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Phosphate solubilization by a wild type strain and UV-induced mutants of Aspergillus tubingensis
ARTICLE IN PRESS
Soil Biology & Biochemistry 39 (2007) 695–699 www.elsevier.com/locate/soilbio
Short communication
Phosphate solubilization by a wild type strain and UV-induced mutants of Aspergillus tubingensis Varenyam Achala, V.V. Savantb, M. Sudhakara Reddya, a
Department of Biotechnology, Thapar Institute of Engineering and Technology, Patiala 147 004, India b Ballarpur Chemicals Ltd., Karwar, Karnataka, India Received 15 January 2006; received in revised form 29 August 2006; accepted 6 September 2006 Available online 12 October 2006
Abstract Phenotypic mutants of Aspergillus tubingensis were obtained by UV irradiation and phosphate solubilization ability of these mutants were studied and compared with the wild type strain. Low phosphate solubilizing mutant was also selected in this study. Among the different mutants, AtM-5 and AtM-2 showed highest P solubilization when tri-calcium phosphate and rock phosphate were used as P source compared to the wild type strain and other mutants. These mutants also showed maximum acid phosphatase and phytase activity. These results suggest that P solubilization by these isolates is due to lowering of pH of the culture filtrate and also the activity of acid phosphatase and phytase. r 2006 Elsevier Ltd. All rights reserved. Keywords: Aspergillus tubingensis; UV mutants; Phosphate solubilization; Phosphate rock; Phytase activity; Acid phosphatase activity
Phosphorus is one of the most essential macroelements required for the growth and development of plants, but in most soils its content is about 0.05% of which only 0.1% is plant available (Scheffer and Schachtschabel, 1988). Deficiency of soil P is one of the most important chemical factors restricting plant growth in soils. Therefore, large quantity of soluble forms of P fertilizers is applied to achieve maximum plant productivity. However, the applied soluble forms of P fertilizers are easily precipitated into insoluble forms and are not efficiently taken up by the plants, which lead to an excess application of P fertilizers to cropland (Omar, 1998). In recent years the possibility of practical use of rock phosphate as fertilizer has received significant interest. In India, it is estimated that about 260 million tons of phosphatic rock deposits are available and this material should provide a cheap source of phosphate fertilizer for crop production (FAI, 2002). Unfortunately, rock phosphate is not plant available in soils with a pH greater than 5.5–6.0 and even when conditions are optimal, plant yields are lower than those obtained with soluble Corresponding author. Tel.: +91 175 2393043; fax: +91 175 2393738.
E-mail address: [email protected] (M.S. Reddy). 0038-0717/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2006.09.003
phosphate (Khasawneh and Doll, 1978). It has been shown that organic acids can greatly increase the concentration of P solubilization through chelation and an exchange reaction (Gadd, 1999). Soil microorganisms that solubilize mineral phosphates can significantly affect phosphorus cycling in both natural and agricultural ecosystems. Phosphorus absorption by plants can be increased by the presence of symbiotic organisms such as mycorrhizal fungi (Azcon-Aguilar et al., 1986) or by the inoculation with the soil mineral phosphate solubilizing fungi particularly black Aspergilli (Vassilev et al., 1997; Narsian and Patel, 2000; Goenadi et al., 2000; Reddy et al., 2002) and some species of Penicillium (Asea and Kucey, 1988; Cunningham and Kuiack, 1992; Whitelaw et al., 1999). The black Aspergilli include Aspergillus tubingensis, Aspergillus niger, Aspergillus awamori and Aspergillus aculateus. The potential mechanism for phosphate solubilization might be acidification either by proton extrusion associated with ammonium assimilation (De Freitas et al., 1997; Reyes et al., 1999b) or by organic acid production (Cunningham and Kuiack, 1992). Acid phosphatases and phytases secreted by these microorganisms also have an important role in phosphate solubilization (Richardson
ARTICLE IN PRESS V. Achal et al. / Soil Biology & Biochemistry 39 (2007) 695–699
with wild type (At-W) isolate for phosphate solubilization and other growth parameters. The P solubilization ability of these isolates was tested both on tri-calcium phosphate and rock phosphate (equivalent to 100 mg P2O5). The rock phosphate used was Rajasthan Rock Phosphate (RRP) which consists of 34.1% P2O5; 52.6% calcium as CaO; 3.85% Fluoride as F and 0.95% acid insoluble. After 4 days of incubation at 30 1C under shaking conditions, the contents of the flasks were filtered through Whatman no. 42 filter paper and soluble P in the culture filtrate was estimated. Acid phosphatase and phytase activity were also measured in the mycelium. Acid phosphatase activity was estimated with the method described by Tabatabai and Bremner (1969) and the phytase activity by the method described by Ullah and Gibson (1987). Triplicates were maintained for all the experiments. Analysis of variance (ANOVA) and the comparison of treatment means (LSD) were conducted by using GraphPad Prism software. The results presented in Fig. 1 showed that the optimum time for maximum solubilization of phosphorus was found at fourth day. The levels of soluble P in culture filtrate increased significantly up to fourth day and decreased at fifth and sixth day. The biomass was also increased significantly up to fourth day. A plausible reason for such an observation could be attributed to the availability of soluble form of phosphate, which has an inhibitory effect on further phosphate solubilization (Narsian et al., 1995). The negative effect of soluble P on microbial acid productivity (Rohr et al., 1983) might also be responsible for final soluble P concentration. Another explanation for this might be formation of an organo-P compound induced by organic metabolites released, which in turn, reduces the amount of available P (Illmer and Schinner, 1992). The experiment carried out to determine the UV exposure time showed that as the time of UV exposure increased the number of colonies decreased significantly. Ten percent survival (compared to control) was obtained when the mycelia was exposed for 15 min (Fig. 2).
