mycological research 111 (2007) 931–938

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Phosphate solubilization potential and stress tolerance of Eupenicillium parvum from tea soil
Pratibha VYAS, Praveen RAHI, Anjali CHAUHAN, Arvind GULATI*
Plant Pathology and Microbiology, Hill Area Tea Sciences, Institute of Himalayan Bioresource Technology, Post Box No. 06, Palampur, Himachal Pradesh 176 061, India

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Article history: Received 6 November 2006 Received in revised form 1 May 2007 Accepted 10 June 2007 Published online 29 June 2007 Corresponding Editor: Stephen W. Peterson Keywords: Aluminium stress Desiccation tolerance Eupenicillium parvum Inorganic phosphate solubilization Iron stress Tea

Eupenicillium parvum was recorded for first time during isolation of phosphate-solubilizing microorganisms from the tea rhizosphere. The fungus developed a phosphate solubilization zone on modified Pikovskaya agar, supplemented with tricalcium phosphate. Quantitative estimation of phosphate solubilization in Pikovskaya broth showed high solubilization of tricalcium phosphate and aluminium phosphate. The fungus also solubilized North Carolina rock phosphate and Mussoorie rock phosphate, and exhibited high levels of tolerance against desiccation, acidity, salinity, aluminium, and iron. Solubilization of inorganic phosphates by the fungus was also observed under high stress levels of aluminium, iron, and desiccation, though the significant decline in phosphate solubilization was marked in the presence of aluminium than iron. The fungal isolate showed 100 % identity with E. parvum strain NRRL 2095 ITS 1, 5.8S rRNA gene and ITS 2, complete sequence; and 28S rRNA gene, partial sequence. ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Tea is an economically important plantation crop. With little scope to increase the area under cultivation, improving tea productivity is imperative to meet the increasing consumer demand. The availability of phosphorus is a limiting factor in the growth and production of tea (Verma & Palani 1997). The acidic conditions of the soil required for tea growth favour the formation of insoluble phosphate complexes through binding of phosphate anions with metal cations. Consequently, a large portion of the inorganic phosphates applied as fertilizers is rendered inaccessible to the plant due to rapid binding into complex forms (Venktesan & Murugesan 2004).

The complex dynamic equilibrium of solubilization and immobilization of both macro- and micronutrients and their availability to the plant are greatly influenced by the activity of microorganisms (van Elsas et al. 1997). The ability of several microorganisms, including actinomycetes, other bacteria, and fungi, to dissolve relatively insoluble phosphate compounds has introduced the possibility that microbial inoculants could be used to induce solubilization of phosphates in the soil (Whitelaw 2000). Although exotic strains of the microbial agents can increase growth and yield of plants, evidence suggests that genotypes of beneficial microbes may be endemic to a biogeographical region (Cho & Tiedje 2000). The endemic microbial pool of a region may contain highly efficient genotypes

* Corresponding author. E-mail address: 0953-7562/$ – see front matter ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2007.06.003

DNA was extracted using Qiagen Plant DNeasy Kit (Qiagen Gmb H. and a final extension at 72  C for 8 min. Screening the fungus for stress tolerance The tolerance of the fungus toward different abiotic factors was studied by growing the fungus for 9 d in potato dextrose broth modified to produce stressful conditions. Vyas et al. 5. and glycine-NaOH buffer for maintaining pHs of 3-7. and 10 %). 30.5.nlm. the fungus was grown over different concentrations of NaCl (2. The fungal colonies producing distinct zones of TCP solubilization were raised into pure cultures. Edison. The evolutionary distances among E. Methods and materials Isolation and identification of the fungus The fungus was isolated from soil samples collected at the Tea Experimental Farm Banuri. Barnett & Hunter 1972). The microorganisms growing in tea soil are subject to acidic conditions. 50. and 100 mM). 