# In Vitro Solubilization of Rock Phosphate by Fungus Isolated from Agricultural Soil

Ajeet Kumar, B.R. Maurya, Shankar Jha

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Ajeet Kumar, B.R. Maurya, Shankar Jha. In Vitro Solubilization of Rock Phosphate by Fungus Isolated from Agricultural Soil. RAU Journal of Research, 2014, 24 (1&2), pp.39-48. hal-04972626

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# *In Vitro* Solubilization of Rock Phosphate by Fungus Isolated from Agricultural Soil

Ajeet Kumar, B. R. Maurya<fnref n="1" /> and Shankar Jha<fnref n="2" />

Regional Research Station, Agwanpur, Saharsa - 852201 (Bihar), India
E-mail: ajeetbau@gmail.com

Thirty fungal species isolated from soil were screened out on the basis of halozone diameter produced on the Pikovskaya's agar media and tested for their ability to solubilize rock phosphate in liquid culture. Most of these fungi were rock phosphate solubilizers, but two isolates, *Aspergillus fomitopsis* and *Aspergillus sulfureous*, had high activity. Liquid culture experiments revealed that both fungi caused a remarkable drop in pH of culture media and solubilized considerable amount of phosphate. Drop in pH varied from 3.9 to 2.7 from initial value of 7.0. Out of thirty, the fifteen most efficient phosphate solubilizing fungi isolated from different crop rhizospheres were further studied for their ability to judge the type and amount of organic acids produced under *in vitro* conditions. Drop in pH of the medium ranged from 7 to 3.9 with the continuous growth of the isolates for 15 days during the estimation of organic acid as produced by the metabolic activity of PSF. At first, second and third week of incubation, the rock phosphate solubilization ranged from 10.54 to 21.11 %, 12.23 to 34.34 % and 21.97 to 46.50 %, respectively. Study showed that more P was solubilized (21.97 – 46.50%) at third week of incubation. Tartaric acid and malonic acid were two common organic acids produced by PSF. Correlation studies suggested that besides organic acids there might be other factors responsible for P solubilization.

**Key words:** Phosphate solubilizing fungus, rock phosphate, organic acids, phosphorus

In fact, despite being abundant phosphorus in soil, both in inorganic and organic forms, it is the major plant growth-limiting nutrient after nitrogen (N). Plant nutrition depends mainly on the phosphorus content of soil, which can be assimilated only as soluble phosphate. Physical and chemical weathering of mineral phosphates is mainly realized along plant roots in the rhizosphere. Phosphorus in soils is present in insoluble form complexed with cations like iron, aluminum, zinc and calcium. Although, use of chemical fertilizers for improving soil fertility is the common approach of increasing agricultural production, but a large portion of inorganic phosphates applied to soil as fertilizer is rapidly immobilized after application and becomes unavailable to plants (Yadav and Dadarwal, 1997). Microorganisms substantially influence the soil productivity by solubilizing this insoluble P through their metabolic processes in soil (Ravikumar et al., 2007; Sahu et al., 2007). The process of microbe

mediated P solubilization is generally ascribed to the production of organic acids by them (Fleischer et al., 1988; Das et al., 2003; Mattey, 1992). Still there are reasonable doubts on the exclusive role of organic acids in solubilization process (Asea et al., 1988; Illmer and Schinner, 1995). Use of rock phosphate (RP) as fertilizer for P-deficient soils has received significant interest in recent years since they are natural, inexpensive and available fertilizers. However its solubilization rarely occurs in non-acidic soils (Caravaca et al., 2004; Ouahmane et al., 2007). Physical and chemical treatment like thermal alteration and partial acidification, for increasing the P availability from RP are cost intensive. A much cheaper and convenient alternative is reclamation through use of P solubilizing microorganisms. In the light of these facts, the present study was undertaken to investigate the process of RP solubilizing activity of isolated fungal species. It is known that potentially useful

<footnote n="1">¹ Department of Soil Science and Agricultural Chemistry, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi-221005</footnote>

<footnote n="2">² Department of Soil Science and Agricultural Chemistry, Rajendra Agricultural University, Pusa-848125 Samastipur, Bihar</footnote>
organisms reported in the scientific literature fail in natural
environment and never appear in commercial market
(Bashan, 1998). Khalil (1995) reported that phosphate
availability from rock phosphate was increased in soil
from 0.67 ppm in control to 17.78 ppm with phosphate
solubilizing microorganism inoculation in 20 days. In an
effort to develop a phosphate solubilizing fungi inoculum
for improving P uptake by wheat crop, numerous
phosphate solubilizing fungi were isolated from the
rhizosphere of different commonly grown crops of
Varanasi region.

# MATERIALS AND METHODS

## Isolation, identification and characterization of phosphate solubilizing fungi

Phosphate solubilizing fungi isolated from
rhizospheric soils of different localities of Varanasi were
cultivated on Pikovskaya's modified medium by serial
dilution and plating technique. Fungal colonies showing
plaque were transferred to agar slants.