35 30
b
25
10
a
a
300 250
a ab
20 15
a
a
200
b
b
150
c c c
Soluble P Biomass
5 0
100 50
Dry biomass (mg/100 ml)
et al., 2000). Reyes et al. (1999a) isolated the mutants of Penicillium rugulosum which showed high phosphate solubilizing activity compared to wild type strain. The present work was aimed to develop phenotypic mutants for enhancing the phosphate solubilization activity and also to understand the mechanism of phosphate solubilization using A. tubingensis. A. tubingensis (AT1) was isolated from the rhizospheric soils of Eucalyptus plantations near Patiala, Punjab, India. The culture is routinely maintained on Potato Dextrose agar in 90 mm diameter Petri plates at 28 1C. To optimize the time for maximum P solubilization, A. tubingensis was grown in YMG broth (Yeast extract: 4.0 g; Malt extract: 10.0 g; Glucose: 4.0 g; Distilled water 1000 ml) for 5 days and macerated the mycelium with the help of a tissue homogenizer (IKA, Ultra Turax T25, France). Then 10 ml of mycelial suspension was added to each 250 ml conical flask (as uniform inoculum source) containing 100 ml of Pikovskaya’s broth (Glucose: 10.0 g; (NH)2SO4: 0.5 g; NaCl: 0.2 g; MgSO4.7H2O: 0.1 g; KCl: 0.2 g; Yeast extract: 0.5 g; MnSO4: 0.1 mg; FeSO4.7H2O: 0.1 mg; water 1000 ml; pH: 7.070.2, Pikovskaya, 1948) amended with tri-calcium phosphate equivalent to 100 mg P2O5. The flasks were incubated at 30 1C under shaking. The mycelium was harvested after 1, 2, 3, 4 and 5 days of incubation. The mycelium was washed repeatedly with distilled water and dried at 70 1C for 48 h. The culture filtrate was analyzed for soluble P by chlorostannous reduced molybdophosphoric acid blue method as described by Jackson (1967). Fungal growth was expressed as organic matter produced per flask containing 100 ml of medium. The pH of the culture filtrate was also recorded. The phenotypic mutants of A. tubingensis were developed by UV irradiation. A. tubingensis was grown in YMG for 5 days and macerated the mycelium with the help of a tissue homogenizer. Then 5 ml of the mycelial suspension (35–40CFU/ml) was transferred to 90 mm Petri plates and exposed to UV irradiation for 2, 4, 6, 8, 10 and 15 min by using Philips 20 W germicidal UV lamp. Ten microliters of irradiated mycelial suspension was spread on potato dextrose agar medium and the plates were incubated at 30 1C for 48 h along with the control (without UV exposure). Survival counts were obtained after 48 hrs of incubation to optimize the UV exposure time where approximately 10% survival was recorded. To develop the UV induced mutants, mycelial suspension was irradiated for 15 min under the above given conditions and was plated on potato dextrose agar medium. Colonies (10–12) were randomly selected from each plate and transferred into Pikovskaya’s medium containing tri-calcium phosphate (equivalent to 100 mg P2O5). From these, seven different colonies (AtM-1, AtM2, AtM-3, AtM-4, AtM-5, AtM-6 and AtM-7) were selected which showed maximum P solubilization zone along with one colony (AtM-8), of poor solubilization zone for further studies. These isolates were cultured at least five times to assure their mitotic stability. These were compared
Soluble P (mg/100 ml)
696
0 1
2
4
3
5
6
Days Fig. 1. Growth and P solubilization kinetics of Aspergillus tubingensis grown in presence of tri-calcium phosphate (values sharing a common letter within treatment are not significant at Po0:05).
ARTICLE IN PRESS V. Achal et al. / Soil Biology & Biochemistry 39 (2007) 695–699
Solubilization of tri-calcium phosphate and rock phosphate differed significantly with wild type and different mutants of A. tubingensis. AtM-5 and AtM-2 isolates showed maximum P solubilization in both tri-calcium phosphate and rock phosphate amended medium. AtM-8 showed poor solubilization compared to other isolates. The pH of the spent medium was drastically reduced in all
mutants except AtM-8 where the pH was significantly higher. The biomass was not well correlated with mutants in respect to P solubilization. Though the biomass was higher in AtM-8, its P solubilization capacity was less when compared to other isolates. The maximum acid phosphatase activity was observed in AtM-5 followed by AtM-2 whereas AtM-8 showed lower activity compared to other isolates in both tri-calcium phosphate and rock phosphate media (Tables 1 and 2). The phytase activity was significantly higher in AtM-5 and AtM-2 in both media compared to other isolates. AtM-8 showed lower phytase activity than other isolates in tri-calcium phosphate medium (Table 1) whereas in rock phosphate medium no significant difference was observed with most of the other isolates (Table 2). From these results it was observed that among the different mutants developed, AtM-5 and AtM-2 isolates showed maximum P solubilization rate and both acid phosphatase and phytase activities compared to other isolates. Reyes et al. (2001) also observed the maximum P solubilization with Mps++ mutant of P. rugulosum in different rock phosphates compared to the wild type and Mps strain. The growth of AtM-8 in presence of rock phosphate did not lower the pH of the culture filtrate compared to other isolates. Reyes et al. (1999a) suggested the presence of an H+ pump mechanism involved in the solubilization of small amounts of P by the Mps mutant
500
Colonies/10µl
400
300
200
100
0 0
5
10 UV exposure time (min)
15
697
20
Fig. 2. Survival counts of Aspergillus tubingensis exposed to UV after different time intervals.