5. The phosphorus in the culture filtrate was estimated on days 3.nih..3b (Copyright Yves van de Peer.85 % NaCl) for preparation of the fungal suspension. MRP.ncbi. North Carolina phosphate (NCRP) or Udaipur rock phosphate (URP). The flasks containing broth were inoculated by 1 ml fungal suspension (105 CFU mlÀ1) and incubated at 28  C in a Innova Model 4230 refrigerated incubator shaker (New Brunswick Scientific.5 % tricalcium phosphate (TCP) as the source of insoluble phosphate (Gupta et al.5 and 36  C. Whitehouse Station. after aligning the sequences with ClustalW. The solubilization of AP.5. The phosphatesolubilizing fungus identified as Eupenicillium parvum at the Microbial Type Culture Collection and Gene Bank. 1998) using Kimura’s two-parameter model. frozen in liquid nitrogen and ground to a fine powder. was selected for further studies on inorganic phosphate solubilization and stress tolerance parameters typical of tea soil.1 Cycle Sequencing Kit. AP.5 mM MgCl2.5. 25.5-11.5 % aluminium phosphate (AP). and 8. 9 and 12 of incubation by the vanadomolybdophosphoric yellow colour method (Jackson 1973). and 7) at 36  C.5. The sequence of the PCR-product was determined by employing the ABI Prism Big Dye Terminator v. 30. 25. 1990). 40.5 mM dNTPs. Tris-HCl. The ITS region sequence of Aspergillus flavipes strain ATCC 1030 was used as an search algorithm and aligned to the nearest neighbours. The optimum temperature for the fungal growth was 36  C and optimum pH was 4. The influence of pH on fungal growth was studied by growing the fungus at 36  C in the medium made in citrate– phosphate buffer. The preliminary experiments on discerning the optimum temperature and pH for fungal growth were performed by measuring the radial growth on solid medium (data not shown).5 and 8. NJ) at 180 rev minÀ1. 1 Â PCR buffer with 1. The Petri dishes were incubated for 12 d at 28  C after point inoculations with the fungus and observed every third day for the presence of a clear zone around the colony indicating phosphate solubilization.8 rRNA gene and ITS 2 was achieved using primers ITS1: 50 TCC GTA GGT GAA CCT GCG G and ITS4: GCT GCG TTC ATC GAT GC (White et al. and 3 U taq polymerase.5. The rock phosphates were washed to remove the soluble phosphate and dried at 40  C for 24 h before use as the phosphate source. Subsequently. 54  C for 1 min and 72  C for 2 min. The acidic pH of medium was adjusted with 1 N HCl. The PCR reaction was performed in 50 ml total volume including 50 ng genomic DNA. 7. 7. Five-day-old mycelium was scraped from the Petri dishes.5. 1994. 5. Hiden). Quantitative estimation of TCP solubilization was undertaken in PVK broth. The present paper reports in vitro solubilization of inorganic mineral phosphates and rock phosphates and tolerance to high levels of desiccation and salt concentration of Eupenicillium parvum. high concentrations of aluminium and iron. 10. 5. Solubilization of . 25. respectively. Palampur in Himachal Pradesh (India) from 15–25 cm depth from the rhizosphere of feeder roots of tea (Camellia sinensis). parvum and related taxa were calculated with TREECON software package version 1. Inc. 1994). 7. FP. 3. 50. which has been isolated from tea rhizosphere. NCRP or URP was studied by replacing TCP (0.5 and pH (4. 20. and 50 %) at pH 4.5. and 36  C) at pH 4. and NCRP by the fungus was studied under different temperatures (15. University of Antwerp. and the indigenous strains are also likely to perform better than the exotic strains. FeCl3 (2. 10 pmol each primer. AlCl3 (2. and 100 mM). Solubilization of phosphate sources by the fungus The solubilization of different inorganic phosphate sources was studied by replacing TCP from modified PVK agar and unmodified PVK agar with 0.5 % w/v) from PVK broth. The amplification of ITS 1. 6487). The thermocycling conditions consisted of an initial denaturation at 94  C for 5 min. Institute of Microbial Technology. little work has been done on the aspects of phosphate solubilization by microorganisms from the tea soil. and desiccation through periodic spells of drought.5.932 P..5. The serial soil dilutions were spread plated on modified Pikovskaya (PVK) agar containing 0. Phosphate solubilization under stress The solubilization of TCP. Chandigarh (MTCC accession no. followed by 35 amplification cycles at 94  C for 1 min. the 5. iron phosphate (FP). The effect of temperature was studied by incubating the cultures over a range of 16-40  C at pH 4. 10. The uninoculated autoclaved medium with different phosphate substrates was incubated under similar conditions as those employed for incubation of the inoculated medium to serve as the controls for solubilization of various phosphate substrates by the fungus. Five-day-old fungal colonies were homogenized and suspended in normal saline (0. 7. on the basis of phenotypic characters. Mussoorie rock phosphate (MRP). maintained on potato dextrose agar slants at 4  C. The results have introduced the feasibility of assessing plant growth-promoting activity through inoculation of the tea soil with phosphate-solubilizing fungus. 0. 6. NJ).5. However. The sequence was analysed using the gapped BLASTn (http://www. and identified on the basis of cultural and microscopic features (Subramanian 1971. The pH of the liquid medium was measured using CyberScan 510 pH meter (Merck and Cp. and polyethylene glycol (PEG) 6000 (20.