## Inoculation and incubation

Each fungal isolate was grown in culture tube broth separately. The sterilized Pikovskaya's broth in 250 ml Erlenmeyer flask was inoculated in triplicate with 0.5 ml suspension of each fungal culture, keeping uninoculated control. The inoculated flasks were incubated at 28 ± 2°C in BOD for 7, 14 and 21 days under static condition and gently shaken at three days interval to provide better aeration for fungal growth

## Measurement of halozone diameter for screening of phosphate solubilising fungi

0.1 ml of each PSF culture preserved in sterile
distilled water was placed on Pikovskaya's agar
(Pikovskaya, 1948) plates [containing insoluble tricalcium
phosphate 2.5 g, glucose 13 g, (NH₄)₂SO₄ 0.5 g, NaCl
0.2 g, MgSO₄·7H₂O 0.1 g, KCl 0.2 g, yeast extract 0.5
g, MnSO₄ trace, FeSO₄·7H₂O trace, agar 15 g, pH
adjusted to 7.2 and dissolved in 1000 ml distilled water]
and incubated for seven days at 28 ± 2°C. The diameters
of halozone formed by fungal isolates of different habitats
were recorded.

## Change in pH of broth

Sterile uninoculated medium served as control. Initial
pH and change in pH was noted for three week at the

weekly interval of 7, 14 and 21 days of incubation by
digital pH meter.

## 'P' Analysis in broth

### Preparation and sterilization of broth containing rock phosphate

100 ml of Pikovskaya's broth was dispensed in 250 ml Erlenmeyer flask and the rock phosphate was added @ 1 g / 100 ml in addition to control. Rock phosphate used in this experiment contained 15.3 % P or 35.03 % P₂O₅. Thus addition of one gram of rock phosphate supplements 350 mg P₂O₅ / 100 ml broth. Entire flask containing rock phosphate was autoclaved at 15lb pressure for 15 min (at 121°C).

### Solubilized phosphorus determination

The quantitative estimation of water soluble
phosphorus released during the solubilisation of rock
phosphate in broth at the end of different incubation
period was done by chlorostannous method following the
standard procedures as described by Jackson (1973).

## Test for available P

Broth P was determined by Ascorbic acid method (Watanabe & Olsen, 1965). One ml of broth sample extract was taken in 50 ml conical flask and 9 ml of distilled water + 2.5 ml of freshly prepared colour reagent [12 g ammonium molybdate {$(NH_4)_6Mo_7.4H_2O$} + 250 ml distilled water and 0.2908 mg antimony potassium tartrate {K(SbO)C₄H₄O₆.1/2 H₂O} were added in 1000 ml of 5N H₂SO₄ (148 ml conc. H₂SO₄ L⁻¹)]. Both the solutions were mixed and the volume was raised upto 2 L. 140 ml of this mixture was added to 0.74 g ascorbic acid and stirred gently. The optical density of the blue colour was measured at 880 nm by spectrophotometer and the concentration of available P (ppm) was calculated.

## Organic acids determination

Organic acids produced by PSF in PB medium were
analyzed by paper chromatography. The PSF were
grown in 100 ml PB in 250 ml sterilized conical flask for
15 days in B.O.D. at 28 ± 2°C. The broths were filtered
through filter paper in a Buchner funnel under vacuum.
The filtrate was concentrated at 90°C on water bath
and evaporated up to 3 ml and then poured in 5 ml
volumetric flask and volume made up with distilled water
and the type and amount of organic acids produced by
fungal culture were identified by paper chromatography
in 20 cm × 5 cm paper strip (Whatman No 1). Two
parallel lines were drawn longitudinally with lead pencil at a distance of 2 cm from both ends of paper strip. Thereafter the concentrated culture sample as well as standard solution of organic acids were applied with the help of capillary tube on a horizontal line from one end of the paper, and were dried at room temperature. After drying of spots, the chromatographic paper was put in the cylindrical developing chamber (containing mixture of n-butanol: formic acid: water: 5:1:4, v/v) for 10-12 hr and made air tight by ground glass cover. After developing spots in chromatographic paper, it was allowed to dry and dried paper was sprayed with bromophenol blue reagent (0.05 % solution in ethanol) with a chromatographic sprayer and the area of yellow coloured spots on sky blue background was immediately marked with lead pencil. The distances travelled by the advancing front in papers were noted for finding out the retention factor (Rf values). The Rf values were calculated for different culture samples and were compared with those of standard Rf values of organic acids (fumaric acid, lactic acid, succinic acid, malonic acid, malic acid, oxalic acid, citric acid, tartaric acid and gluconic acid), and organic acids produced by fungal culture were identified through comparing standard Rf values of organic acids.

## Quantification of organic acids

For quantification, the yellow coloured spots were striped out from paper with the help of scissor and kept in petriplate. About 3 ml of n-butanol was spread over the cutting part of chromatograph in order to dissolve the compound of yellow spots in n-butanol. When the paper strip became colourless, the filtrate was poured into 5 ml of volumetric flask and final volume made up to 5 ml with n-butanol. The intensity of light yellow coloured spots of the entire sample was measured at 426 nm wavelength, where it gives maximum absorbance (%). As most of the fungal isolates produced tartaric acid so the standard was prepared with this acid. The standard curve were plotted with the reaction product of tartaric acid (LR grade) and bromophenol blue of different strength of tartaric acid viz. 2.5, 5.0, 10, 15, 20, and 25 mg L⁻¹ dissolved in butanol. The percentage absorbance of sample was measured and its actual amount produced was calculated from standard curve.

