Formation of bio-hybrid nanocomposite [P. oxalicum/P 2 O 5 +Ca 3 (PO 4 ) 2 ] BHC by Penicillium oxalicum and its application on the growth of canola

doi:10.21203/rs.3.rs-1666294/v1

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Formation of bio-hybrid nanocomposite [P. oxalicum/P 2 O 5 +Ca 3 (PO 4 ) 2 ] BHC by Penicillium oxalicum and its application on the growth of canola

https://doi.org/10.21203/rs.3.rs-1666294/v1

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The novel synthesis of biohybrid nanocomposite [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC by Penicillium oxalicum in presence of tricalcium phosphate and phosphorus pentoxide for phosphate rocks. Phosphorus elemental solubilization from insoluble rock phosphate by utilizing microorganisms is a promising approach for lowering production costs and increasing environmental sustainability. P. oxalicum was isolated and identified in NRC, Cairo, Egypt, and proved its ability of P highest phosphate solubilization potential by estimating clear zone and solubilization index in Pikovskaya’s agar medium and available P and phosphatase enzyme in broth media. The highest dry shoot is the inoculation treatment P. oxalicum + Extract (52.59 g) in presence of TCP and the highest dry root is the inoculation treatment P. oxalicum (6.72 g) in presence of RP. The highest phosphate content and uptake are extremely in the presence of rock phosphate and tricalcium phosphate. the results showed that significantly increased the total counts of PSM at all sources of mineral phosphate fertilizer and PSF count is increased in presence of RP and TCP more than SSP compared with control. The biohybrid nanocomposite showed an absorbance of 0.26 and 2.23 at the wavelength (λ) 378 nm and 465 nm. Also, significant changes in the absorption index and indirect/direct bandgap of the biohybrid nanocomposite have been detected. To perform frequency calculations for crystal models and isolated molecules and optimize the molecular structure, the materials Studio 7.0 program using TDDFT/DMol³ and CASTEP methodologies were utilized. The DFT-Gaussian09W-vibration values are quite similar to the experimental data either in the structure or in the optical properties.

Penicillium oxalicum

Biohybrid nanocomposite

Canola

Rock Phosphate

Tricalcium phosphate

TD-DFT calculations

Phosphorous is an essential macronutrient that is required for the formation of critical structural components as well as the catalysis of a variety of chemical plant functions [1]. Phosphorus shortage in crops causes slowed development and aberrant bluish-green discoloration in the early stages of growth, as well as a reduction in yield at maturity [2]. phosphorus of soil is found as organic and inorganic sources that have been chemically chelated to form calcium, ferrous, manganese, and aluminum hydrate oxides, making it insoluble and difficult to access for plants [3]. Recommended huge quantities of soluble-P fertilizers are commonly used to boost agricultural yield. Furthermore, much of the soluble P can bind adsorb on the soil particles surface and react with soil cations, particularly Ca, Fe, and Al, to generate insoluble forms such as CaHPO4, Ca₃(PO₄)₂, FePO₄, and AlPO₄, which are less accessible to plants., resulting in pollution with phosphorus which causes soil disturbance and runs off [4, 5]. Furthermore, traditional P fertilizer production procedures result in unacceptable and unsustainable environmental and economic concerns, as well as a decline in overall soil fertility [6]. As a result, the need to develop cost-effective and environmentally friendly technology is rapidly growing [7]. Phosphate-bearing minerals, especially RP, are sprayed directly into the soil and usually contain some type of mineral apatite. as an alternate technique, with varied agronomic efficiency depending on the type of soil and crop [8]. The most common microorganisms that can solubilize unavailable P to available and soluble form to plants are Aspergillus, Pseudomonas, Rhizobium, Enterobacter, Penicillium, Leif-Sonia, Bacillus, and Burkholderia [8–10]. Acidification, chelation, and exchange processes are among the strategies used by microbes to solubilize phosphate. A drop in pH is caused by the synthesis of organic acids, mainly gluconic acid, and there is a strong link between final pH and soluble phosphate, with a drop in pH directly influencing rock phosphate solubilization.[11, 12]. Alternatively, microbial activity can cause Ca²⁺, Fe²⁺, and Al³⁺ ions to be chelated and exchanged, converting Pinto accessible forms.[13]. Canola is a great model plant for researching the rhizosphere microbiome since it does not form symbiotic connections with mycorrhizae and produces antimicrobial isocyanates, resulting in simplified rhizosphere ecosystems [14, 15].