The solubilization increased significantly with increases in the incubation period from 3-9 d. However. However. and MRP in the decreasing order. Fungus growth under stress The results of the effects of temperature. All values are means of three replicates. surface texture dense.5. The data were found to be normally distributed. and 28S rRNA gene partial sequence. and aluminium and iron concentrations of 2. Solubilization of URP showed the lowest reduction in the pH of the medium. followed by the solubilization of FP. not observed below 24  C. centrally maize yellow. 10 % NaCl. USA 2004). . AP. based on the ITS region sequences. Likewise. The phylogenetic tree constructed with the TREECON software is shown in Fig 1. somewhat irregular. pH. followed by the solubilization of NCRP. followed by a significant decline at 12 d of incubation (Table 1). The decline in the pH of the culture medium was highest in the solubilization of AP. stipes nonvesiculate. no significant difference was recorded in the solubilization of FP and URP. reddish brown soluble pigment produced. 100 mM aluminium. and NCRP. Results Identification of the fungus The fungal colonies were radially sulcate. The maximum fungal biomass was obtained at 36  C and pH 4. with deep floccose overlay. In the tree. Conidia sparse. no phosphate solubilization zone was observed in the medium supplemented with AP.02 substitutions per site. Distance 0. URP. The sequence of 439 bp ITS region of the fungus showed 100 % identity with the Eupenicillium parvum strain NRRL 2095 ITS 1. FP. a significant increase in the reduction of the pH of the medium was also registered with advancement in the incubation up to 9 d. reverse deep reddish brown. and salt concentrations on fungal growth. MRP. and iron are given in Fig 2. cleistothecia abundant and submerged at 37  C. and 100 mM iron for up to 9 d of incubation in potato dextrose broth. sequences of reference strains were obtained from the NCBI GenBank. The data were checked for normality and subjected to two-way analysis of variance (ANOVA) using the STATISTICA data analysis software system version 7 (StatSoftÒ Inc.02 Eupenicillium parvum strain NRRL 2095 Eupenicillium parvum strain FIHB 539 Penicillium vinaceum strain NRRL 739 Eupenicillium rubidurum strain NRRL 6033 Eupenicillium erubescens strain NRRL 6223 Eupenicillium hirayamae strain NRRL 143 Penicillium indicum strain NRRL 3387 Aspergillus flavipes strain ATCC 1030 Fig 1 – Phylogenetic tree showing the relationships among Eupenicillium parvum (FIHB 539) and representatives of related taxa. as well as ability to tolerate desiccation. Solubilization of phosphate sources by the fungus The zone of TCP solubilization by Eupenicillium parvum appeared on third day. more on PVK and modified PVK than potato dextrose agar. Tulsa.Phosphate solubilization potential and stress tolerance 933 inorganic phosphates was also quantified under desiccation of 30 % PEG.8S rRNA gene and ITS 2 complete sequence..5 and 36  C. conidia and ascospores small. which became prominent and sharp after 7 d of incubation both on PVK agar and modified PVK agar. The mean of the treatments were compared by CD value at P ¼ 0. margins low. 5. Experimental design and data analysis Randomized block design with two factor factorial arrangement was adopted for conducting the experiments. The newly generated sequence was deposited at GenBank (accession no. DQ536524). Bar [ 0. The reduction in the pH of the medium was comparable during the solubilization of TCP and NCRP. The fungus was able to grow in up to 40 % PEG 6000.01. aluminium. mycelium white at the margins. A single band of ca 600 bp was obtained on amplification of the ITS region of the fungus. The incubation period also exhibited significant influence on the quantities of phosphate solubilization. The quantification of the phosphate liberated showed that TCP solubilization was significantly higher. deep red-brown exudates abundant. followed by a significant decline at 12 d of incubation.5-5 mM at pH 4. and monoverticillate. as compared with FP and URP (Table 1).

