2.1 Study area
Soil samples were collected from Beni-Suef governorate as shown in Fig. 1 (28° 36 N–29° 26' latitude and 30° 36' E–31° 21' E), Egypt. The climate of this region is arid, with annual rainfall of 1 L/m2.
2.1.1 Isolation and screening of P-solubilizing fungi (Qualitative method)
Thirty soil samples were obtained from different localities and habitats as shown in Fig. 1. Soil samples were taken from rhizospheres of Alhagi graecorum Boiss., Allium cepa L., Ehrharta calycina Sm., Ludwigia stolonifera (Guill. & Perr.) Raven, Mentha longifolia L., Phragmites communis Trin., Triticum aestivum L. and Zea mays L. Phosphate solubilizing fungi (PSF) were isolated by the dilution plate method, this method was modified by Johnson et al. [17]. 10 g of rhizosphere soil samples were taken at profile (0–10 cm) by using sterile auger and air dried for 24 h then passed through a 0.5 mm soil sieve to remove large particles. Pikovskaya medium was used in the isolation of PSF.
The compositions of the medium (g/l) are glucose, 10 g; Ca3(PO4)2, 5 g; (NH4)2SO4,0.5 g; NaCl,0.2 g; MgSO4·7H2O,0.1 g; KCl, 0.2 g; yeast extract, 0.5 g; MnSO4·7H2O, 0.003 g; FeSO4·7H2O, 0.003, agar, 15 g and 1000 ml distilled water. The pH of the medium was adjusted to 7 before autoclaving. Rose Bengal as a bacteriostatic agent was added to the medium (10 ml/l) at concentration 1/15000 [18]. One ml for each dilution was put in sterilized petri-dishes. Four replicates for each dilution were used. Finally, the melted PVK medium was poured in the plates containing samples. The plates were incubated at 25–28 °C for 2–7 days in inverted position. After incubation, fungal colonies which showed clear zones around the colonies isolated and purified.
2.2 Purification of phosphate solubilizing fungi
Pikovskaya,s medium without Rose Bengal addition was prepared for purification. Streak plate method was used to purify the fungal isolates. Monospore technique or hyphal tip method [19] was used to obtain monospore fungal colony. Two drops of tween 80 were added to spore suspension of pure culture of PSF. The streak plate method was used to spread the spore suspension on PDA media by sterile cotton swab. The plates were incubated at 25 °C for 24 h. The plates were examined under light microscope to choose hyphal tip. The microscopic hyphal tip as shown in Fig. 2 was cut by sterile forceps and transferred to plates containing PDA media to complete its growth. The pure cultures were preserved on Potato Dextrose Agar (PDA) slant at 4 °C for further investigation.
2.2.1 Calculation of solubilization index of phosphate solubilizing fungi (PSF)
The isolates were assayed for phosphate solubilization under in vitro condition by the method described by Iman [20] on Pikovskaya’s agar medium. The fungal isolates were put onto the center of plates by pin point inoculation in triplicate under aseptic condition. The plates were incubated at 25–28 °C for 7 days. The clear zone and colony diameters of the fungal isolates were measured in centimeters to determine the solubilization index. The Phosphate solubilization index was calculated by The ratio of the total diameter (colony + halo zone) and the colony diameter.
2.2.2 Morphological identification of phosphate solubilizing fungi
The fungal isolates were grown on Dox agar medium for 2–3 days at 25 ± 2 °C and identified according to Moubasher [21].
2.2.3 Extraction of genomic DNA, PCR amplification and sequencing
The molecular identification was conducted in Microbiology and Applied Genomics Group, Institute of Chemical, Environmental & Bioscience Engineering Vienna, University of Technology, Austria. PSF were grown on malt extract agar medium and the extraction of DNA was carried by the plant DNeasy Minikit (QIAgen GmbH, Hilden, Germany) according to the manufacturer’s instructions. The nuclear DNA region containing the ITS1 and 2 regions of the rRNA gene cluster was amplified by PCR. Secondary markers were used in addition to ITS, namely calmodulin (CaM) for genus Aspergillus and β-tubulin (BenA) for genus Penicillium. PCR products were purified with the QIAquick PCR Purification Kit (QIAgen) and were subjected to automated sequencing at MWG (Martinsried, Germany).The obtained sequences were subjected to sequence similarity comparison of the sequences from the NCBI Gene Bank database (www.ncbi.nlm.nih.gov).