In the present study, studying the ability of P. oxalicum for phosphate solubilization potential by estimating the clearing zone of P solubilization on Pikovskaya’s agar and phosphate solubilization index and confirming with available P and phosphatase activity in liquid media in the presence of TCP and RP. Materials Studio 7.0 program optimizes molecular structure and performs frequency calculations for crystal models and isolated molecules using TDDFT/DMol3 and CASTEP methods. The DFT-Gaussian09W-vibration values and the optical properties were studied. Studying the effect of salinity on the solubilization of phosphate and applying the ability of P. oxalicum to solubilize RP and TCA in pot experiment and utilize the solubilized P in the growth of Canola by estimating plant height, plant dry weight, shoot and root dry weight, available P and the effect of PSF in the solubilization phosphate comparing with superphosphate.

2.1. Materials and reagents

Table 1 provides information on the chemicals, metals, and reagents utilized in these experiments. All chemicals were utilized exactly as they were supplied, with no further purification.

Table 1

The molecular formula and supplier for chemicals, reagents, and metals used.
Chemical	Molecular formula	Supplier
Yeast Extract	-	Merck
Glucose	C₆H₁₂O₆	Sigma
Ammonium Sulphate	(NH₄)₂SO₄	Sigma
Potassium Chloride	KCl	Sigma
Magnesium Sulphate	MgSO₄	Merck
Manganese Sulphate	MnSO₄·H₂O	Aldrich
Ferrous Sulphate	FeSO₄	Merck
Tri Calcium Phosphate	Ca₃(PO₄)₂	Merck
Agar	-	Sigma &Aldrich
Nutrient agar medium	-	Sigma
Potato Dextrose Agar medium	-	Sigma
Ethanol	C₂H₅OH	Sigma
Chromic acid and nitric acid	H₂CrO₄ and HNO₃	Merck
choro molybdic	MoO₃·2H₂O	Sigma
choro stannous acid	SnCl₂	Sigma
Potassium dihydrogen phosphate	KH₂PO₄	Sigma
p- nitrophenyl phosphate	C₆H₄NO₆P^-2	Sigma
Sodium hydroxide	NaOH	Sigma

2.2. Synthesis of biohybrid nanocomposite of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC.

2.2.1. Fungal isolate

P. oxalicum was obtained from the Agricultural Microbiology Department., NRC, Egypt. The strains identified Based on their 18S rRNA gene sequences and phylogenetic positions, P. oxalicum strain was recorded under number Po-5 No. HQ680452.

2.2.2. Testing the ability of P. oxalicum for phosphate solubilization.

P. oxalicum was examined for their ability of phosphate solubilization (PS) by growing on Pikovskaya’s agar medium supplemented with tricalcium phosphate (TCP) which composed from (Yeast Extract 0.50 g, Glucose, 10 g, Ammonium Sulphate 0.5 g, Magnesium Sulphate 0.1 g, Potassium Chloride 0.2 g, Manganese Sulphate 0.002 g, Ferrous Sulphate 0.002 g, Tri Calcium Phosphate 5.0 g, and agar. The incubation occurred at 37^ᴼC for 3, 5, and 7 days. The colony's diameter and the clear zone's area were both measured. [18]. The clear zone to colony ratios was computed by dividing the clear zone's area by the colony's area, and the phosphate solubilization index was derived using the following formula: the colony diameter divided by the overall diameter (colony + halo zone) [19].

