research Institute' phosphate growth medium (NBRIP) which contained per liter : glucose, 10 g; Ca3 (PO4 )2 , 5 g; MgCl2 W6H2 O, 5 g; MgSO4 W7H2 O, 0.25 g; KCl, 0.2 g; and (NH4 )2 SO4 , 0.1 g at 30³C in the presence of 0% salt (NaCl) at pH 7. Autoclaved, uninoculated batch cultures served as negative controls. Final pH of the growth medium is given within the parentheses, and variation (S.D.) was within þ 0.2, for triplicate samples. b The absolute value of phosphate solubilization (Wg ml31 ) of control corresponding to 100%. Values are means þ S.D. for triplicate samples. c NBRIP was modi¢ed with various amounts of salt (NaCl, w/v), pH or temperature as indicated.

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Table 3 E¡ect of various calcium supplements on TCP solubilization by NBRI0603, NBRI2601, NBRI3246 and NBRI4003 Calcium

a

Control Absolute valueb CaClc2 0.25 0.5 1.0 2.5 5.0 CaSO4 0.25 0.5 1.0 2.5 5.0 CaCO3 0.25 0.5 1.0 2.5 5.0 Ca(OH)2 0.25 0.5 1.0 2.5 5.0

Phosphate solubilization compared to control (%) NBRI0603

NBRI2601

NBRI3246

NBRI4003

100 (3.5) 250 þ 11.5

100 (3) 450 þ 21.7

100 (3.5) 290 þ 13.5

100 (3.2) 200 þ 9.8

200 (3.2) 200 (3.4) 181 (3.6) 169 (3.8) 88 (4.2)

113 (2.9) 115 (3) 121 (3.2) 101 (3.2) 78 (3)

186 157 154 143 100

(3.6) (3.4) (3.5) (3.5) (3.6)

138 (3.3) 143 (3.4) 125 (3.5) 88 (3.6) 63 (3.4)

125 132 156 138 100

131 (3.2) 121 (2.9) 90 (3.1) 85 (3.2) 75 (3.2)

143 (3.5) 150 (3.5) 157 (3.6) 105 (3.4) 86 (3.5)

125 (3.5) 113 (3.4) 100 (3.5) 75 (3.3) 38 (3.4)

213 (3.4) 200 (3.5) 193 (3.8) 0 (7.5) 0 (7.3)

94 (3.2) 97 (3.5) 63 (4) 0 (4.8) 0 (6.3)

222 (3.6) 165 (3.5) 137 (4.2) 0 (5.1) 0 (6.3)

63 (3.8) 43 (3.4) 5 (4) 0 (7) 0 (7.4)

75 (3.8) 13 (4.3) 0 (4.9) 0 (6.6) 0 (9.4)

69 (4) 50 (4.3) 38 (4.8) 0 (7.5) 0 (9.5)

186 (4) 137 (4.2) 69 (5) 0 (7.3) 0 (9.4)

30 (3.5) 0 (3.6) 0 (4) 0 (7.2) 0 (8.8)

(3.5) (3.4) (3.3) (3.6) (4)

a

Control strains were grown for 3 days in National Botanical Research Institute's phosphate growth medium (NBRIP) which contained per liter: glucose, 10 g; Ca3 (PO4 )2 , 5 g; MgCl2 36H2 O, 5 g; MgSO4 37H2 O, 0.25 g; KCl, 0.2 g; and (NH4 )2 SO4 , 0.1 g at 30³C in the presence of 0% salt (NaCl) at pH 7. Autoclaved, uninoculated batch cultures served as negative controls. Final pH of the growth medium is given within the parentheses, and variation (S.D.) was within þ 0.2, for triplicate samples. b The absolute value of phosphate solubilization (Wg ml31 ) of control corresponding to 100%. Values are means þ S.D. for triplicate samples.