Discussion The previously unreported fungus from tea soil was identified as Eupenicillium parvum on the basis of morphological features and ITS region sequencing.4 11. the development of clear halos was not observed around the colonies of E.6 CD (P ¼ 0.9 URP 5.5 22.6 6.6 19. The fungus was also able to solubilize inorganic phosphates in the presence of 30 % PEG 6000.3 S. with aluminium phosphate or iron phosphate as the sole phosphate source.5 Æ 0. Kang et al. ferrous phosphate. In our study. and iron was accompanied by a smaller decrease in the pH as compared with the decrease in pH during phosphate solubilization in the absence of stress conditions (Table 4).8 150.M. The appearance of a clear halo around the colony of E.6 S. The pH of the medium also decreased significantly on solubilization of phosphate substrates (Table 2).3 110. with a significantly higher reduction at higher concentrations of these metals in the medium (Table 4). .4 24.6 URP 2 3. Fungi are generally more tolerant to acidity than bacteria and account for most of the highly aluminium-resistant microorganisms (Myrold & Nason 1992.7 20.5 31. tricalcium phosphate.1 NCRP 17. which are abundant in tea soils.2 12.5 63.9 51.5 213.01) 3.7 5. 1994).4 72. Kucey et al.9 7. Quantitative estimation of phosphate solubilization by the fungus in PVK broth containing TCP showed high phosphate solubilization (Table 1). although E. with the minimum reduction at 15  C and the maximum reduction at 36  C.M. shearli from the soils of Brazil has been reported to solubilize phosphate (Nahas 1996). The presence of aluminium and iron at stressful concentrations caused a reduction in phosphate solubilization. 1994. This is the first report on the solubilization of a phosphate source by E. The pH of the culture medium influenced phosphate solubilization by the fungus.1 79.6 21. aluminium. Vyas et al.5 51. Reduction in pH of medium (%) Mean 27. iron. The increase in incubation temperature also influenced the reduction in the pH of the culture medium.1 26. which is at par with 30  C (Table 3). The phosphate-solubilizing microbial isolates from tea soils are likely to be more successful as microbial inoculants than the microorganisms isolated from other soils because of their ability to survive the stress factors that occur in tea culture. Kanazawa & Kunito 1996). parvum. 1989). North Carolina rock phosphate. aluminium. 1968.2 6. NCRP. 2002).9 25. parvum isolated from tea soil.E. The fungus was isolated from the soil around tea rhizospheres.7 4. AP 35. aluminium phosphate. grown on PVK agar and modified PVK agar. Maximum phosphate solubilization occurred at 36  C. Kang et al. which were acidic and had high aluminium levels. The solubilization of various substrates was significantly higher in the medium with a neutral pH than those with acidic pHs (Table 2).4 8. as also reported previously for many microorganisms (Ahmad & Jha.9 48.2 13.3 0.9 20. parvum from cultivated and forest soils has not been previously recorded. MRP.01) 3 2. FP.E. Udaipur rock phosphate. the isolate exhibited solubilization of both aluminium phosphate and iron phosphate in PVK broth (Table 1).3 50 52.3 170. Thus solubilization of aluminium phosphate and iron phosphate in the broth was independent of the appearance of a phosphate solubilization zone on the solid medium. render the applied phosphorus unavailable to the plants by forming insoluble complexes (Ranganathan 1976).6 45.3 FP 1. and other phosphates (Gaur 1990. Table 1 – Effect of incubation on solubilization of phosphate substrates by Eupenicillium parvum at 28  C in Pikovskaya broth with initial pH 7 P source Days 3 6 9 12 Mean Variant P source Days Interactions Phosphate solubilization over control (mg mlÀ1) TCP 120.3 2.7 Æ FP 25 35.6 NCRP 6. 