عنوان مقاله [English]
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.