## Determination of titratable acidity

It is a measurement of the concentration of total dissociated plus undissociated H⁺. The titratable acidity (m mol H⁺ Liter⁻¹) was determined by titrating 10 ml of

the culture filtrate with 0.05 M NaOH solutions using phenolphthalein indicator.

## Statistical analysis

Simple correlation was run to determine correlation coefficients through MSTAT statistical package and Microsoft Excel computer programme.

## RESULTS AND DISCUSSION

### Halozone diameter

Halozone diameter produced by each PSF isolate is presented in Table I. Numerous fungal isolates were observed which produced halozone due to solubilization of tricalcium phosphate on Pikovskaya's agar medium but only those fungal isolates were screened for further research work whose halozone diameter was ≥ 2.5 cm on Pikovskaya's agar medium. Results showed that among all PSF, the *Aspergillus sulfureous* (F₁₅) isolated from rhizosphere of parwal showed the maximum and significantly greater diameter of halozone (6.9 cm) than others. Studies on agar plate revealed that phosphate solubilizing fungi formed clear zones by solubilizing suspended tricalcium phosphate which varied from 3.2-6.9 cm. Generally, halozone increased with increase in colony diameter but in few isolates fluctuations in halozone diameter were observed during the seven days of incubation period. Similar results were found by Kucey (1983) and Edi-Premono *et al*. (1996). In general, the performance of *Aspergillus sulfureous* isolated from different rhizospheric soil showed greater halozone diameter than other species. The efficiency of *Penicillium* species in respect to halozone diameter was similar to *Aspergillus sulfureous*. *Rhizopus stolonifer* isolated from rhizosphere of pigeonpea differed significantly from the rhizosphere of sugarcane and nonsignificantly from tomato rhizosphere. The *Penicillium fuscum* isolated from pea rhizosphere differed significantly from the *Penicillium fuscum* of maize rhizosphere but nonsignificantly from the radish rhizosphere.

### pH

All phosphates solubilizing fungi studied lowered the pH of Pikovskaya's broth as compared to uninoculated sterile control incubated for 7, 14 and 21 days under conditions as inoculated (Table 2). Fungi were found to be active in lowering the pH. Drop in pH by phosphates solubilizing fungi ranged from 3.9 to 2.7 at the end of incubation period. Few phosphates solubilizing fungal isolates although showed larger halozones but did not
<table><caption>**Table 1.** Phosphate solubilising fungal isolates screened *in vitro* on the basis of halozone diameter</caption><thead><tr><th>Sl. No</th><th>Crop rhizosphere</th><th>Notation of Fungal isolates</th><th>Fungal isolates</th><th>Halozone diameter (cm)</th></tr></thead><tbody><tr><td>1</td><td>Lobia</td><td>F<sub>1</sub></td><td>Acrothecium</td><td>4.8</td></tr><tr><td>2</td><td>Sugarcane</td><td>F<sub>2</sub></td><td>Aspergillus awamori</td><td>3.8</td></tr><tr><td>3</td><td>Potato</td><td>F<sub>3</sub></td><td>Aspergillus awamori</td><td>4.6</td></tr><tr><td>4</td><td>Okra</td><td>F<sub>4</sub></td><td>Aspergillus flavus</td><td>3.9</td></tr><tr><td>5</td><td>Gram</td><td>F<sub>5</sub></td><td>Aspergillus flavus</td><td>3.7</td></tr><tr><td>6</td><td>Sugarcane</td><td>F<sub>6</sub></td><td>Aspergillus flavus</td><td>3.2</td></tr><tr><td>7</td><td>Wheat</td><td>F<sub>7</sub></td><td>Aspergillus fomitopsis</td><td>4.2</td></tr><tr><td>8</td><td>Okra</td><td>F<sub>8</sub></td><td>Aspergillus niger</td><td>4.7</td></tr><tr><td>9</td><td>Cucumber</td><td>F<sub>9</sub></td><td>Aspergillus niger</td><td>4.9</td></tr><tr><td>10</td><td>Radish</td><td>F<sub>10</sub></td><td>Aspergillus niger</td><td>4.5</td></tr><tr><td>11</td><td>Lobia</td><td>F<sub>11</sub></td><td>Aspergillus niger</td><td>5.1</td></tr><tr><td>12</td><td>Pigeon pea</td><td>F<sub>12</sub></td><td>Aspergillus niger</td><td>4.4</td></tr><tr><td>13</td><td>Rajmah</td><td>F<sub>13</sub></td><td>Aspergillus sulfureous</td><td>6.4</td></tr><tr><td>14</td><td>Lobia</td><td>F<sub>14</sub></td><td>Aspergillus sulfureous</td><td>6.4</td></tr><tr><td>15</td><td>Parwal</td><td>F<sub>15</sub></td><td>Aspergillus sulfureous</td><td>6.9</td></tr><tr><td>16</td><td>Wheat</td><td>F<sub>16</sub></td><td>Aspergillus sulfureous</td><td>4.9</td></tr><tr><td>17</td><td>Pea</td><td>F<sub>17</sub></td><td>Aspergillus sulfureous</td><td>5.9</td></tr><tr><td>18</td><td>Potato</td><td>F<sub>18</sub></td><td>Aspergillus wentii</td><td>3.9</td></tr><tr><td>19</td><td>Tomato</td><td>F<sub>19</sub></td><td>Fusarium</td><td>3.9</td></tr><tr><td>20</td><td>Parwal</td><td>F<sub>20</sub></td><td>Penicillium bilaii</td><td>5.4</td></tr><tr><td>21</td><td>Mustard</td><td>F<sub>21</sub></td><td>Penicillium bilaii</td><td>4.9</td></tr><tr><td>22</td><td>Okra</td><td>F<sub>22</sub></td><td>Penicillium digitatum</td><td>6.5</td></tr><tr><td>23</td><td>Rajmah</td><td>F<sub>23</sub></td><td>Penicillium digitatum</td><td>5.1</td></tr><tr><td>24</td><td>Radish</td><td>F<sub>24</sub></td><td>Penicillium fuscum</td><td>4.9</td></tr><tr><td>25</td><td>Maize</td><td>F<sub>25</sub></td><td>Penicillium fuscum</td><td>3.9</td></tr><tr><td>26</td><td>Pea</td><td>F<sub>26</sub></td><td>Penicillium fuscum</td><td>4.9</td></tr><tr><td>27</td><td>Maize</td><td>F<sub>27</sub></td><td>Phoma exigua</td><td>4.1</td></tr><tr><td>28</td><td>Tomato</td><td>F<sub>28</sub></td><td>Rhizopus stolonifer</td><td>3.9</td></tr><tr><td>29</td><td>Pigeon pea</td><td>F<sub>29</sub></td><td>Rhizopus stolonifer</td><td>3.8</td></tr><tr><td>30</td><td>Sugarcane</td><td>F<sub>30</sub></td><td>Rhizopus stolonifer</td><td>4.3</td></tr></tbody></table>