25.7 55. and 36  C.8 0.4 Mean 18. Gaur & Gaind 1983.8 58.5 46.5 41. The majority of phosphate-solubilizing microorganisms are able to solubilize calcium-phosphorus complexes but only a few can solubilize iron–phosphorus and aluminium-phosphorus complexes (Banik & Dey 1983.6 22.4 89. The microbial solubilization of phosphates is not restricted to calcium salts as microorganisms also act upon iron.4 MRP 5.8 13.4 50.3 55. there was no significant difference in the pH reduction at 25. A significant increase in the solubilization of phosphate substrates was recorded in the cultures incubated at various temperature intervals through 15-30  C.8 0. However.1 27.6 CD (P ¼ 0. The tea soil is also subject to desiccation under periodic spells of drought.9 65. However.9 27. Aluminium.3 24.2 16. URP. AP. and 30  C. Mussoorie rock phosphate.8 7.6 1.7 197. Phosphate solubilization by the fungus under different stress conditions The fungus was tested for the ability to solubilize inorganic phosphates at different pHs and temperatures. The percent reduction at 15  C was statistically comparable with the reduction at 20.2 AP 27.9 MRP 8.1 2. and clay.934 P.9 7.5 23.4 6 TCP.8 TCP 17. The lowered phosphate solubilization in the presence of PEG 30 %.7 1. parvum indicated phosphate solubilization by the fungus (Gupta et al.4 58. 2002). Gupta et al. 30. The reduction in phosphate solubilization was significantly higher in the presence of aluminum as compared with the presence of iron in the growth medium.3 3 3. Phosphate solubilization by isolates of E.

4 3 10 5.5 15.8 CD (P ¼ 0.8 55.8 0.7 AP 52.4 36.E.2 6.2 1 0.M.6 0.5 10 25 50 100 Al Fe Fungal Dry Wt.5 5 7. and pH (E) on growth of Eupenicillium parvum in potato-dextrose broth after 9 d of incubation.9 25. salt concentration (D). Table 2 – Effect of pH of the medium on the solubilization of phosphate substrates by Eupenicillium parvum at 36  C in Pikovskaya broth after 9 d of incubation P source pH 4.2 1 0.2 1 0.2 0 0 D 2.7 11. error bars indicate standard deviation. (g/50ml broth) C 1.M.2 0 16 20 24 28 32 36 40 Fungal Dry Wt.8 0.2 10. metal concentration (C).6 0.5 7 Mean Variant P source pH Interactions Phosphate solubilization over control (mg mlÀ1) TCP 190.6 0.4 0.4 2.6 TCP.1 14.5 5. (g/50ml broth) 1. PEG 6000 (desiccant) (B).4 84.5 1.01) 5.4 0.4 0. tricalcium phosphate.4 207 231.2 CD (P ¼ 0.3 5.4 71.2 1 0. (g/50ml broth) 1.6 S.9 92. aluminium phosphate.6 0. (g/50ml broth) 1.01) 6. NCRP.4 209. The results are mean of three replicates.7 Mean 14.4 0. AP.4 Æ 1. Reduction in pH of medium (%) Mean 102.Phosphate solubilization potential and stress tolerance 935 Fungal Dry Wt.5 S.8 0.8 0.E.7 116.5 111. .4 1. (g/50ml broth) 1 0.9 NCRP 4.2 Æ 1.4 50 36.6 0.5 2. AP 24.5 5 7.2 0 0 B 20 30 40 50 Temperature (ºC) Fungal Dry Wt.5 10 Al3+/Fe3+ Concentration (mM) Fungal Dry Wt.4 0.7 5.5 146.7 9.8 0.9 TCP 13.2 0 0 2.2 0 3 4 5 6 7 8 9 10 11 E pH Fig 2 – Effect of temperature (A).2 A 1.3 67 NCRP 64. North Carolina rock phosphate.