2.2.3. The solubilization of tricalcium and rock phosphates (TCP and RP) in liquid medium

P. oxalicum was examined for their ability of phosphate solubilization (PS) by growing on Pikovskaya’s broth medium supplemented with TCP and RP replaced by TCP (50 mg P₂O₅100 mL^− 1) [20]. The inoculation ratio of 10⁶ spores ml^− 1 was prepared to suspend to each isolate with 0.85% w/v of physiological saline. In a 250 ml Erlenmeyer flask, one milliliter of the spore suspension was added to 50 ml Pikovskaya's medium. For 7 days, the triplicate repetition was incubated at 37°C with 200 rpm. The uninoculated PKV medium was used as a control. By using a Whatman paper filter no. 42, the cultures were collected.[16]. The dry mycelium was determined by oven-dried at 65°C for 24 hours[17]. The supernatants were diluted to 100.0ml with autoclaved distilled water and tested for available phosphate(P₂O₅) using the molybdovanadate-phosphate technique. Then 5.0ml of each dilution was transferred to a 50.0 ml volumetric flask. Then 10.0 ml choro molybdic acid was poured along the flask's sides. With distilled water, the contents of the flask were diluted to 40.0 ml. The choro stannous acid was then added in 5 drops. With distilled water, the volume was increased to 50.0 ml after mixing. A spectrophotometer was used to detect the solution's blue color intensity at 660nm O.D. [18–20]. A standard curve of KH₂PO₄ (0–2 ppm) was made against O.D. 600 nm to estimate the soluble [21], Phosphatase activity was measured using p-nitrophenol[23, 24] and final pH was determined with a glass electrode[25]. In the flask, one ml of supernatant, four ml of buffer (pH 5), and one ml of filter-sterilized 0.115M p-nitrophenyl phosphate solution were added. To mix the contents, the tubes were twirled for a few seconds. The tubes were capped and incubated at 37°C for 1 hour in the dark before being stopped with 4 ml of 0.5M NaOH. At 410 nm, the intensity of the yellow color produced was measured. The above method was carried out without the sample as a control, and 1 ml p-nitrophenyl phosphate (PNP) was added after 0.5N NaOH. Potassium dihydrogen phosphate was used as a standard of available phosphate and PNP was used as the standard of phosphatase activity [25]. By adding the composite released from RP and released from TCP the biohybrid composite is former[26, 27] .

2.2.4. Phosphate solubilization in liquid medium under salt stress.

Phosphate solubilization by P. oxalicum was tested under different concentrations of NaCl. various concentrations of sodium chloride (0, 2, 4, 6, and 8%) added to PKV broth medium. Media was inoculated with 1 ml of spore’s suspension containing 106 spores ml-1 to 50 ml Pikovskaya’s medium in a 250 ml Erlenmeyer flask. The triplicate repetition was incubated at 37^ᴼC in an orbital shaker at 200 rpm for 3, 5, and 7 days. The uninoculated medium was used as a control. Filtration with a Whatman paper filter no. 42 was used to harvest the cultures. The dry mycelium was a determination by oven-dried at 65°C for 24 hours [22]. The supernatants were analyzed for available phosphate (P₂O₅) with the molybdovanadate-phosphate method [23, 24] and final pH was determined (with glass electrode).

2.3. Computational study of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC as an isolated molecule in the cell unit

For the crystal models and isolated molecules, the Materials Studio 7.0 program using TDDFT/DMol³ and CASTEP methodologies was utilized to perform frequency calculations and optimize the molecular structure. TDDFT/DMol³ and CASTEP approaches were used to complete the norm-conserving pseudopotential generalized gradient approximation at Perdew–Burke–Ernzerh of correlation functional, as well as the double numerical plus polarization (DNP) basis set for isolated molecules in the gaseous state. [28, 29]. The plane-wave cut-off energy was 830 eV, and geometry properties were estimated at the gamma point using the Gaussian 09W software program from DMol³ frequency calculations. [30]. The ground state option was used after the optimization + frequency task type was chosen, and then the TDDFT default and the WBX97XD/6-311 G spin and basis set were chosen [31, 32].

2.4. Evaluation of efficient strains under Pot Experiment conditions

During the winter of 2021, a pot experiment was done to determine to what extent the influence of TCP and RP solubilization by P. oxalicum on plant growth and soil fertility. Canola was selected for the pot study under salinity-affected soil. The pot experiments were carried out in plastic pots with a diameter of 21cm and unsterile soil from El-Santa. Gharbia Governorate, Egypt. Some chemical, physical, and microbiologically properties of soil used before and after treatments were shown as pH, EC, available phosphate as mentioned above and some biological parameters as the total count of microorganisms, the total count of fungi, and PSF by making ten-fold serial dilution to place 1 ml of dilutions (1/10, 1/100, 1/1000,1/10⁵, 1/10⁷, 1/10¹⁰ on plates of potato dextrose agar medium for the total count and T.F and Pikovskaya’s agar media for PSF. Under aerobic conditions, the plates were incubated at 30°C for two days for bacteria and five days for fungi, after that the colonies of bacteria and fungi were counted [17, 33].