lower the pH accordingly. Fluctuations in pH drop were also noted. pH studies showed a drop of pH from 7±0.2 (control) to 2.7 (F<sub>15</sub>). Similar results were observed by Bar - Yosef *et al.* (1999), and Khalil and Sultan (2000).

## Solubilized P

The rock phosphate solubilization was closely related to the magnitude of acid produced in broth. The fungal population notably increased the solubilization of rock

phosphate. The extent of rock phosphate solubilization was lower as compared to tricalcium phosphate solubilization (data not shown in table) which may be due to complexity of structures. The rock phosphate solubilization varied from 10.54 - 46.50 % where as tricalcium phosphate solubilization varied from 30.7 - 66.55 % (Data not shown in table). At seven days of incubation, the maximum amount of rock phosphate (73.9
<table><caption>**Table 2.** Efficacy of screened phosphate solubilising fungal isolates on lowering of pH and percent solubilisation of rock phosphate in broth</caption><thead><tr><th rowspan="3">Fungal isolate</th><th colspan="6">Week of incubation</th></tr><tr><th colspan="2">First week</th><th colspan="2">Second week</th><th colspan="2">Third week</th></tr><tr><th>pH</th><th>P<sub>2</sub>O<sub>5</sub> solubilized (mg/100 mL)</th><th>pH</th><th>P<sub>2</sub>O<sub>5</sub> solubilized (mg/100 mL)</th><th>pH</th><th>P<sub>2</sub>O<sub>5</sub> solubilized (mg/100 mL)</th></tr></thead><tbody><tr><td>F<sub>1</sub></td><td>5.8</td><td>51.4 (14.69)</td><td>4.9</td><td>68.8 (19.66)</td><td>3.9</td><td>89.2 (25.49)</td></tr><tr><td>F<sub>2</sub></td><td>5.7</td><td>55.3 (15.80)</td><td>4.0</td><td>65.8 (18.80)</td><td>3.1</td><td>118.6 (33.89)</td></tr><tr><td>F<sub>3</sub></td><td>5.3</td><td>68.2 (19.49)</td><td>3.9</td><td>95.7 (27.34)</td><td>3.2</td><td>129.8 (37.09)</td></tr><tr><td>F<sub>4</sub></td><td>5.4</td><td>48.6 (13.89)</td><td>4.4</td><td>72.7 (20.77)</td><td>3.7</td><td>123.7 (35.34)</td></tr><tr><td>F<sub>5</sub></td><td>5.7</td><td>53.5 (15.29)</td><td>4.9</td><td>66.4 (18.97)</td><td>3.3</td><td>125.6 (35.89)</td></tr><tr><td>F<sub>6</sub></td><td>5.6</td><td>63.4 (18.11)</td><td>4.3</td><td>88.9 (25.40)</td><td>3.3</td><td>143.4 (40.97)</td></tr><tr><td>F<sub>7</sub></td><td>5.3</td><td>68.4 (19.54)</td><td>4.1</td><td>120.6 (34.46)</td><td>3.5</td><td>154.8 (44.23)</td></tr><tr><td>F<sub>8</sub></td><td>5.4</td><td>44.3 (12.66)</td><td>4.0</td><td>63.9 (18.26)</td><td>3.8</td><td>112.4 (32.11)</td></tr><tr><td>F<sub>9</sub></td><td>4.5</td><td>45.8 (13.09)</td><td>3.4</td><td>51.7 (14.77)</td><td>2.9</td><td>113.4 (32.40)</td></tr><tr><td>F<sub>10</sub></td><td>5.3</td><td>63.8 (18.23)</td><td>4.7</td><td>120.2 (34.34)</td><td>3.3</td><td>145.3 (41.51)</td></tr><tr><td>F<sub>11</sub></td><td>4.3</td><td>63.5 (18.14)</td><td>3.3</td><td>89.8 (25.66)</td><td>2.8</td><td>144.8 (41.37)</td></tr><tr><td>F<sub>12</sub></td><td>4.7</td><td>46.7 (13.34)</td><td>3.8</td><td>82.8 (23.66)</td><td>3.4</td><td>119.8 (34.23)</td></tr><tr><td>F<sub>13</sub></td><td>5.0</td><td>65.2 (18.63)</td><td>4.6</td><td>88.9 (25.40)</td><td>3.7</td><td>148.4 (42.40)</td></tr><tr><td>F<sub>14</sub></td><td>4.7</td><td>64.6 (18.46)</td><td>3.6</td><td>105.1 (30.03)</td><td>3.3</td><td>146.7 (41.91)</td></tr><tr><td>F<sub>15</sub></td><td>4.1</td><td>65.7 (18.77)</td><td>3.5</td><td>88.9 (25.40)</td><td>2.7</td><td>151.2 (43.20)</td></tr><tr><td>F<sub>16</sub></td><td>4.9</td><td>71.8 (20.51)</td><td>4.7</td><td>96.3 (27.51)</td><td>3.4</td><td>158.2 (45.20)</td></tr><tr><td>F<sub>17</sub></td><td>4.1</td><td>67.3 (19.23)</td><td>3.4</td><td>105.1 (30.03)</td><td>3.3</td><td>156.2 (44.63)</td></tr><tr><td>F<sub>18</sub></td><td>4.3</td><td>36.9 (10.54)</td><td>3.6</td><td>42.8 (12.23)</td><td>3.1</td><td>76.9 (21.97)</td></tr><tr><td>F<sub>19</sub></td><td>4.2</td><td>48.5 (13.86)</td><td>3.1</td><td>62.7 (17.91)</td><td>3.0</td><td>108.9 (31.11)</td></tr><tr><td>F<sub>20</sub></td><td>5.2</td><td>66.5 (19.00)</td><td>4.4</td><td>108.6 (31.03)</td><td>3.8</td><td>131.4 (37.54)</td></tr><tr><td>F<sub>21</sub></td><td>5.3</td><td>49.6 (14.17)</td><td>4.1</td><td>76.5 (21.86)</td><td>3.6</td><td>111.3 (31.80)</td></tr><tr><td>F<sub>22</sub></td><td>5.5</td><td>69.6 (19.89)</td><td>4.2</td><td>98.6 (28.17)</td><td>3.1</td><td>159.4 (45.54)</td></tr><tr><td>F<sub>23</sub></td><td>5.8</td><td>59.9 (17.11)</td><td>4.1</td><td>88.2 (25.20)</td><td>3.4</td><td>162.76 (46.50)</td></tr><tr><td>F<sub>24</sub></td><td>5.7</td><td>51.2 (14.63)</td><td>4.7</td><td>78.4 (22.40)</td><td>3.3</td><td>110.4 (31.54)</td></tr><tr><td>F<sub>25</sub></td><td>5.7</td><td>73.9 (21.11)</td><td>4.3</td><td>115.4 (32.97)</td><td>3.0</td><td>161.9 (46.26)</td></tr><tr><td>F<sub>26</sub></td><td>5.6</td><td>50.3 (14.37)</td><td>4.1</td><td>80.4 (22.97)</td><td>3.7</td><td>120.6 (34.46)</td></tr><tr><td>F<sub>27</sub></td><td>5.8</td><td>55.4 (15.83)</td><td>4.5</td><td>78.6 (22.46)</td><td>3.4</td><td>119.7 (34.20)</td></tr><tr><td>F<sub>28</sub></td><td>5.3</td><td>48.5 (13.86)</td><td>4.1</td><td>70.9 (20.26)</td><td>3.8</td><td>110.5 (31.57)</td></tr><tr><td>F<sub>29</sub></td><td>5.5</td><td>46.7 (13.34)</td><td>4.6</td><td>81.3 (23.23)</td><td>3.5</td><td>123.8 (35.37)</td></tr><tr><td>F<sub>30</sub></td><td>5.1</td><td>63.6 (18.17)</td><td>4.3</td><td>115.4 (32.97)</td><td>3.4</td><td>145.3 (41.51)</td></tr><tr><td>SEm ±</td><td>0.08</td><td>1.77</td><td>0.06</td><td>2.99</td><td>0.06</td><td>2.93</td></tr><tr><td>C D (P=0.05)</td><td>0.25</td><td>5.39</td><td>0.20</td><td>8.97</td><td>0.15</td><td>9.09</td></tr></tbody></table>