Table 1 Fungal growth, soluble P, pH, acid phosphatase and phytase activity of mutants of Aspergillus tubingensis in tri-calcium phosphate (equivalent to 100 mg P2O5) amended medium Mutants
Dry biomass (mg/100 ml)
Soluble P (mg/100 ml)
pH
Acid phosphatase activity (mM/g/h)
Phytase activity (mM/g/h)
At-W AtM-1 AtM-2 AtM-3 AtM-4 AtM-5 AtM-6 AtM-7 AtM-8
273d 289abc 283c 295ab 286bc 296a 289abc 296a 283c
35.1cd 34.0bc 46.6ab 35.6c 41.2bc 47.4a 41.3bc 37.2c 29.1d
3.8ab 2.5c 2.3c 2.8c 2.6c 2.3c 2.6 c 3.5b 4.1a
45.0cd 51.4ab 52.3a 49.8c 50.7b 52.3a 50.0c 49.2bc 29.0d
37.8cd 43.0b 45.1a 40.7bc 42.6c 46.8a 41.5c 39.4bc 33.2d
Means sharing a common letter within the column are not significant at Po0:05. Table 2 Fungal growth, soluble P, pH, acid phosphatase and phytase activity of mutants of Aspergillus tubingensis in rock phosphate (equivalent to 100 mg P2O5) amended medium Mutants
Dry biomass (mg/100 ml)
Soluble P (mg/100 ml)
pH
Acid phosphatase activity (mM/g/h)
Phytase activity (mM/g/h)
At-W AtM-1 AtM-2 AtM-3 AtM-4 AtM-5 AtM-6 AtM-7 AtM-8
230e 278d 292ab 275d 289abc 300a 292ab 287bc 277cd
27.7e 38.9c 44.0ab 33.63d 40.9bc 46.0a 39.5bc 36.5cd 26.3e
4.1ab 2.7c 2.5c 3.8b 2.5c 2.5c 2.7c 3.8b 4.3a
33.0cd 39.9bc 45.7a 34.8c 42.5b 46.0a 38.2c 35.0c 25.0d
32.0c 36.7b 40.0a 34.7c 35.9 c 42.4a 35.2c 34.0c 30.9c
Means sharing a common letter within the column are not significant at Po0:05.
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which allowed the fungus to develop a high biomass in media. They also reported less biomass in wild type and Mps++ mutant and suggested that these isolates might have translocated carbon to produce different types and amounts of organic acids instead of fungal biomass to solubilize more P. The results obtained in the present study support the hypothesis proposed by Reyes et al. (1999a). A decrease in the pH of the culture filtrate with all the mutants of A. tubingensis was invariably observed after four days of incubation. The lowest pH was recorded in case of AtM-2 and AtM-5 isolates, which showed highest phosphate solubilization, whereas AtM-8 isolate showed highest pH in media containing tri-calcium phosphate and rock phosphate. A significant relationship could be established between the quantity of phosphate solubilized and drop in pH of the culture filtrate (Tables 1 and 2). This finding is in contrary to the other reports where they did not establish a relationship between the quantity of phosphate solubilized and drop in pH of the culture filtrate (Sethi and Subba Rao, 1968; Narsian and Patel, 2000). The phosphate solubilization and change in pH showed better relationship than with P solubilization and dry biomass, which suggests that lowering the pH has a significant role in phosphate solubilization. A fall in pH during the growth of Aspergillus species in liquid medium containing insoluble phosphates has often been reported due to production of organic acids (Sperber, 1958; Illmer and Schinner, 1995; Reyes et al., 1999b). Assimilation of ammonium present in Pikovskaya’s medium may be one of the causes of lower pH due to higher acid production. Higher acid production and P solubilization from ammonium assimilation has been reported for the solubilization of fluoroapatite by A. niger (Cerezine et al., 1988) and tricalcium phosphate by A. aculeatus (Narsian et al., 1995). The major portion of the P that is applied to soil rapidly becomes ‘fixed’ into inorganic and organic fractions which are poorly available to plants. These fixed fractions of soil P accounts for a major component of the large pool of P occurring in most soils (Sanyal and De Datta, 1991). Of particular significance is the occurrence of soil organic P as phytate. Anderson (1980) reported that phytates (derivatives of inositol hexaphosphates) account for large component of the organic P (some 20–50% of the total soil organic P), yet appear to be only poorly utilized by plants (Hayes et al., 2000; Richardson et al., 2000). Phytates may readily undergo physical and chemical reactions in soil environments, rendering them unavailable for plant uptake (McKercher and Anderson, 1989). Phosphatases (phytase, acid phosphatase, etc.) produced by soil microorganisms play a major role in mineralization of organic forms of soil P to release phosphate (Raghothama, 1999). Isolates of Aspergillus showed high specific activity towards phytate (Gargova et al., 1997). This may be one of the causes of highest P solubilization in case of AtM-2 and AtM-5. Also, the activity of this enzyme might play a role in lowering the pH of the medium. Acid phosphatase participates in the total dephosphorylating