In each pot, five canola seeds were sowed at a depth of 3 cm [34], filled with a mixture of sandy soil and loamy clay soil (1:1, v: v). This soil of the experiment has a moisture content of around 44%. Urea was used as a source of nitrogen at 80 kg N ha^− 1 in two-separate doses (initially at sowing and during the flowering stage)[35]. Depending on the treatments, 20 kg P ha-1 was applied in the form of SSP, RP, or TCP. The following treatments were used in the experiment: The soil treatments (1. Soil Control (without phosphate, 2. Soil treated with single super phosphate, 3. Soil treated with Rock phosphate, 4. Soil treated with tricalcium phosphate) and the seed treatments are (1-Control (without fungi, 2. Seed treated with spores of P. oxalicum, 3. Seed treated with supernatant of P. oxalicum and Seed treated with spores and supernatant of P. oxalicum). Bio-inoculants were applied by the seed soaking in the spore’s suspension (2.6xl0⁶ spore ml^− 1) of P. oxalicum or by the supernatant of each fungus for 15 min and then sowed immediately in the presence of Arabic gum as an adhesive agent. In the control, the seeds are those that have been treated with a spore-free solution [36]. Three replicates of each treatment were made. Irrigation was done once before canola was sown in the experiment to ensure adequate soil water storage for seedling establishment. The plants were watered regularly. [37]. All of the canola crop's required agricultural practices were followed. [34]. Before sowing, phosphate fertilization in the form of superphosphate (SSP), RP, or TCP was applied to the soil at the full recommended dose, based on the P₂O₅ level. The treatments were set up in a three-replication randomized block design. The plants were harvested 60 days after sowing, and the growth data of the plants were recorded.

Rhizospheric soil samples were obtained after 30, 45, and 60 days following sowing for counting total bacteria on NB, total fungus on PDA, and fungi phosphate solubilizers on modified Pikovskaya's agar medium as mentioned above. Various growth indicators, such as plant height, shoots, and root length (cm) of canola plants, were measured at the end of the period of cultivation. The plants were dried in an oven for 24 hours at 65°C and the dry weight of the shoots and roots were measured. After digestion with H₂SO₄, the procedure was used to determine the P content, the acid phosphatase activity and available phosphate (P₂O₅) in the soil samples were assessed using the above method, and the final pH was evaluated using a glass electrode.

2.5. Tricalcium phosphate (TCP) and Rock Phosphate (RP)

Tricalcium phosphate (TCP) and Rock Phosphate (RP) were kindly provided by the Soils, Water, and Environment Research Institute, Agricultural Research Center, Egypt, and their P content was determined (7.97%). Instead of TCP, rock phosphate in the amount of 50 mg P₂O₅ /100 mL^− 1 was added to the growth medium.[38].

2.6. Data analysis

The results were given as a mean with a standard deviation. The study is carried out using a completely randomized experimental design[34]. The variance of one-way analysis of variance(ANOVA) was used to examine the significant differences between the values [39]. The Minitab 15 Statistical Software was used to examine the significant differences using Duncan's multiple range tests.

Table 2 shows the instruments used to characterize the solution and powder samples, as well as the fabricated [P. oxalicum /P₂O₅ + Ca₃(PO₄)₂]^BHC.

Table 2

List of analyses methods and equipment.
Analytical methods	Types and models
FT-IR	Perkin-Elmer FT-IR type 1650 spectrophotometer.
XRD	A RIGAKUU Ultimo IV XRD / Cu Kα radiation ( λ = 1.5418 Å)
Optical measurements	Shimadzu UV-3600 UV–vis NIR spectrophotometer.
pH	TOA, pH meter- HM-14P
EC	Mahita multi-paramer Analyzer, model 917
Sterilization	Autoclave- Vertical type –Shimadzu- Model SS-325
Distillation equipment	Fistreem international Ltd –UK— Model (WSC044.MH3.4)- serial NO: FI-P070003
Drying	Oven, SIBATA-Shimadzu- model VS-300
Incubation	Shimadzu- Model (C1-610)
Fungi cultivation	Clean BenchShimadzu – model (SCB-1000 AS)