2.3 Antagonistic potential of phosphate solubilizing fungi
Nine PSF isolates were tested for inhibiting the growth of A. alternata, F.solani, G. candidum, R. solani and S. rolfisii by dual culture on PDA in petri dishes according to Helrich [22]. The test pathogenic fungi were collected from Agricultural research center, Giza. The tested PSFs and fungal pathogen were separately cultured by streak plate method in PDA for 7 days. Five mm disc from pathogens and PSFs were grown in two polar side positions in plates of PDA. The treatments were replicated three. The dual cultures were incubated at 25–30 °C for 7 days. The diameters of colonies of both phosphate solubilizing fungi and fungal pathogens were measured after 7 days of incubation. The percentage of growth reduction of the pathogens was calculated using
$$\% \;{\text{growth inhibition}} = \frac{{R_{c} - R_{t} }}{{R_{c} }} \times 100$$
where Rc is colony diameter of pathogen (control) and Rt is colony diameter of pathogen inoculated with tested fungi.
2.4 Effect of different carbon sources and glucose concentration on phosphate solubilization by Aspergillus japonicus 2
The used carbon sources (1%) were xylose, sucrose, starch and mannitol instead of glucose in PKV medium according to Jayaraman and Ilyas [23]. PKV medium was prepared without carbon source and different glucose concentrations (0–5%) were added. Five mm diameter discs from 5 days old colony of Aspergillus japonicus 2 grown on PDA medium was inoculated to 100 ml Pikovskya broth medium with different carbon sources and different glucose concentrations. Sterilized Pikovaskaya medium without carbon source was used as control. Culture of Aspergillus japonicus 2 was incubated at 28 °C ± 2 °C, for 7 days under shaking conditions (121 rpm). The culture was harvested after 7 days of incubation, centrifuged at 15,000 rpm for 10 min to remove the cells and debris, and then subjected to analysis. The solubilized phosphorus was estimated by ascorbic acid method [24].
2.5 Effect of different nitrogen sources on the phosphate solubilization by Aspergillus japonicus 2
The nitrogen source (0.05%) ammonium sulphate in basel PVK medium was replaced by ammonium chloride, sodium nitrate, urea and tryptophan according to Jayaraman and Ilyas [23]. Sterilized Pikovaskaya medium without nitrogen source was used as control. Culture of A. japonicus 2 was incubated at 28 °C ± 2 °C, for 7 days under shaking conditions (121 rpm).The culture was harvested after 7 days of incubation, centrifuged at 15,000 rpm for 10 min to remove the cells and debris and then subjected to analysis. The solubilized phosphorus was estimated by ascorbic acid method.
2.6 Effect of different pH values on the solubilization activity of Aspergillus japonicus 2
To determine the optimum pH for tricalcium phosphate (TCP) solubilization by A. japonicus 2, fungal discs (5 mm) from four days, old culture were inoculated into 100 ml broth PKV medium. The initial pH of the culture medium was adjusted by adding 1 N HCL or 1 N NaoH solution. The different initial pH (3, 4, 5, 6, 7 and 8). Culture of A.japonicus 2 incubated at 28 °C ± 2 °C, for 7 days under shaking conditions (121 rpm) for 7 days. The solubilized phosphorus was estimated as mentioned above.
2.7 Effect of incubation periods on the phosphate solubilization by Aspergillus japonicus 2
The fungal discs of A. japonicus 2 were incubated at 28 °C ± 2 °C on different time intervals (0, 2, 4, 6, 8 and 10 days) under shaking conditions (121 rpm) to study the effect of time on solubilization activities by A. japonicus 2.The culture was harvested at each time intervals and centrifuged at 15,000 rpm for 10 min to remove the cells and debris, and then subjected to analysis. The solubilized phosphorus was estimated as mentioned above.
2.8 Statistical analysis
The experiments were designed in three replicates. Statistical analysis was operated using one-way analysis of variance (ANOVA). Post-hoc mean comparison was performed by Duncan’s multiple range tests at p ≤ 0.05 by using SPSS software version 20.