action of this enzyme group and due to the production of acids, pH lowers. AtM-8 and wild type strain (At-W) showed lowest phosphatase secretion in the liquid medium. The production of the acid phosphatase and phytase is favored by low pH values. The lower phytase activity of AtM-8 compared to other mutants and the wild type was better correlated with decrease in pH of medium in case of tri-calcium phosphate than the rock phosphate medium. Gargova et al. (1997) also reported that low pH values favour the production of acid phosphatase and phytase activity in Aspergillus sp. 307. Hence, apart from acidification (or pH decrease), another mechanism which may aid microbial P solubilization is acid phosphatase and phytase activity. A significant increase in soluble P level was observed in case of UV induced mutants of A. tubingensis compared with the wild type. There might be a possibility of alteration at genetic level in case of mutants. In this work, the wild type A. tubingensis (At-W) and its mutants (AtMs) solubilized the tri-calcium phosphate and rock phosphates tested mainly by lowering the pH and also due to production of acid phosphatase and phytase enzymes. The over-expression of the mineral phosphate solubilizing activity in the phenotypic mutants caused an important increase in the P solubilization activity. Our long-term aim is the biosolubilization of poorly soluble phosphate rocks using A. tubingensis. Future work is required based on the plant growth promoting activities of these isolates under pot culture as well as field conditions in presence of rock phosphates to increase soil P availability for different crops before they are recommended as biofertilizers. The authors are thankful to TIFAC-CORE for facilities and Dr. S.C. Saxena, Director, TIET for encouragement. References Anderson, G., 1980. Assessing organic phosphorus in soils. In: Khasawneh, FE., Sample, EC., Kamprath, EJ. (Eds.), The Role of Phosphorus in Agriculture. American Agronomy, Madison, USA, pp. 411–432. Asea, P.E.A., Kucey, R.M.N., 1988. Inorganic phosphate solubilization by two Penicillium species in solution culture and soil. Soil Biology & Biochemistry 20, 459–464. Azcon-Aguilar, C., Gianinazzi-Pearson, V., Fardeau, J.C., Gianinazzi, S., 1986. Effect of vesicular–arbuscular mycorrhizal fungi and phosphatesolubilizing bacteria on the growth and nutrition of soybean in a neutral-calcareous soil amended with 32P–45Ca–tri-calcium phosphate. Plant and Soil 96, 3–15. Cerezine, P.C., Nahas, E., Banzatto, D.A., 1988. Soluble phosphate accumulation by Aspergillus niger from fluoroapatite. Applied Microbiology and Biotechnology 29, 501–505. Cunningham, J.E., Kuiack, C., 1992. Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium billai. Applied and Environmental Microbiology 52, 1451–1458. De Freitas, J.R., Banerjee, M.R., Germida, J.J., 1997. Phosphatesolubilizing rhizobacteria enhance the growth and yield but not phosphorus uptake of canola (Brassica napus L.). Biology and Fertility of Soils 24, 358–364. FAI, A., 2002. Fertilizer statistics 2001–2002 I. The Fertilizer Association of India, New Delhi, pp. 39–47.
ARTICLE IN PRESS V. Achal et al. / Soil Biology & Biochemistry 39 (2007) 695–699 Gadd, G.M., 1999. Fungal production of citric and oxalic acid: importance in metal speciation, physiology and biogeochemical processes. Advances in Microbial Physiology 41, 47–92. Gargova, S., Roshkova, Z., Vancheva, G., 1997. Screening of fungi for phytase production. Biotechnology Techniques 11, 221–224. Goenadi, D.H., Siswanto, Sugiaro, Y., 2000. Bioactivation of poorly soluble phosphate rocks with a phosphorus-solubilizing fungus. Soil Science Society of America Journal 64, 927–932. Hayes, J.E., Simpson, R.J., Richardson, A.E., 2000. The growth and phosphorus utilization of plants in sterile media when supplied with inositol hexaphosphate, glucose 1-phosphate or inorganicphosphate. Plant and Soil 220, 165–174. Illmer, P., Schinner, F., 1992. Solubilization of inorganic phosphates by microorganisms isolated from forest soils. Soil Biology & Biochemistry 24, 389–395. Illmer, P., Schinner, F., 1995. Solubilization of inorganic calciumphosphatessolubilization mechanisms. Soil Biology & Biochemistry 27, 257–263. Jackson, M.L., 1967. Soil Chemical Analysis. Prentice-Hall of India, New Delhi. Khasawneh, F.E., Doll, E.C., 1978. The use of phosphate rock for direct application to soils. Advances in Agronomy 39, 159–206. McKercher, R.B., Anderson, G., 1989. Organic phosphate sorption by neutral and basic soils. Communications in Soil Science and Plant Analysis 20, 723–732. Narsian, V., Patel, H.H., 2000. Aspergillus aculeatus as a rock phosphate solubilizer. Soil Biology & Biochemistry 32, 559–565. Narsian, V., Thakkar, J., Patel, H.H., 1995. Mineral phosphate solubilization by Aspergillus aculeatus. Indian Journal of Experimental Microbiology 33, 91–93. Omar, S.A., 1998. The role of rock phosphate solubilizing fungi and vesicular arbuscular mycorrhiza (VAM) in growth of wheat plants facilitated with rockphosphate. World Journal of Microbiology and Biotechnology 14, 211–218. Pikovskaya, R.I., 1948. Mobilization of phosphorus in soils in connection with vital activity of some microbial species. Microbiologiya 17, 362–370. Raghothama, K.G., 1999. Phosphate acquisition. Annual Review of Plant Physiology 50, 665–693. Reddy, M.S., Kumar, S., Khosla, B., 2002. Biosolubilization of poorly soluble rock phosphates by Aspergillus tubingensis and Aspergillus niger. Bioresource Technology 84, 187–189.