3.1. The ability of P. oxalicum for phosphate solubilization.

Clear zone and phosphate solubilization index are the essential parameter for solubilization of P. More than a 5 mm zone of solubilization was seen in fungus strains, indicating P solubilization. [18]

Table 3

Clearing zone (mm) of P solubilization on Pikovskaya’s media by selected fungal isolates under different incubation periods (day).
Incubation period (days)	Clear zone(mm) of P. oxalicum	Phosphate solubilization index
3days	15.0	2.1
5 days	25	2.5
7 days	38.0	3.8

3.2. The solubilization of tricalcium and rock phosphates (TCP and RP) in liquid medium

Phosphate solubilizing activity of P. oxalicum on a PVK solid medium was confirmed, so P. oxalicum should be tested for its ability on and liquid medium in the presence of RP and TCP [40, 41].

Data presented in Table 4 present the amount of solubilized P (µg/ml) in PVK liquid medium containing TCP or RP and RP (equivalent to 100 mg P₂O₅/100 ml). P. oxalicum has demonstrated the ability to solubilize P in a liquid media. The inorganic, insoluble phosphate had been efficiently transformed into a soluble form by P. oxalicum. The acid phosphatase activity for P. oxalicum was determined and recorded in Table 5. The maximum phosphatase activity released, varied between the isolates from 3rd to 9th day. The amount of phosphatase released by P. oxalicum ranged from (200 to 310 EU ml^− 1) either in presence of RP or TCP. It was seen that the production of phosphatase decreased from 3rd to 5th day. This proves that the P. oxalicum produced a high amount of phosphatase on 9th day (310 and 300 EU, in TCP, and RP respectively).

After 1 week of incubation, the fungal mycelium was filtered and dried at 45°C followed by measuring its dry weight and pH. The results showed that Increasing P solubilization resulting a significant decrease in PH value and no relation between dry way and p solubilization (Table 4).

Table 4

Phosphate solubilizing ability of *P. oxalicum* in PKV broth supplemented with TCP or RP on day 7th of incubation
TCP			RP
pH	P-released in medium (mg P ml^-1)	Mycelium DW (g 50ml^-1)	pH	P-released in medium (mg P ml^-1)	Mycelium DW (g 50ml^-1)
3.90 ± 0.9	396 ± 13	0.225 ± 0.023	4.24 ± 0.04	295 ± 7	0.355 ± 0.83

Values are Mean ± SD (n = 3) DW: dry weight TCP: tricalcium phosphate RP: rock phosphate

Table 5

production of phosphatase activity (µg PNP ml^− 1 l h^− 1) in PKV liquid medium after different incubation periods (day).
Incubation periods (day)
	3	5	7	9
TCP	240	200	240	310
RP	210	200	225	300

3.3. Phosphate solubilization in liquid medium under salt stress

From the results of clearing zone, pH, phosphatase activity, and phosphatase activity in a liquid medium, P. oxalicum showed its ability of p solubilization. Now P. oxalicum can be applied in a liquid medium under different salinity levels. Pe. oxalicum was tested in Pikovskaya’s medium when different concentrations of NaCl as a source of salinity (0, 2, 4, 6, and 8%) were used at 30°C for 5 days. The calculation of variance revealed that salt had a significant effect on the isolates' ability to solubilize phosphate and that the isolates differed in their ability to do so (Table 6). P. oxalicum demonstrated a slower rate of solubilization at the highest concentration of NaCl (8%). P. oxalicum gave the best results at a concentration of NaCl (2%), which was (78.73 mg/ml). When the concentration of NaCl is increased, the activity decreases. This is owing to salt-reduced solubilization efficiency by suppressing cell multiplication, lowering solubilization efficiency, or chloride ions (Cl-) sequester or neutralizing protons or acids produced in the media. [42]. The solubilization of phosphate in the liquid media was usually accompanied by a drop in the pH of the medium. For all isolates, the analysis of variance noted there is a significant effect on pH medium reached to (P < 0.01) (Table 6).