and Ellis [20] suggested the calcium activity as an important factor controlling the rate and extent of dissolution of rock phosphate. Inhibition of phosphate solubilization in the presence of the calcium salts CaCO3 and Ca(OH)2 at 2.5 and 5.0 mg ml31 as Ca was associated with a higher pH as compared to the control (Table 3). Contrary to the growth of strains in CaCl2 and CaSO4 , the growth was poor in the presence of CaCO3 and Ca(OH)2 at 2.5 and 5.0 mg ml31 . The presence of CaCO3 , due to its bu¡ering nature, may have provided resistance to the decrease in pH normally caused by the bacterial production of acid. At 5.0 mg ml31 of CaCO3 , the ¢nal pH for all four strains was close to neutrality and the level of phosphate solubilization was negligible. The absence of phosphate solubilization in the presence of Ca(OH)2 at 2.5 and 5.0 mg ml31 on the other hand, could have been attributed to its basic nature, which caused the medium pH to be highly alkaline, which inhibited the growth of the bacteria and phosphate solubilization. The data apparently indicate the role of acid for phosphate solubilization. However, acid production was not always correlated with phosphate solubilization activity. For example, the growth and ¢nal pH of the strains in the presence of CaCO3 and Ca(OH)2 at 0.25,

0.5, and 1.0 mg ml31 were comparable, contrary to diverse level of phosphate solubilization (Table 3). The results of the study further support our earlier observations that acid production was not the only reason for phosphate release into the medium. 3.5. E¡ect of EDTA on phosphate solubilization The e¡ect of chelation on solubilization of phosphate from TCP was studied by adding 0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0 EDTA salt (mg ml31 ) to the strains. Addition of 0.5 mg ml31 EDTA caused maximal increase of 76, 28, 48 and 15% phosphate solubilization by NBRI0603, NBRI2601, NBRI3246 and NBRI4003, respectively, compared to control strains (Fig. 1). This could be due to chelation of Ca2‡ produced during TCP dissolution. Further increase in the EDTA caused a linear decrease in the solubilization of phosphate (Fig. 1). The results suggest that the bacterial strains NBRI0603, NBRI2601, NBRI3246 and NBRI4003 isolated from alkaline soils have evolved to solubilize phosphate in the presence of high salt, high pH or high temperature. The strains should serve as an excellent model to study the physiolog-

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References

Fig. 1. E¡ect of addition of increasing concentrations of EDTA on phosphate solubilization by PSB strains of NBRI0603, NBRI2601, NBRI3246 and NBRI4003. Solubilization of phosphate (Wg ml31 ) by phosphate solubilizing 72 h grown strains of NBRI0603 (a), NBRI2601 (b), NBRI3246 (P) and NBRI4003 (S) was determined using National Botanical Research Institute's phosphate growth medium (NBRIP) medium which contained per liter: glucose, 10 g; Ca3 (PO4 )2 , 5 g; MgCl2 W6H2 O, 5 g; MgSO4 W7H2 O, 0.25 g; KCl, 0.2 g; and (NH4 )2 SO4 , 0.1 g at 30³C in the presence of 0% salt (NaCl) at pH 7. Addition of 0.5 mg ml31 EDTA caused maximal increase in phosphate solubilization, compared to control strains. Further increase in the EDTA caused a linear decrease in the solubilization of phosphate. Variation (S.D.) was within symbol dimensions.

ical, biochemical and molecular mechanism of phosphate solubilization in stressed ecosystems. The strains may also be used as a bacterial host to propagate new or improved characters for ecosystems having problems of high salt, high pH and high temperature. Since the conditions in soil are much more complex than those in vitro, further study of the environmental factors a¡ecting phosphate solubilization in alkaline soils should suggest the basis for obtaining inoculants that are able to give greater phosphate solubilization for crops of economic or agricultural importance in tropical and subtropical areas. Acknowledgements Financial assistance in part was provided by Super Special Grant from the Director General, Council of Scienti¢c and Industrial Research, New Delhi, and Department of Biotechnology, Ministry of Science and Technology, New Delhi, Grant No. BT/PR0322/R and D/12/19/96, awarded to C. Shekhar Nautiyal.