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Reyes, I., Bernier, L., Simard, R., Tanguay, P.H., Antoun, H., 1999a. Characteristics of phosphate solubilization by an isolate of a tropical Penicillium rugulosum and two UV-induced mutants. FEMS Microbiology Ecology 28, 291–295. Reyes, I., Bernier, L., Simard, R.R., Antoun, H., 1999b. Effect of nitrogen source on the solubilization of different inorganic phosphates by an isolate of Penicillium rugulosum and two UV- induced mutants. FEMS Microbiology Ecology 28, 291–295. Reyes, I., Bernier, L., Baziramakenga, R., Antoun, H., 2001. Solubilization of phosphate rocks and minerals by a wild-type strain and two UV-induced mutants of Penicillium rugulosum. Soil Biology & Biochemistry 33, 1741–1747. Richardson, A., Hadobas, P.A., Hayes, J.E., 2000. Acid phosphomonoesterase and phytase activities of wheat (Triticum aestivum L.) roots and utilization of organic phosphorus substrates by seedlings grown in sterile culture. Plant, Cell and Environment 23, 397–405. Rohr, M., Kubicek, C.P., Kominek, J., 1983. Gluconic acid. In: Rehm, HJ., Reed, G. (Eds.), Biotechnology III. Verlag Chemie, Wienheim, pp. 456–465. Sanyal, S.K., De Datta, S.K., 1991. Chemistry of phosphorus transformations in soil. Advances in Soil Science 16, 1–120. Scheffer, F., Schachtschabel, P., 1988. Lehrbuch der Bodenkunde. Enke, Stuttgart. Sethi, R.P., Subba Rao, N.S., 1968. Solubilization of tri-calcium phosphates and calcium phosphate by soil fungi. Journal of General and Applied Microbiology 14, 329–331. Sperber, J.I., 1958. Solution of apatite by soil microorganisms producing organic acids. Australian Journal of Agricultural Research 9, 778–781. Tabatabai, M.A., Bremner, J.M., 1969. Use of p-nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biology & Biochemistry 1, 301–307. Ullah, A.H.J., Gibson, D.M., 1987. Extracellular phytase (E.C. 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization. Preparative Biochemistry 17, 63–91. Vassilev, N., Vassileva, M., Azcon, R., 1997. Solubilization of rock phosphate by immobilized Aspergillus niger. Bioresource Technology 59, 1–4. Whitelaw, M.A., Harden, T.J., Heylar, K.R., 1999. Phosphate solubilization in solution culture by the soil fungus Penicillium radicum. Soil Biology & Biochemistry 31, 655–665.
Soil Biology & Biochemistry 39 (2007) 695–699 www.elsevier.com/locate/soilbio
Short communication
Phosphate solubilization by a wild type strain and UV-induced mutants of Aspergillus tubingensis Varenyam Achala, V.V. Savantb, M. Sudhakara Reddya, a
Department of Biotechnology, Thapar Institute of Engineering and Technology, Patiala 147 004, India b Ballarpur Chemicals Ltd., Karwar, Karnataka, India Received 15 January 2006; received in revised form 29 August 2006; accepted 6 September 2006 Available online 12 October 2006
Abstract Phenotypic mutants of Aspergillus tubingensis were obtained by UV irradiation and phosphate solubilization ability of these mutants were studied and compared with the wild type strain. Low phosphate solubilizing mutant was also selected in this study. Among the different mutants, AtM-5 and AtM-2 showed highest P solubilization when tri-calcium phosphate and rock phosphate were used as P source compared to the wild type strain and other mutants. These mutants also showed maximum acid phosphatase and phytase activity. These results suggest that P solubilization by these isolates is due to lowering of pH of the culture filtrate and also the activity of acid phosphatase and phytase. r 2006 Elsevier Ltd. All rights reserved. Keywords: Aspergillus tubingensis; UV mutants; Phosphate solubilization; Phosphate rock; Phytase activity; Acid phosphatase activity
Phosphorus is one of the most essential macroelements required for the growth and development of plants, but in most soils its content is about 0.05% of which only 0.1% is plant available (Scheffer and Schachtschabel, 1988). Deficiency of soil P is one of the most important chemical factors restricting plant growth in soils. Therefore, large quantity of soluble forms of P fertilizers is applied to achieve maximum plant productivity. However, the applied soluble forms of P fertilizers are easily precipitated into insoluble forms and are not efficiently taken up by the plants, which lead to an excess application of P fertilizers to cropland (Omar, 1998). In recent years the possibility of practical use of rock phosphate as fertilizer has received significant interest. In India, it is estimated that about 260 million tons of phosphatic rock deposits are available and this material should provide a cheap source of phosphate fertilizer for crop production (FAI, 2002). Unfortunately, rock phosphate is not plant available in soils with a pH greater than 5.5–6.0 and even when conditions are optimal, plant yields are lower than those obtained with soluble Corresponding author. Tel.: +91 175 2393043; fax: +91 175 2393738.