After 1 week of incubation, the fungal mycelium was filtered and dried at 65 °C followed by measuring their dry weight. P. oxalicum exhibited an increase in the dry weight at 8% NaCl, (Table 6 and Fig. 2). The solubilization of phosphate and the decreasing pH of the medium had a negative significant correlation reached to (r = -0.6758) (Table 6).

Table 6

Amounts of solubilized phosphate (µg ml^− 1) in Pikovskaya’s medium under different concentrations of NaCl (%).
parameters	NaCl concentration (%)
parameters	0	2	4	6	8
Amounts of solubilized phosphate (µg ml^-1)	64.75 ± 4.06^a	78.73 ± 5.67^a	75.80 ± 8.57^a	45.67 ± 1.31^b	43.50 ± 1.10^b
pH	5.17 ± 0.09^b	5.15 ± 0.06^b	5.22 ± 0.11^ab	5.14 ± 0.08^b	5.72 ± 0.38^a
Dry weight	0.580 ± 0.06^b	0.580 ± 0.05^c	0.615 ± 0.08^b	0.595 ± 0.04^a	0.470 ± 0.05^b
phosphatase activity (µg PNP ml^-1 l h^-1)	290	340	300	260	230

Means amounts of P205 ± the SD. Figures in the same Row followed by the same letters are not significantly different (p > 0.05) based on Duncan’s multiple range tests

Table 7

The correlation coefficient between salinity, phosphate solubilization, and pH.
Source of variation	Df	P₂O₅ (mg ml^-1)	pH	r²
Salinity(S1)	4	9798.11912**	2.09014**	-0.6758**
Strains (S)	9	3193.41812**	1.71681**
S1xS	36	1240.80964**	0.26392**
Error	90	63.49535	0.05576

3.4. Characterization [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC.

3.4.1. Raman spectrum of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC thin films.

The other active Raman bands were attributed to P–O stretching (1016 cm^− 1 and 988 cm^− 1), P–O–P deformation (460 cm^− 1 and 377 cm^− 1), absorption bands at 959 and 974 cm^− 1 throughout the spectra. The ν₁symmetric stretching mode of PO₄ ions causes these bands, whereas antisymmetric ν₃ vibrations cause bands of intermediate strength at 1020 and 1082 cm^− 1[43]. Mono substituted benzene near appears 1003 − 999 cm^− 1 stretching band. In [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC Raman band appears at -999 cm^− 1 which is assigned to trigonal ring breathing mode [44]. weak beak appears at 339 and 397which is assigned to C-C aliphatic chain and 530 cm^− 1 which is attributed to S-S. The medium band appears between 490–660 which is attributed to C-I. Medium bands appear in [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC at 800–950 cm^− 1 which attributed to C-O-C [45]. The little deviation between experiential and stimulation is that the studied calculation was done in a vacuum, Although the calculations of experiential were done in a solid-state. Because the ligands analyzed contain complicated vibrational modes, and ring modes degrade at the same rate as imitative modes, they contribute to low symmetry, making it harder to assign to torsion and all plane modes.. [17].

3.4.2. Geometry study for [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC as an isolated molecule

The most stable highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO) in the ground gaseous state were determined using M062X/6–31 + G(d,p) calculations. and presented in Fig. 4. The difference in energy between the fragment molecular orbitals theory (FMOs) determines the equilibrium state of the molecule, which is important for determining the electrical conductivity and grasping electricity transit. Isolated compounds are stable if their entropy values are completely negative [46]. An aromatic compound electrophilic sites may be deduced using the observed FMOs. When dimer molecule bonds (DMB) grew and bond length reduced, the Gutmann at variance technique was used on the DMB sites to enhance the HOMO energy (\({E}_{H}\)) [47]. These properties were determined by looking at the optimized energy gap (\({E}_{g}^{Opt})\), as well as the reactivity and stability of the molecular system. Softness and hardness are the most critical factors in determining the stability and responsiveness [48, 49]. From the calculated electronegativity equation (χ) \(= ({E}_{H}+{E}_{L})/2\), and the energy bandgap which explains the link between charge transport in the molecule were demonstrated in Table 8.