[1] Zou, K., Binkley, D. and Doxtader, K.G. (1992) New method for estimating gross P mineralization and mobilization rates in soils. Plant Soil 147, 243^250. [2] Abd-Alla, M.H. (1994) Phosphatases and the utilization of organic P by Rhizobium leguminosarum biovar viceae. Lett. Appl. Microbiol. 18, 294^296. [3] Yadav, K.S. and Dadarwal, K.R. (1997) Phosphate solubilization and mobilization through soil microorganisms. In: Biotechnological Approaches in Soil Microorganisms for Sustainable Crop Production (Dadarwal, K.R., Ed.), pp. 293^308. Scienti¢c Publishers, Jodhpur. [4] Jones, D.L. and Darrah, P.R. (1994) Role of root derived organic acids in the mobilization of nutrients from the rhizosphere. Plant Soil 166, 247^257. [5] Subba Rao, N.S. (1982) Phosphate solubilization by soil microorganisms. In: Advances in Agricultural Microbiology (Subba Rao, N.S., Ed.), Butterworth Sci., London. [6] Chonker, P.K. and Taraeder, J.C. (1984) Accumulation of phosphates in soils. J. Indian Soc. Soil Sci. 32, 266^272. [7] Venkateswarlu, B., Rao, A.V. and Raina, P. (1984) Valuation of P solubilization by microorganisms isolated from aridsoils. J. Indian Soc. Soil Sci. 32, 273^277. [8] Yahya, A.I. and Al-Azawi, S.K. (1989) Occurence of phosphate-solubilizing bacteria in some Iraqi soils. Plant Soil 117, 135^141. [9] Pal, S.S. (1998) Interactions of an acid tolerant strain of phosphate solubilizing bacteria with a few acid tolerant crops. Plant Soil 198, 169^171. [10] Surange, S., Wollum II, A.G., Kumar, Nikhil and Nautiyal, C.S. (1997) Characterization of Rhizobium from root nodules of leguminous trees growing in alkaline soils. Can. J. Microbiol. 43, 891^894. [11] Nautiyal, C.S. (1997) A method for Selection and Characterization of Rhizosphere-Competent Bacteria of Chickpea. Curr. Microbiol. 34, 12^17. [12] Nautiyal, C.S. (1999) An e¤cient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol. Lett. 170, 265^270. [13] Fiske, C.H. and Subbarow, Y. (1925) A colorimetric determination of P. J. Biol. Chem. 66, 375^400. [14] Kpomblekou-A, K. and Tabatabai, M.A. (1994) E¡ect of organic acids on release of P from phosphate rocks. Soil Sci. 158, 442^443. [15] Halder, A.K., Mishra, A.K. and Chakarbarthy, P.K. (1991) Solubilization of inorganic phosphates by Bradyrhizobium. Ind. J. Exp. Biol. 29, 28^31. [16] Abd-Alla, M.H. (1994) Solubilization of rock phosphates by Rhizobium and Bradyrhizobium. Folia Microbiol. 39, 53^56. [17] Roos, W. and Luckner, M. (1989) Relation between proton extrusion and £uxes of ammonium ions and organic acids in Penicillium cyclopium. J. Gen. Microbiol. 130, 1007^1014. [18] Thomas, G.V. (1985) Occurence and ability of phosphate-solubilizing fungi from coconut plant soils. Plant Soil 87, 357^364. [19] Asea, P.E.A., Kucey, R.M.N. and Stewart, J.W.B. (1988) Inorganic phosphate solubilization by two penicillium species in solution culture and soil. Soil Biol. Biochem. 20, 459^464. [20] Wilson, M.A. and Ellis, B.G. (1984) In£uence of calcium solution activity and surface area on the solubility of selected rock phosphates. Soil Sci. 138, 354^359.

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