E-mail address: [email protected] (M.S. Reddy). 0038-0717/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2006.09.003
phosphate (Khasawneh and Doll, 1978). It has been shown that organic acids can greatly increase the concentration of P solubilization through chelation and an exchange reaction (Gadd, 1999). Soil microorganisms that solubilize mineral phosphates can significantly affect phosphorus cycling in both natural and agricultural ecosystems. Phosphorus absorption by plants can be increased by the presence of symbiotic organisms such as mycorrhizal fungi (Azcon-Aguilar et al., 1986) or by the inoculation with the soil mineral phosphate solubilizing fungi particularly black Aspergilli (Vassilev et al., 1997; Narsian and Patel, 2000; Goenadi et al., 2000; Reddy et al., 2002) and some species of Penicillium (Asea and Kucey, 1988; Cunningham and Kuiack, 1992; Whitelaw et al., 1999). The black Aspergilli include Aspergillus tubingensis, Aspergillus niger, Aspergillus awamori and Aspergillus aculateus. The potential mechanism for phosphate solubilization might be acidification either by proton extrusion associated with ammonium assimilation (De Freitas et al., 1997; Reyes et al., 1999b) or by organic acid production (Cunningham and Kuiack, 1992). Acid phosphatases and phytases secreted by these microorganisms also have an important role in phosphate solubilization (Richardson
ARTICLE IN PRESS V. Achal et al. / Soil Biology & Biochemistry 39 (2007) 695–699
with wild type (At-W) isolate for phosphate solubilization and other growth parameters. The P solubilization ability of these isolates was tested both on tri-calcium phosphate and rock phosphate (equivalent to 100 mg P2O5). The rock phosphate used was Rajasthan Rock Phosphate (RRP) which consists of 34.1% P2O5; 52.6% calcium as CaO; 3.85% Fluoride as F and 0.95% acid insoluble. After 4 days of incubation at 30 1C under shaking conditions, the contents of the flasks were filtered through Whatman no. 42 filter paper and soluble P in the culture filtrate was estimated. Acid phosphatase and phytase activity were also measured in the mycelium. Acid phosphatase activity was estimated with the method described by Tabatabai and Bremner (1969) and the phytase activity by the method described by Ullah and Gibson (1987). Triplicates were maintained for all the experiments. Analysis of variance (ANOVA) and the comparison of treatment means (LSD) were conducted by using GraphPad Prism software. The results presented in Fig. 1 showed that the optimum time for maximum solubilization of phosphorus was found at fourth day. The levels of soluble P in culture filtrate increased significantly up to fourth day and decreased at fifth and sixth day. The biomass was also increased significantly up to fourth day. A plausible reason for such an observation could be attributed to the availability of soluble form of phosphate, which has an inhibitory effect on further phosphate solubilization (Narsian et al., 1995). The negative effect of soluble P on microbial acid productivity (Rohr et al., 1983) might also be responsible for final soluble P concentration. Another explanation for this might be formation of an organo-P compound induced by organic metabolites released, which in turn, reduces the amount of available P (Illmer and Schinner, 1992). The experiment carried out to determine the UV exposure time showed that as the time of UV exposure increased the number of colonies decreased significantly. Ten percent survival (compared to control) was obtained when the mycelia was exposed for 15 min (Fig. 2).
35 30
b
25
10
a
a
300 250
a ab
20 15
a
a
200
b
b
150
c c c
Soluble P Biomass
5 0
100 50
Dry biomass (mg/100 ml)
et al., 2000). Reyes et al. (1999a) isolated the mutants of Penicillium rugulosum which showed high phosphate solubilizing activity compared to wild type strain. The present work was aimed to develop phenotypic mutants for enhancing the phosphate solubilization activity and also to understand the mechanism of phosphate solubilization using A. tubingensis. A. tubingensis (AT1) was isolated from the rhizospheric soils of Eucalyptus plantations near Patiala, Punjab, India. The culture is routinely maintained on Potato Dextrose agar in 90 mm diameter Petri plates at 28 1C. To optimize the time for maximum P solubilization, A. tubingensis was grown in YMG broth (Yeast extract: 4.0 g; Malt extract: 10.0 g; Glucose: 4.0 g; Distilled water 1000 ml) for 5 days and macerated the mycelium with the help of a tissue homogenizer (IKA, Ultra Turax T25, France). Then 10 ml of mycelial suspension was added to each 250 ml conical flask (as uniform inoculum source) containing 100 ml of Pikovskaya’s broth (Glucose: 10.0 g; (NH)2SO4: 0.5 g; NaCl: 0.2 g; MgSO4.7H2O: 0.1 g; KCl: 0.2 g; Yeast extract: 0.5 g; MnSO4: 0.1 mg; FeSO4.7H2O: 0.1 mg; water 1000 ml; pH: 7.070.2, Pikovskaya, 1948) amended with tri-calcium phosphate equivalent to 100 mg P2O5. The flasks were incubated at 30 1C under shaking. The mycelium was harvested after 1, 2, 3, 4 and 5 days of incubation. The mycelium was washed repeatedly with distilled water and dried at 70 1C for 48 h. The culture filtrate was analyzed for soluble P by chlorostannous reduced molybdophosphoric acid blue method as described by Jackson (1967). Fungal growth was expressed as organic matter produced per flask containing 100 ml of medium. The pH of the culture filtrate was also recorded. The phenotypic mutants of A. tubingensis were developed by UV irradiation. A. tubingensis was grown in YMG for 5 days and macerated the mycelium with the help of a tissue homogenizer. Then 5 ml of the mycelial suspension (35–40CFU/ml) was transferred to 90 mm Petri plates and exposed to UV irradiation for 2, 4, 6, 8, 10 and 15 min by using Philips 20 W germicidal UV lamp. Ten microliters of irradiated mycelial suspension was spread on potato dextrose agar medium and the plates were incubated at 30 1C for 48 h along with the control (without UV exposure). Survival counts were obtained after 48 hrs of incubation to optimize the UV exposure time where approximately 10% survival was recorded. To develop the UV induced mutants, mycelial suspension was irradiated for 15 min under the above given conditions and was plated on potato dextrose agar medium. Colonies (10–12) were randomly selected from each plate and transferred into Pikovskaya’s medium containing tri-calcium phosphate (equivalent to 100 mg P2O5). From these, seven different colonies (AtM-1, AtM2, AtM-3, AtM-4, AtM-5, AtM-6 and AtM-7) were selected which showed maximum P solubilization zone along with one colony (AtM-8), of poor solubilization zone for further studies. These isolates were cultured at least five times to assure their mitotic stability. These were compared
Soluble P (mg/100 ml)
696
0 1
2
4
3
5
6
Days Fig. 1. Growth and P solubilization kinetics of Aspergillus tubingensis grown in presence of tri-calcium phosphate (values sharing a common letter within treatment are not significant at Po0:05).
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Solubilization of tri-calcium phosphate and rock phosphate differed significantly with wild type and different mutants of A. tubingensis. AtM-5 and AtM-2 isolates showed maximum P solubilization in both tri-calcium phosphate and rock phosphate amended medium. AtM-8 showed poor solubilization compared to other isolates. The pH of the spent medium was drastically reduced in all
mutants except AtM-8 where the pH was significantly higher. The biomass was not well correlated with mutants in respect to P solubilization. Though the biomass was higher in AtM-8, its P solubilization capacity was less when compared to other isolates. The maximum acid phosphatase activity was observed in AtM-5 followed by AtM-2 whereas AtM-8 showed lower activity compared to other isolates in both tri-calcium phosphate and rock phosphate media (Tables 1 and 2). The phytase activity was significantly higher in AtM-5 and AtM-2 in both media compared to other isolates. AtM-8 showed lower phytase activity than other isolates in tri-calcium phosphate medium (Table 1) whereas in rock phosphate medium no significant difference was observed with most of the other isolates (Table 2). From these results it was observed that among the different mutants developed, AtM-5 and AtM-2 isolates showed maximum P solubilization rate and both acid phosphatase and phytase activities compared to other isolates. Reyes et al. (2001) also observed the maximum P solubilization with Mps++ mutant of P. rugulosum in different rock phosphates compared to the wild type and Mps strain. The growth of AtM-8 in presence of rock phosphate did not lower the pH of the culture filtrate compared to other isolates. Reyes et al. (1999a) suggested the presence of an H+ pump mechanism involved in the solubilization of small amounts of P by the Mps mutant
500
Colonies/10µl
400
300
200
100
0 0
5
10 UV exposure time (min)
15
697
20
Fig. 2. Survival counts of Aspergillus tubingensis exposed to UV after different time intervals.
Table 1 Fungal growth, soluble P, pH, acid phosphatase and phytase activity of mutants of Aspergillus tubingensis in tri-calcium phosphate (equivalent to 100 mg P2O5) amended medium Mutants
Dry biomass (mg/100 ml)
Soluble P (mg/100 ml)
pH
Acid phosphatase activity (mM/g/h)
Phytase activity (mM/g/h)
At-W AtM-1 AtM-2 AtM-3 AtM-4 AtM-5 AtM-6 AtM-7 AtM-8
273d 289abc 283c 295ab 286bc 296a 289abc 296a 283c
35.1cd 34.0bc 46.6ab 35.6c 41.2bc 47.4a 41.3bc 37.2c 29.1d
3.8ab 2.5c 2.3c 2.8c 2.6c 2.3c 2.6 c 3.5b 4.1a
45.0cd 51.4ab 52.3a 49.8c 50.7b 52.3a 50.0c 49.2bc 29.0d
37.8cd 43.0b 45.1a 40.7bc 42.6c 46.8a 41.5c 39.4bc 33.2d
Means sharing a common letter within the column are not significant at Po0:05. Table 2 Fungal growth, soluble P, pH, acid phosphatase and phytase activity of mutants of Aspergillus tubingensis in rock phosphate (equivalent to 100 mg P2O5) amended medium Mutants
Dry biomass (mg/100 ml)
Soluble P (mg/100 ml)
pH
Acid phosphatase activity (mM/g/h)
Phytase activity (mM/g/h)
At-W AtM-1 AtM-2 AtM-3 AtM-4 AtM-5 AtM-6 AtM-7 AtM-8
230e 278d 292ab 275d 289abc 300a 292ab 287bc 277cd
27.7e 38.9c 44.0ab 33.63d 40.9bc 46.0a 39.5bc 36.5cd 26.3e
4.1ab 2.7c 2.5c 3.8b 2.5c 2.5c 2.7c 3.8b 4.3a
33.0cd 39.9bc 45.7a 34.8c 42.5b 46.0a 38.2c 35.0c 25.0d
32.0c 36.7b 40.0a 34.7c 35.9 c 42.4a 35.2c 34.0c 30.9c
Means sharing a common letter within the column are not significant at Po0:05.
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which allowed the fungus to develop a high biomass in media. They also reported less biomass in wild type and Mps++ mutant and suggested that these isolates might have translocated carbon to produce different types and amounts of organic acids instead of fungal biomass to solubilize more P. The results obtained in the present study support the hypothesis proposed by Reyes et al. (1999a). A decrease in the pH of the culture filtrate with all the mutants of A. tubingensis was invariably observed after four days of incubation. The lowest pH was recorded in case of AtM-2 and AtM-5 isolates, which showed highest phosphate solubilization, whereas AtM-8 isolate showed highest pH in media containing tri-calcium phosphate and rock phosphate. A significant relationship could be established between the quantity of phosphate solubilized and drop in pH of the culture filtrate (Tables 1 and 2). This finding is in contrary to the other reports where they did not establish a relationship between the quantity of phosphate solubilized and drop in pH of the culture filtrate (Sethi and Subba Rao, 1968; Narsian and Patel, 2000). The phosphate solubilization and change in pH showed better relationship than with P solubilization and dry biomass, which suggests that lowering the pH has a significant role in phosphate solubilization. A fall in pH during the growth of Aspergillus species in liquid medium containing insoluble phosphates has often been reported due to production of organic acids (Sperber, 1958; Illmer and Schinner, 1995; Reyes et al., 1999b). Assimilation of ammonium present in Pikovskaya’s medium may be one of the causes of lower pH due to higher acid production. Higher acid production and P solubilization from ammonium assimilation has been reported for the solubilization of fluoroapatite by A. niger (Cerezine et al., 1988) and tricalcium phosphate by A. aculeatus (Narsian et al., 1995). The major portion of the P that is applied to soil rapidly becomes ‘fixed’ into inorganic and organic fractions which are poorly available to plants. These fixed fractions of soil P accounts for a major component of the large pool of P occurring in most soils (Sanyal and De Datta, 1991). Of particular significance is the occurrence of soil organic P as phytate. Anderson (1980) reported that phytates (derivatives of inositol hexaphosphates) account for large component of the organic P (some 20–50% of the total soil organic P), yet appear to be only poorly utilized by plants (Hayes et al., 2000; Richardson et al., 2000). Phytates may readily undergo physical and chemical reactions in soil environments, rendering them unavailable for plant uptake (McKercher and Anderson, 1989). Phosphatases (phytase, acid phosphatase, etc.) produced by soil microorganisms play a major role in mineralization of organic forms of soil P to release phosphate (Raghothama, 1999). Isolates of Aspergillus showed high specific activity towards phytate (Gargova et al., 1997). This may be one of the causes of highest P solubilization in case of AtM-2 and AtM-5. Also, the activity of this enzyme might play a role in lowering the pH of the medium. Acid phosphatase participates in the total dephosphorylating
action of this enzyme group and due to the production of acids, pH lowers. AtM-8 and wild type strain (At-W) showed lowest phosphatase secretion in the liquid medium. The production of the acid phosphatase and phytase is favored by low pH values. The lower phytase activity of AtM-8 compared to other mutants and the wild type was better correlated with decrease in pH of medium in case of tri-calcium phosphate than the rock phosphate medium. Gargova et al. (1997) also reported that low pH values favour the production of acid phosphatase and phytase activity in Aspergillus sp. 307. Hence, apart from acidification (or pH decrease), another mechanism which may aid microbial P solubilization is acid phosphatase and phytase activity. A significant increase in soluble P level was observed in case of UV induced mutants of A. tubingensis compared with the wild type. There might be a possibility of alteration at genetic level in case of mutants. In this work, the wild type A. tubingensis (At-W) and its mutants (AtMs) solubilized the tri-calcium phosphate and rock phosphates tested mainly by lowering the pH and also due to production of acid phosphatase and phytase enzymes. The over-expression of the mineral phosphate solubilizing activity in the phenotypic mutants caused an important increase in the P solubilization activity. Our long-term aim is the biosolubilization of poorly soluble phosphate rocks using A. tubingensis. Future work is required based on the plant growth promoting activities of these isolates under pot culture as well as field conditions in presence of rock phosphates to increase soil P availability for different crops before they are recommended as biofertilizers. The authors are thankful to TIFAC-CORE for facilities and Dr. S.C. Saxena, Director, TIET for encouragement. References Anderson, G., 1980. Assessing organic phosphorus in soils. In: Khasawneh, FE., Sample, EC., Kamprath, EJ. (Eds.), The Role of Phosphorus in Agriculture. American Agronomy, Madison, USA, pp. 411–432. Asea, P.E.A., Kucey, R.M.N., 1988. Inorganic phosphate solubilization by two Penicillium species in solution culture and soil. Soil Biology & Biochemistry 20, 459–464. Azcon-Aguilar, C., Gianinazzi-Pearson, V., Fardeau, J.C., Gianinazzi, S., 1986. Effect of vesicular–arbuscular mycorrhizal fungi and phosphatesolubilizing bacteria on the growth and nutrition of soybean in a neutral-calcareous soil amended with 32P–45Ca–tri-calcium phosphate. Plant and Soil 96, 3–15. Cerezine, P.C., Nahas, E., Banzatto, D.A., 1988. Soluble phosphate accumulation by Aspergillus niger from fluoroapatite. Applied Microbiology and Biotechnology 29, 501–505. Cunningham, J.E., Kuiack, C., 1992. Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium billai. Applied and Environmental Microbiology 52, 1451–1458. De Freitas, J.R., Banerjee, M.R., Germida, J.J., 1997. Phosphatesolubilizing rhizobacteria enhance the growth and yield but not phosphorus uptake of canola (Brassica napus L.). Biology and Fertility of Soils 24, 358–364. FAI, A., 2002. Fertilizer statistics 2001–2002 I. The Fertilizer Association of India, New Delhi, pp. 39–47.
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