Data in parenthesis show the % solubilization of RP
<table><caption>**Table 3.** Low molecular weight organic acids produced by PSF in broth and their impact on titratable acidity and lowering of pH</caption><thead><tr><th>Isolate number</th><th>Fungal strains</th><th>Organic acid produced</th><th>Colour (yellow) *intensity of organic acid spots in chromatograph.</th><th>Total organic acid (mg/L)</th><th>Titratable acidity (m mol H<sup>+</sup> liter<sup>-1</sup>)</th><th>pH</th></tr></thead><tbody><tr><td rowspan="2">F<sub>3</sub></td><td rowspan="2">Aspergillus awamori</td><td>Tartaric</td><td>++</td><td rowspan="2">8.612</td><td rowspan="2">16.0</td><td rowspan="2">5.5</td></tr><tr><td>Fumaric</td><td>+</td></tr><tr><td rowspan="3">F<sub>6</sub></td><td rowspan="3">Aspergillus flavus</td><td>Malonic</td><td>+</td><td rowspan="3">2.272</td><td rowspan="3">17.5</td><td rowspan="3">4.5</td></tr><tr><td>Tartaric</td><td>++</td></tr><tr><td>Gluconic</td><td>+</td></tr><tr><td rowspan="2">F<sub>7</sub></td><td rowspan="2">Aspergillus fomitopsis</td><td>Gluconic</td><td>+++</td><td rowspan="2">8.068</td><td rowspan="2">23.0</td><td rowspan="2">4.3</td></tr><tr><td>Tartaric</td><td>++</td></tr><tr><td rowspan="2">F<sub>10</sub></td><td rowspan="2">Aspergillus niger</td><td>Gluconic</td><td>+++</td><td rowspan="2">4.544</td><td rowspan="2">24.0</td><td rowspan="2">4.8</td></tr><tr><td>Tartaric</td><td>++</td></tr><tr><td>F<sub>11</sub></td><td>Aspergillus niger</td><td>Tartaric</td><td>++</td><td>4.408</td><td>23.0</td><td>4.7</td></tr><tr><td rowspan="2">F<sub>13</sub></td><td rowspan="2">Aspergillus sulfureous</td><td>Gluconic</td><td>+</td><td rowspan="2">6.272</td><td rowspan="2">21.0</td><td rowspan="2">4.3</td></tr><tr><td>Tartaric</td><td>++</td></tr><tr><td rowspan="2">F<sub>14</sub></td><td rowspan="2">Aspergillus sulfureous</td><td>Tartaric</td><td>+++</td><td rowspan="2">4.238</td><td rowspan="2">28.0</td><td rowspan="2">4.6</td></tr><tr><td>Fumaric</td><td>+</td></tr><tr><td rowspan="2">F<sub>15</sub></td><td rowspan="2">Aspergillus sulfureous</td><td>Tartaric</td><td>+++</td><td rowspan="2">7.932</td><td rowspan="2">21.0</td><td rowspan="2">4.3</td></tr><tr><td>Fumaric</td><td>+</td></tr><tr><td rowspan="2">F<sub>16</sub></td><td rowspan="2">Aspergillus sulfureous</td><td>Tartaric</td><td>++</td><td rowspan="2">9.360</td><td rowspan="2">17.5</td><td rowspan="2">4.5</td></tr><tr><td>Gluconic</td><td>+</td></tr><tr><td>F<sub>17</sub></td><td>Aspergillus sulfureous</td><td>Tartaric</td><td>+++</td><td>7.258</td><td>34.5</td><td>3.9</td></tr><tr><td>F<sub>20</sub></td><td>Penicillium bilaii</td><td>Tartaric</td><td>+++</td><td>7.364</td><td>21.0</td><td>4.5</td></tr><tr><td rowspan="2">F<sub>22</sub></td><td rowspan="2">Penicillium digitatum</td><td>Citric</td><td>++</td><td rowspan="2">2.816</td><td rowspan="2">22.0</td><td rowspan="2">5.1</td></tr><tr><td>Lactic</td><td>+</td></tr><tr><td rowspan="2">F<sub>23</sub></td><td rowspan="2">Penicillium digitatum</td><td>Citric</td><td>++</td><td rowspan="2">5.768</td><td rowspan="2">18.5</td><td rowspan="2">5.3</td></tr><tr><td>Fumaric</td><td>+</td></tr><tr><td rowspan="2">F<sub>25</sub></td><td rowspan="2">Penicillium fuscum</td><td>Tartaric</td><td>++</td><td rowspan="2">8.918</td><td rowspan="2">22.0</td><td rowspan="2">3.9</td></tr><tr><td>Citric</td><td>+</td></tr><tr><td>F<sub>30</sub></td><td>Rhizopus stolonifer</td><td>Tartaric</td><td>+++</td><td>3.36</td><td>14.5</td><td>5.4</td></tr><tr><td>SEm ±</td><td>—</td><td>—</td><td>—</td><td>0.213</td><td>1.21</td><td>0.23</td></tr><tr><td>CD</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>(P=0.05)</td><td>—</td><td>—</td><td>—</td><td>0.893</td><td>3.38</td><td>0.68</td></tr></tbody></table>