The HOMO level is frequently found on Ca₃(PO₄)₂ which are prime targets for nucleophilic attacks. From Fig. 4, we observed that the HOMO energy of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂] ^BHC in gaseous state is-5.738 (which is an extremely large value), respectively. This value indicated high excitation energies and, high stability for [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC. On the other hand, the lower value energy LUMO= -2.796 eV can be due to the softer and more polarizable which concentrated on πfor [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC in a gaseous state, respectively. Because soft molecules can supply electrons to an acceptor, they are referred to as reactive molecules rather than hard molecules. The most intriguing descriptor is the quantified compounds index electrophilicity (ω). As the device absorbs external electronic charges, it predicts energy stability [50, 51].

There were numerous conformers studied for the ground state geometry in quantum-chemical calculations and the conformer with the lowest energy was selected, which was validated by the harmonic vibrational frequency. The dimer's binding energies were adjusted for the basis set superposition error using the counterpoise correction technique BSSE. The binding energies of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC dimers and single molecules are − 5,023.472 and − 2494.236 kcal/mol [52]. The binding energies (\({\Delta }{\text{E}}_{\text{b}}\)) of dimers were calculated using the following formula at the same level of theory: \({\Delta }{\text{E}}_{\text{b}}={E}_{dimer}-2{E}_{monomer}\). To gain a better understanding of the nature of intermolecular interactions, the TDDFT/DMOl³ approach was used on the examined compounds and their dimers. [53]. Figure 5 illustrates the examined molecule's intermolecular interactions, which include ionic and coordinated bonds, Ca----P, Ca—O, and O—P. The lengths of the lowest different type bonds are 3.645 Å, 4.50 Å, and 4.418 Å for O—P, Ca----P, and Ca—O for [P. oxalicum/P₂O₅+Ca₃(PO₄)₂]^BHC dimers, respectively. On the other hand, the centroid lengths of the dimer are 1.493 Å, 1.704 Å, and 2.088 Å for biohybrid nanocomposite dimers [54, 55]. Because the intermolecular distance between the two dimers is greater than 3.50 Ả, the rings of both molecules are unable to rotate around the single bonds. Whereas the centroid length of the dimer is less than 3.50 Å, the molecule rings rotate around the centroid point [56]. The dihedral angles (\(\varTheta\))of (O-P-Ca), (O-P-O), and (O-Ca-O) for [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC dimers isolated molecule is \(67.246^\circ \le \varTheta \le 112.955^\circ\). It has been concluded that when two isolated molecules are attached (as polymerization case) by π bonding in phenylene diamine moiety, the lengths bond, centroid lengths, centroid angle, and dihedral angle [57].

Table 8

Calculated \({E}_{H}\), \({E}_{L}\), global hardness (η), chemical potential (µ), electronegativity (χ), global softness (S), and global electrophilicity index (ω), \({N}_{max}\) and σ for [*P. oxalicum*/P₂O₅ + Ca₃(PO₄)₂]^BHC as an isolated molecule.
Compound	\({E}_{H}\)	\({E}_{L}\)	\(\varDelta {E}_{g}^{Opt.}\)	χ (eV)	µ (eV)	η (eV)	S (eV)	ω (eV)	\({\varDelta N}_{max}\)	σ(eV)⁻¹	\({\left({\Delta }{\text{E}}_{\text{b}}\right)}^{}\)
[1]	-5.738	-2.796	2.942	4.267	-4.267	-1.471	-0.340	-6.189	-2.901	-0.680	35
Dimer	-5.251	-2.345	2.906	3.798	-3.798	-1.453	-0.344	-4.964	-2.614	-0.688

[1]= [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC

3.4.3. Optical properties of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC.

Understanding the basic absorption edge in the UV region is critical to our film's transition and band structure. Figure 6 indicates the recorded experimental absorption spectra of the thin [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC where two bands at 378 nm and 465 nm as the main peak, and 517 nm as minor peak were observed. As can be observed, absorption reduces significantly as the UV wavelength increases [58]. This drop might be due to the observed decrease in the crystallinity of the films in this condition [59]. The TD-DFT analysis was used to examine the theoretical optical response of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC as an isolated molecule at 250 > λ(nm) > 600. The TD-DFT/CASTEP findings were used to compare the absorption bands of the fabricated thin films with a thickness of 100 ± 5 nm developed at 298 K is indicated in Fig. 6. The theoretical and experimental photo-absorption spectrum showed the main absorption attributed to π–π* electronic transitions in phenylene diamine matric structure [60–62].