6 6.8 62.8 12.936 P.4 13.6 S.4 10.1 NCRP 2. Phosphate solubilization was accompanied by the reduction in the pH of the medium.1 11. rock phosphates could be used through the action of microbial solubilization. AP. .9 4.7 3. Kang et al.1 5.9 1.E.6 7.4 8.01) 2. The ability of E. The solubilization of rock phosphates have been reported to depend on their structural complexity and particle size as well as the nature and quantity of organic acids secreted by the microorganisms (Gaur 1990. aluminium phosphate. North Carolina rock phosphate.6 14.01) 4.1 15.5 0.E.8 6.1 8. FP and AP are less amenable to microbial solubilization than TCP (Gaur 1990.4 32 4.8 1.5 after 9 d of incubation at 36  C P source Stress level Controla 2.M.3 13.2 17. 1978.2 52.4 Mean 8.9 11. Pradhan & Sukla 2005).3 TCP. which could be attributed to the depletion of nutrients in the culture medium (Ortuno et al.E.5 mM iron 5 mM iron 30 % polyethylene glycol 6000 Mean Variant P source Stress level Interactions Phosphate solubilization over control (mg mlÀ1) TCP 190 125.6 35.M.2 37.4 24. which decreased with further incubation (Table 1).9 10.7 TCP 14.7 54. Goenadi et al.2 2 Æ 1.5 4.4 25 CD (P ¼ 0. 27.1 TCP. a Medium without stress.26 20. solubilization of URP was very low. whereas the solubilization of iron phosphate was low. In acidic soils with a low availability of phosphorus.2 5.7 100.9 117.6 AP 22.4 8.8 41 NCRP 29.4 16.1 11. Table 3 – Effect of temperature on the solubilization of phosphate substrates by Eupenicillium parvum in Pikovskaya broth with initial pH 4. with the highest reduction coinciding with the day of highest solubilization of phosphate source (Table 1).1 CD (P ¼ 0.1 14.1 1. Reduction in pH of medium (%) Mean 63.6 NCRP 64. Narsian & Patel 2000.4 22. North Carolina rock phosphate.3 52.8 165.7 NCRP 11. parvum from the different inorganic phosphates tested (Table 1).9 24.3 34.9 TCP 2.4 9 4. There was a significant variation in the quantities of phosphorus liberated by E.4 12.4 9.2 186.5 7.8 CD (P ¼ 0.5 26.2 CD (P ¼ 0.3 50.2 23. tricalcium phosphate.4 Æ 0.9 79.2 11.6 12. was evident from the results (Table 1).2 7. 2002).1 S.1 9.5 56. Vyas et al.2 17.4 20.5 Reduction in pH of medium (%) Mean 100.2 22.E. 2000. aluminium phosphate.2 4. A significant reduction in the pH of the Table 4 – Effect of different stress levels on the solubilization of phosphate substrates by Eupenicillium parvum in Pikovskaya broth with initial pH 4.7 1 2 Æ 0.9 91.6 190. AP. The highest solubilization was obtained for NCRP among the three rock phosphates tested. AP 22. tricalcium phosphate.9 73.2 2.6 10. MRP was found to be more vulnerable to solubilization as compared with URP (Illmer & Schinner 1992).8 57.1 64. Shin et al.3 S.3 16. Although solubilization of NCRP and MRP was appreciable.4 2.3 102.3 AP 46.8 135.1 51.4 143. A similar trend of decreasing phosphate solubilization with advancement in the incubation period has been reported for some microorganisms.6 88.M. which contains rock phosphates as the sole phosphate source.2 3.4 22. The results on solubilization of inorganic phosphates by the test fungus agreed with reports that rock phosphates.M.4 9.8 51. The quantity of phosphate solubilized was by far the highest for TCP among the inorganic phosphates.3 Æ 0.01) 2.58 32.2 24. NCRP.5 mM aluminium 5 mM aluminium 2.1 S. parvum to solubilize various rock phosphates from PVK broth.4 165. NCRP. The maximum solubilization of different phosphate sources was obtained at day 9 of incubation.5 8. 20.2 AP 22. The solubilization of aluminium phosphate was also high.7 15 12.5 after 9 d of incubation P source Temperature 15  C 20  C 25  C 30  C 36  C Mean Variant P source Temperature Interactions Phosphate solubilization over control (mg mlÀ1) TCP 139.8 10.4 9.6 125.01) 3.6 Mean 15.7 166.7 53. 2006).

Prentice Hall. Gaind S. 2000. The phosphate-solubilization by the fungus appears to be affected by the availability of phosphorus at the time of inoculations in the culture medium.7 and 3.76 MPa of osmotic pressures. Saxena RK. Biodiversity Division. Tolerance to acidity. .and Al-tolerant microorganisms. New Delhi. Kunito T. 1972. Soil Science Society of America Journal 64: 927–932. Illmer P. though a decline was observed in the growth with an increasing salt concentration. Previously six strains identified as Aspergillus flavus. The results revealed the ability of E. 1983. whereas the growth of most microorganisms was almost completely inhibited by 1-2 mM inorganic monomeric aluminium (Kawai et al. the fungus could also tolerate high salt concentrations. Cryptococcus humicola and Rhodotorula glutinis from tea fields were reported to tolerate 100-200 mM aluminium under strongly acidic conditions. The ability to adapt to desiccation and temperature stresses may also be important in the survival of the microorganisms during droughts. Biogeography and degree of endemicity of fluorescent pseudomonas strains in soil. Phosphate Solubilizing Microorganisms as Biofertilizers. and stress levels. 236 pp. Institute of Himalayan Bioresource Technology and for their help in statistical analysis. 1996. Longman Group.. Phosphate solubilization potentiality of the microorganisms capable of utilizing AlPO4 as sole phosphate source. Singh. The acidic culture conditions were found suitable for fungal growth. parvum to solubilize phosphate substrates over a wide range of pHs. An understanding of the physiology of E. The results are in agreement with earlier studies on the effect of the concentration of soluble phosphate on the solubilization of fluorapatite by Aspergillus niger (Nahas & Assis 1992). Kuhad RC. Gaur AC. However. Soil Science and Plant Nutrition 42: 165–173. Barnett HL. parvum under similar stress conditions is required for the successful application as a bioinoculant in tea. Likewise. This is evident from the results obtained in the present study regarding the solubilization of phosphate substrates at acidic pH. Siswanto S. Aluminium and iron added to the medium under strongly acidic conditions are toxic to microorganisms (Haug 1984). Trichoderma asperellum. The fungus also showed tolerance to aluminium and iron under strongly acidic conditions of the culture. Minneapolis. Dey BK. Department of Statistics. Science and Culture 49: 110–112. The low phosphate solubilization by the fungus under acidic pH could be attributed to the availability of higher initial content of soluble phosphate in the medium. although a decline was registered in solubilization under stress conditions for the fungal growth (Tables 2–4). The fungus exhibited good growth over a temperature range of 20-36  C (Fig 2). The results have shown the potential for further testing the fungus as a phosphatesolubilizing inoculant in soil where conditions are much more complex than those prevailing in vitro. which is ideal for tea growth (Eden 1976). Solubilization of inorganic phosphates by microorganisms isolated from forest soils. Soil Biology and Biochemistry 24: 389–395. Gaur AC. 2000). as a significantly higher reduction in the pH of the medium was observed in AP and FP solubilization. 1984. AP and NCRP at pH 7.Phosphate solubilization potential and stress tolerance 937 culture filtrates containing various inorganic phosphates suggested secretion of organic acids by the fungal strain (Nahas 1996. references Ahmad N.5 in comparison with 25.7 and 0. Penicillium janthinellum. 1994. Burgess Publishing. Tiedje JM. coinciding with the pH of the soil required for good tea growth (Eden 1976). Jackson ML. though the phosphate solubilization was significantly low. third edn.6 mg mlÀ1 for TCP. in comparison with the other substrates (Table 1). for his help in identification of the fungus. 2. The fungus also showed high tolerance to drought as it could grow in the presence of 20-40 % PEG 6000 in broth. Shanker A. as it could grow at pH 4. Goenadi DH.8 mg mlÀ1 for TCP. Critical Reviews In Plant Sciences 1: 345–373. enumeration of acid-tolerant and Al-resistant microorganisms in acid soils. Illustrated Genera of Imperfect Fungi. New Delhi. Solubilization of rock phosphate by microorganisms isolated from Bihar soils. Journal of General and Applied Microbiology 14: 89–95. Mathematics and Physics. respectively. 1983. 1992. Gaur AC. 1976. Applied and Environmental Microbiology 66: 5446–5448. AP and NCRP at pH 4. Hunter BB. 1990. in the present study. Earlier findings on the solubilization of rock phosphates by some efficient strains of Aspergillus and Penicillium also indicated that phosphate solubilization varies greatly with the nature of phosphate substrates (Bardiya & Gaur 1974). Banik S. Isolation and characterization of acid. 1968. 2000. London. 1973. FEMS Microbiology Letters 189: 143–147. Pradhan & Sukla 2005). 1974. Tea. Singal R. which was estimated at 153.49 and -1. Schinner F. Eden T. Acknowledgements We acknowledge Dharamrajan Ananthapadamanabhan. Preparation of pH 3 agar plate. and Kamlesh Singh. Kawai F. Kanazawa S. Sugiarto Y.3. Cho JC. Penicillium sp. 2000. the phosphate substrates appear to influence the solubilization irrespective of the corresponding reduction in the pH of the medium. Jha KK. Omega Scientific. Journal of General and Applied Microbiology 40: 255–260. Himachal Pradesh Krishi Vishvavidyalaya and Rakesh Deosharan. Isolation and screening of microorganisms dissolving low grade rock phosphate. Gupta R. respectively (Michel & Kaufmann 1973). Microbial solubilization of phosphates with particular reference to ferrous phosphate and aluminum phosphate. aluminium. and iron is important in the growth. Eupenicillium parvum appears to adapt well to the stress conditions and has the ability to solubilize inorganic phosphates under high stress conditions. which generates -0. Molecular aspects of aluminium toxicity. temperatures. A fairly good growth of the fungus was recorded in aluminiumsupplemented medium at concentrations of exchangeable aluminium reported for the acidic soils of tea (Sharma & Tripathi 1989). A modified plate assay for screening phosphate-solubilising microorganisms. Soil Chemical Analysis. Institute of Himalayan Bioresource Technology for providing necessary facilities. Bardiya S.5 in the presence of 100 mM aluminium and 100 mM iron. Zentraalblatt fu ¨ r Bakteriologie 138: 17–23. Folia Microbiology 19: 386–389. Institute of Microbial Technology. establishment and survival of microorganisms in tea soils. Haug A. Thanks are also due to Paramvir Singh Ahuja. Bioactivation of poorly soluble phosphate rocks with phosphorus solubilizing fungus. 12. Zhang D. Sugimoto M.

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