* Key: + = Low intensity

++ = moderate intensity and

+++ = strong intensity
mg/100 ml broth) was solubilized by Penicillium fuscum (F25) (Fig. 1). This fungal isolate produces tartaric acid and citric acid and lowered the pH up to 3.9 from initial 7.0. The total organic acid produced by F25 was 8.918 mg/1000ml of broth at 15 day of incubation. Surprisingly at 14 day of incubation the maximum solubilization of rock phosphate (120.6 mg/100 ml broth) was done by Aspergillus fomitopsis (F7) followed by Aspergillus niger (F10) (120.2 mg/100 ml broth). This is due to huge amount of total organic acid (8.068 mg/1000 ml broth) produced. The solubilization extent of rock phosphate at third week of incubation was comparatively more as compared to first week (TCP solubilization data not shown), this may be because of complex structure of rock phosphate which needs a longer time for reaction with organic acids. The higher titratable acidity of 34.5

m mol H+ liter⁻¹ was reached with Aspergillus sulfureous (F17) in conical flask experiment. F25 initially showed good performance in rock phosphate solubilization because of citric acid production, which is the active agent for solubilization. But due to its low intensity (+) (Table 3), the solubilization action did not prolong for long period of time. The growth of phosphate solubilizers was correlated with the trends of phosphate solubilized. Thus, the main mechanism of phosphate solubilization in broth culture was acid production leading to a decrease in pH of the broth. The similar findings were reported by Whitelaw *et al.*, 1999. At first, second and third week of incubation the rock phosphate solubilization ranged from 10.54 to 21.11 %, 12.23 to 34.34 %, and 21.97 to 46.50 %, respectively. Gluconic acid and tartaric acid were registered to be the principal

<table><caption>**Table 4.** Correlation coefficient of different biochemical characters of PSF isolated from rhizospheric soil</caption><thead><tr><th>Parameter</th><th>Titratable acidity</th><th>Organic acid</th><th>Halo zone</th><th>pH<sub>1</sub></th><th>pH<sub>2</sub></th><th>pH<sub>3</sub></th><th>TCP<sub>1</sub></th><th>TCP<sub>2</sub></th><th>TCP<sub>3</sub></th><th>RP<sub>1</sub></th><th>RP<sub>2</sub></th><th>RP<sub>3</sub></th></tr></thead><tbody><tr><td>Titratable acidity</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Organic acid</td><td>0.04</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Halo zone</td><td>0.41</td><td>0.03</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>pH<sub>1</sub></td><td>-0.52*</td><td>-0.15</td><td>-0.58*</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>pH<sub>2</sub></td><td>-0.49</td><td>0.01</td><td>-0.35</td><td>0.64**</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>pH<sub>3</sub></td><td>-0.08</td><td>-0.02</td><td>-0.04</td><td>0.31</td><td>0.63**</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TCP<sub>1</sub></td><td>0.08</td><td>0.67**</td><td>0.11</td><td>0.27</td><td>0.13</td><td>0.09</td><td>1</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TCP<sub>2</sub></td><td>0.12</td><td>0.67**</td><td>0.28</td><td>0.14</td><td>0.08</td><td>0.11</td><td>0.93**</td><td>1</td><td></td><td></td><td></td><td></td></tr><tr><td>TCP<sub>3</sub></td><td>0.03</td><td>0.42</td><td>-0.09</td><td>0.28</td><td>-0.10</td><td>-0.18</td><td>0.77**</td><td>0.74**</td><td>1</td><td></td><td></td><td></td></tr><tr><td>RP<sub>1</sub></td><td>0.07</td><td>0.59*</td><td>-0.06</td><td>0.06</td><td>0.18</td><td>-0.13</td><td>0.57*</td><td>0.51*</td><td>0.39</td><td>1</td><td></td><td></td></tr><tr><td>RP<sub>2</sub></td><td>0.41</td><td>-0.11</td><td>0.12</td><td>-0.01</td><td>0.07</td><td>0.28</td><td>-0.07</td><td>0.06</td><td>0.17</td><td>-0.07</td><td>1</td><td></td></tr><tr><td>RP<sub>3</sub></td><td>0.25</td><td>0.01</td><td>0.11</td><td>0.015</td><td>0.00</td><td>-0.15</td><td>0.37</td><td>0.36</td><td>0.31</td><td>0.19</td><td>-0.09</td><td>1</td></tr></tbody></table>

pH<sub>1</sub> : pH at 1<sup>st</sup> week of incubation

pH<sub>2</sub> : pH at 2<sup>nd</sup> week of incubation

pH<sub>3</sub> : pH at 3<sup>rd</sup> week of incubation

TCP<sub>1</sub> : TCP solubilization at 1<sup>st</sup> week of incubation

TCP<sub>2</sub> : TCP solubilization at 2<sup>nd</sup> week of incubation

TCP<sub>3</sub> : TCP solubilization at 3<sup>rd</sup> week of incubation

RP<sub>1</sub> : RP solubilization at 1<sup>st</sup> week of incubation

RP<sub>2</sub> : RP solubilization at 2<sup>nd</sup> week of incubation

RP<sub>3</sub> : RP solubilization at 3<sup>rd</sup> week of incubation

<footnote>* Significant (p < 0.05); **highly significant (p < 0.01)</footnote>
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Effect of PSF on solubilization of rock phosphate</figcaption></figure>

organic acids formed during rock phosphate solubilization by *Aspergillus fomitopsis* and *Aspergillus sulfureous*, respectively.