3.5 Pot experiment:

After 60 days of seeding, the effect of PSF inoculation on canola was assessed by evaluating different growth characteristics such as shoot and root lengths, as well as their fresh and dry weights (Tables 5 and 6). PSF inoculation in the presence of TCP and RP increased plant growth parameters more than commercial SSP alone. The canola plants, on the other hand, had a considerable increase in growth parameters when TCP was used as the phosphate source, which was comparable to the SSP treatments. When RP was used as a phosphate source, the canola plants showed an increase in all parameters, but not as much as the SSP and TCP treatments. P. oxalicum as SPF cultures has the potential in enhancing the growth parameters of canola in the presence of TCP and RP.

3.5.1. Soil sample physiochemical analyses.

Some physical and chemical parameters of soil used in the pot experiment were studied as salinity, Electric conductivity (EC), pH, Total P, and Available P as in Table 9.

Table 9

Some chemical, physical and microbiological properties of soil used with various treatments
Treatments	pH	EC (dS m^− 1)	Total P. (%)	Available P. (%)	T.C. (x10⁶)	T.F (x10³⁾)	PSF (x10⁴)
Control	7.80	2.6	0.156	0.048	1.50	3.5	1.5
Salinity	7.40	3.74	0.236	0.051	0.15	11.0	2.5
RP	7.70	3.28	0.266	0.082	3.50	12.0	2.0
TCP	7.70	3.25	0.182	0.074	3.5	19.5	1.0
SSP	7.70	4.11	0.158	0.096	3.00	8.0	1.0

3.5.2. Effect of RP and TCP on canola height.

In the presence of diverse phosphor sources as TCP, RP, and SSP, the effects of P. oxalicum inoculation with the presence of NaCl as a source of salinity on root and shoot length were found to be substantial (Table 5). Furthermore, significant differences with the root and shoot length of canola were observed among the treatments at 60 DAP due to various composite inoculation treatments. The plant height, shoot, and root length of canola plants increased significantly with dual inoculation with fungal supernatant and fungal spore suspension compared with single inoculation and control at 60 days after planting. IT was observed the highest plant height and shoot length were noted in the treatment RP inoculated with P. oxalicum (148and 120 cm, respectively), whereas slight differences were recorded between other treatments. Meanwhile, a non-significant rise was found in the treatments either in root length or canola height, but there is a significant increase in the presence of SSP. In general, the results of this study demonstrate that salinity stress affects some physiological processes in canola. The enhancement of salinity decreased shoot and root length for control plants (Table 10). It is founded that salinity appears to have a greater impact on root length than on shoot length. The extract of P. oxalicum yielded the shortest shoot and root lengths compared to P. oxalicum and P. oxalicum + its extract.

Table 10

The influence of PSF on plant height, shoot, and root length of canola plants fertilized with various sources of P after 60 days of sowing.
Treatments	Control			SSP			TCP
Treatments	(A)	(B)	(C)	(A)	(B)	(C)	(A)	(B)	(C)	(A)	(B)	(C)
Control	46^e	10^e	50^f	106^b	27^a	133^a	70^e	18^b	88^e	76^f	50^d	15^c
P. oxalicum	83^b	23^a	106^b	105^b	22^b	127^c	102^a	27^a	129^a	120^a	28^a	148^a
EX P. oxalicum	60^d	12^d	72^e	56^d	15^e	71^e	68^e	14^c	82^f	58^e	11^d	69^f
P. oxalicum + EX	87^b	20^b	107^b	110^a	22^b	132^a	89^c	25^a	114^c	103^c	21^b	124^c

(A): Shoot length (cm); (B): Root Length (cm) and (C): Plants height (cm), Mean was done and there is not significantly different at P = 0.05 (Duncan's test) the same letter within columns.

3.5.3. Effect of RP and TCP on canola dry weight.

Due to P.oxalicum incubation, significant differences in shoot and root dry weight of the Canola plant were identified among the treatments. (Table 11). The inoculation treatment with P. oxalicum + Ex in presence of TCP is (52.59 g) and gives the maximum shoot dry weight. followed by P. oxalicum + