Effects of Fungal Phytases (Aspergillus Strains) on Availability of Phosphorus from Sodium Phytate and Phosphorus Uptake in Corn Plant

Document Type : Research Article

Authors

• T. Valizadeh ¹
• A. Lakzian ¹
• A. Halajnia ¹
• M. Mazhari ²

¹ Soil Science Department, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran

² Department of Soil Science, Agriculture and Natural Resources Faculty, Karaj Branch, Islamic Azad University

https://doi.org/10.22067/jsw.2021.14925.0

Abstract

Introduction: The deficiency of phosphorus has attracted a lot of attention as one of the most important nutrients for agricultural plants especially in calcareous soils. However, in some soils, organic phosphorous containing 80 percent of total phosphorus in some soils but in most cases, that form of phosphorus is not available for plant uptake. The availability of phosphorus from both organic and inorganic sources by phosphate-solubilizing microorganisms (PSMs) as bio-inoculants are promising substitutes for chemical fertilizer and other agrochemicals amendments. Both arbuscular mycorrhizal fungi (AMF) and phosphate solubilizing bacteria (PSB) play a key role in providing phosphorus for agricultural plants. Among several phosphate-solubilizing fungal isolates, Aspergillus sp. is able to solubilize calcium phosphates by secreting various organic acids, e.g., oxalic and formic acids, and producing phytase enzyme. The present study aimed to evaluate the ability of different strains of Aspergillus for phytase production. The second aim of this study was the purification and application of purified phytase and its efficiency in the phosphorus availability from hexaphosphorylated inositols.
Materials and Methods: Two separate experiments were carried out in two different stages. In the first one phytase was isolated from three strains of Aspergillus (Aspergillus niger provided by the department of plant protection, Agricultural college, Ferdowsi University of Mashhad), Aspergillus flavus, and Aspergillus fumigatus strains were collected from the Iranian biological resources center, Tehran). All Isolates were recultured on PDA (potato dextrose Agar) medium for 5 days at 30 ^oC in an incubator. Quality evaluation of phytase production by three strains of Aspergillus tested using hydrolysis of phytate sodium on PSM (phytase screen medium) medium.  Solubility index was calculated for all three strains (Solubility index = (Colony diameter + Hallow diameter)/ Colony diameter). Phytase production was carried out on fermentation media (Shieh and Ware 1968) but starch was substituted by dextrin. Fermentation media inoculated by fungal strains for 14 days at 30 ^oC.  Fermentation media was centrifuged (10,000 g) for 30 minutes and supernatant was collected. Purification of phytases was done against Tris-HCl 25mM, pH=7.2 for 12 hours. Phytase activities were evaluated in a completely randomized design with three replications. Then purified phytase from three Aspergillus strains was applied in a pot experiment using a completely randomized design with the factorial arrangement and three replications. The experimental factors included two levels of hexaphosphorylated inositols (and 50 mg/kg) and four types of phytase (Control, phytase isolated from Aspergillus niger, Aspergillus flavus, and Aspergillus fumigatus. In the greenhouse experiment, the effects of different phytase types on phosphorus availability from sodium phytate (hexaphosphorylated inositols) and phosphorus uptake by maize plant was evaluated. Corn plants (Zea maize 704 single cross) were grown in 5 kg pots at 70 % of water holding capacity for 60 days. Plant height, root dry weight, shoot dry weight, phosphorus concentration in shoot and root were evaluated.   
Results and Discussion: The results showed that Aspergillus niger and Aspergillus flavus had the highest (4.96) and the lowest (1.23) solubility index among the tested strains, respectively. The results from the laboratory experiment showed that phytase isolated from Aspergillus niger had the maximum amount of phytase activity (16.48 µmol/ min.ml) and phytase isolated from Aspergillus flavus had the minimum phytase activity (4.67 µmol/ min.ml). Aspergillus niger phytase was more effective compared to Aspergillus flavus and Aspergillus fumigatus phytases. The results of the greenhouse experiment represented that the highest amount of phosphorous in the shoot (0.125 percent), root (0.0102 percent), and shoot dry weight (46.08 g/pot) belonged to the maize plants treated by phytase isolated from Aspergillus niger in the presence of 50 mg/kg of sodium phytate. Generally, the results showed that Aspergillus niger strain was more effective than the other two strains in both laboratory and greenhouse experiments. Phytase enzymes isolated from strains had positive effects on phosphorous concentration in a different parts of maize plant and growth characteristics of maize. Phosphatase and phytase generally improve the availability of phosphorus from different phosphorus sources. It should be kept in mind that phytase also increases the bioavailability of other essential minerals such as Ca²⁺, Mg²⁺, P, Zn²⁺, Fe³⁺, which are bound to phytic acid. Since the phytase production by fungi has been attained by different cultivation methods (solid-state, semisolid, and submerged fermentation) it seems that different cultivation methods can affect the phytase efficiency. Therefore, we suggested that phytates from different cultivation methods can be tested for phosphorus bioavailability from different sources.

Keywords

• Aspergillus niger
• Aspergillus flavus
• Aspergillus fumigatus
• Phosphorus availability

20.1001.1.20084757.1400.35.4.4.5

Main Subjects

• Soil science

References

• Bremner J.M., and Mulvaney C.S. 1982. Nitrogen-Urea. In: Miller RH and Keeney, DR (eds). Method of Soil Analysis. Chemical and Microbiological Properties. American Society of Agronomy USA 699-708.
• Chabot R., Cescas M.P., and Antoun H. 1993. Microbiological solubilization of inorganic P-fraction normally encountered in soils. Phosphorus, Sulfur, Silicon 77: 329-336.
• Chapman H.I., and Pratt P.F. 1961. Methods of Analysis for Soils, Plants and Waters. The University of California's Division of Agricultural Science, Berkeley, California, USA.
• Chuang C.C., Kuo Y.L., Chao C.C., and Chao W.L. 2007. Solubilization of inorganic phosphates and plant growth promotion by Aspergillus niger. Biology and Fertility of Soils 43: 575-584.
• Findenegg G.R., and Neiemans J.A. 1993. The effect of phytase on the availability of from myo-inositol hexaphosphate (phytate) for maize roots. Plant and Soil 154: 189-196.
• Gee G.W., and Bauder J.W. 1986. Particle-size analysis. Methods of soil analysis: Part 1—Physical and mineralogical methods. Klute A. Ed. Chap. 15. American Society of Agronomy. Soil Science Society of America 383–411.
• George T.S., Richardson A.E., Li S.S., Gregory P.J., and Daniell T.J. 2009. Extracellular Release of a Heterologous Phytase from Roots of Transgenic Plants: Does Manipulation of Rhizosphere Biochemistry Impact Microbial Community Structure? FEMS Microbiology Ecology 70: 433-445.
• Ghorashi L., Haghnia Gh.H., Lakzian A., and Khorasani R. 2012. Effect of phosphorus and organic matter on availability and uptake of Fe (II) in maize (Zea mays. L). Journal of Agroecology 4(1): 12-19. (In Persian)
• Grant C., Bittman S., Montreal M., Plenchette C., and Morel C. 2005. Soil and fertilizer phosphorus: Effects on plant P supply and mycorrhizal development. Canadian Journal of Plant Science 85(1): 3-14.
• Hariprasada, P., and Niranjana, S.R. 2009. Isolation and characterization of phosphate solubilizing rhizobacteria in improving plant health of tomato. Plant and Soil 316: 13–24.
• Heinonen J.K., and Lahti R.J. 1981. A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal Biochemistry 113: 313-317.
• Idriss E.E., Makarewicz O., Farouk A. … and Borriss R. 2002. Extracellular Phytase Activity of Bacillus amyloliquefaciens FZB45 Contributes to Its Plant-Growth-Promoting Effect. Micro- Biology 148: 2097-2109.
• Javaheri T., Lakzian A., Khorasani R., and Taheri P. 2013. The Effect of Aspergillus Isolates on Hydrolysis of Soil Organic Phosphorus (Phytic Acid and Sodium Glycerophosphate). Journal of Water and Soil 27(6): 24-32. (In Persian with English abstract)
• Karimian N., and Ghanbari A. 1990. Evaluation of different extractants for prediction of Plant response to applied P fertilizer in highly calcareous soils. Abstract, p. 25, 10th Word fertilizer congress, CIEC, Nicosia, Cyprus.
• Khan M.S., Ziadi A., Ahemad M., Oves M., and Wani P.A. 2010. Plant growth promotion by phosphate solubilizing fungi- current perspective. Archives of Agronomy and Soil Science 56: 73-98.
• Khan M.S., Ziadi A., and Wani P.A. 2007. Role of phosphate solubilizing microorganisms in sustainable agriculture - A review. Agronomy for Sustainable Development 27: 29-43.
• Li H., Smith S.E., Holloway R.E., Zhu Y., and Smith F.A. 2006. Arbuscular mycorrhizal fungi contribute to P uptake by wheat grown in a phosphorus-fixing soil even in the absence of positive-growth responses. New Phytology 172: 536–543.
• Loeppert R.H., and Suarez D.L. 1996. Carbonate and gypsum. p. 437-474. In: Sparks, D.L. (ed.) Methods of soil analysis. Part 3. Chemical methods. SSSA Book Series No. 5. SSSA and ASA, Madison, WI.
• Malakoti M.J., and Homaiy M. 2004. Fertility of arid and semi-arid soils (problems and solutions). Second edition with complete revision, Tarbiat Modares University publishers, Tehran.
• Menezes-Blackburn D., Paredes C., Zhang H., Giles C.D., Darch T., Stutter M., George T.S., Shand C., Lumsdon D., Cooper P., Wendler R., Brown L., Blackwell M., Wearing C., and Haygarth P.M. 2016. Organic acids regulation of chemical–microbial phosphorus transformations in soils. Environmental Science and Technology 50: 11521-11531.
• Olsen S.R., and Sommers L.E. 1982. Phosphorus, In: Page A.L. ed. Methods of soil analysis, part 2, Chemical and Microbiological properties, Soil Science Society of American Journal Madison 403–430.
• Ponmurugan P., and Gopi C. 2006. In vitro production of growth regulators and phosphatase activity by phosphate solubilizing bacteria. African Journal of Biotechnology 5: 348-350.
• Rahi P., Vyas P., Sharma S., Gulati A., and Gulati A. 2009. Plant growth promoting potential of the fungus Discosia sp. FIHB 571 from tea rhizosphere tested on chickpea, maize and pea. Indian Journal of Microbiology 49(2): 128–133.
• Richardson A.E., Barea J., McNeill A.M., and Prigent-Combaret C. 2009. Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms. Plant Soil 339: 305–339.
• Richardson A.E., and Simpson Richard J. 2011. Soil microorganisms mediating phosphorus availability. Plant Physiology 156: 989-996.
• Richardson A.E., Hadobas P.A., and Hayes J.E. 2001. Extracellular Secretion of Aspergillus Phytase from Arabidopsis Roots Enables Plants to Obtain Phosphorus from Phytate. The Plant Journal 25: 641-649.
• Rodríguez H., and Fraga R. 1999. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances 17: 319–339.
• Shieh T.R., and Ware J.H. 1968. Survey of microorganisms for the production of extracellular phytase. Appl Microbiol 16: 1348-1351.
• Sarikhani M.R., Malboobi M.A., Aliasgharzad N., and Greiner R. 2019. Identification of two novel bacterial phosphatase‐encoding genes in Pseudomonas putida strain P13. Journal of Applied Microbiology 127(4): 1113-1124.
• Sarikhani M.R., Malboubi M.A., and Ebrahimi A. 2014. Phosphate-solubilizing bacteria: Isolation of bacteria and genes encoding phosphate-solubilizing, mechanism and genetics of phosphate dissolution. Agricultural Biotechnology 6(1): 76-110. (In Persian)
• Singh B., and Satyanarayana T. 2011. Microbial Phytases in Phosphorus Acquisition.
• Walkley A., and Black I.A. 1934. An examination of digestion method for determining soil organic matter and proposed modification of the chromic acid titration. Soil Scince 37: 29-38.
• Wang X., Wang C., Sui J., Liu Z., Li Q., Ji C., Song X., Hu Y., Wang C., Sa R., and Zhang J. 2018. Isolation and characterization of phosphofungi, and screening of their plant growth-promoting activities. AMB Express 8: 63-70.
• Wasaki J., Maruyama H., Tanaka M., Yamamura T., Dateki H., Shinano T., Ito S., and Osaki M. 2009. Overexpression of the LASAP2 gene for secretory acid phosphatase in white lupin improves the phosphorus uptake and growth of tobacco plants. Soil Science and Plant Nutrition 55: 107–113.
• Yadav R.S., Tarafdar J.C. 2003. Phytase and phosphatase producing fungi in arid and semi-arid soils and their efficiency in hydrolyzing different organic P compounds. Soil Biol Biochem 35(6): 745–751.
• Yadav B.K., and Tarafdar J.C. 2009. "Extracellular acid phosphatase and phytase activities of Buffel grass (Cenchrus ciliaris) genotypes." Journal of the Indian Society of Soil Science 57(3): 332-337.