## Organic acids

The best 15 screened phosphate solubilizing fungi (Table 3) have been tested for production of organic acids. Results indicated that the organic acids produced by the fungal isolates were gluconic, tartaric, malonic, fumaric, citric and lactic acids. Highest acid producing phosphates solubilizing fungi were *Aspergillus sulfureous* (9.36 mg L⁻¹), *Penicillium fuscum* (8.92 mg L⁻¹), *Aspergillus awamori* (8.61 mg L⁻¹), and *Aspergillus fomitopsis* (8.07 mg L⁻¹) while lowest was *Aspergillus flavus* (2.27 mg L⁻¹). Most common acids produced were tartaric (by 13 isolates) and gluconic acid (by 5 isolates) while malonic and lactic acid were produced by only *Aspergillus flavus* and *Penicillium digitatum*, respectively. *Penicillium digitatum* and *Penicillium fuscum* produced citric acid in moderate (+) and low (+) intensity, respectively. Studies related to the production of low molecular organic acids have shown that tartaric, gluconic and fumaric acids were major organic acids produced by phosphates solubilizing fungi. This finding is similar to Illmer and Schinner (1995) indicating small quantities of gluconic acid produced from *Aspergillus niger*. However, it contradicts the reports by Illmer and Schinner (1992) and Whitelaw *et al*. (1999)

that gluconic acid was not produced by PSM strains used in their experiments. Venkateswarlu *et al*. (1994) detected lactic acid from *Aspergillus niger* while none of the two isolates produced lactic acid in the present study. *Aspergillus flavus* produced malonic, tartaric and gluconic acid with low, moderate and low intensity, respectively and total amount of organic acid produced was 2.272 mg L⁻¹. The strong yellow colour intensity of gluconic acid spots in paper chromatogram was observed with *Aspergillus niger* and *Aspergillus fomitopsis* and low intensity of spot with *Aspergillus flavus* and *Aspergillus sulfureous*. All the phosphate solubilizing fungi biosynthesized tartaric acid except *Penicillium digitatum*. Contradiction or similarities may be explained on the basis that type and quantity of acid produced depends on the PSM strain, media composition, growth conditions and several other factors. Phosphate solubilizing fungi used glucose as carbon source to produce organic acids. These results are similar to Nautiyal *et al*. (1999). They stated that more organic acids are produced by microorganisms when glucose was used as carbon source. On the basis of the production of total amount of organic acids and quality of organic acids produced by the phosphate solubilizing fungal isolates *Penicillium digitatum* was appeared to be the effective isolate. Calcium was chelated to a small extent with aliphatic monobasic acids (gluconic acid) more strongly with dibasic acids (malic acid, tartaric acid) and most
strongly with a tribasic acids (citric acid). Thus, citric acid produced by Penicillium digitatum was most powerful chelator of calcium than other organic acids

## Correlation coefficient

Correlation coefficient (Table 4) values showed significant (p < 0.05) correlation between rock phosphate solubilization at first week of incubation, organic acid, TCP₁, TCP₂, TCP₃ and RP₁. This result is similar to Illmer and Schinner (1995). But TCP₁, TCP₂, TCP₃ and RP₁ solubilization was non-significantly correlated with titratable acidity. Significant (p < 0.05) and negative correlation was found between pH₁ and titratable acidity and halozone produced. Similar correlation was reported by Kumar and Narula (1999) and Whitelaw (2000). Least correlation was found with pH₂ and RP₃ (r = 0.0). Non-significant positive correlation was found between titratable acidity and halozone produced, RP₂. This result is similar to Kucey's (1983) study but opposite to the findings of Edi-Premono *et al.* (1996) and Kumar and Narula (1999). This is because most PSM show fluctuations in their behaviours. Production of halozone on solid medium is important but it is not necessary that PSM with larger halozones would solubilize more phosphorus. Johri *et al.* (1999) confirmed that criterion for isolation of efficient PSM based on formation of a visible zone on agar plates was not a reliable technique. It has also been studied that some PSM lost their ability to produce halozone on sub culturing (Kucey, 1983). Highest positive correlation was obtained with organic acids produced and TCP₁ and TCP₂ (r = 0.67). This is due to the fact that perhaps organic acids are the main factor responsible for P solubilization. A nonsignificant positive correlation was observed between P solubilized and organic acids produced by PSM suggesting that organic acids may play a important role but are not the only possible mechanism for P solubilization (Illmer & Schinner, 1992). pH and organic acids were also found to be non significantly correlated with each other. Other reasons may be involved for drop in pH e.g. microbial respiration (Illmer & Schinner, 1992).

## Conclusion

It is concluded from the present study that PSF showed variation in their biochemical characteristics. Organic acid production was perhaps not the only possible reason for phosphorus solubilization. On the basis of the production of total amount of organic acids and quality of organic acids produced by the phosphate solubilizing fungal isolates, Penicillium digitatum was appeared to

be the effective isolate. Thus, Penicillium digitatum is the most efficient isolates on the basis of their P solubilizing activity as well as their biochemical characteristics. These may be used for inoculum production.

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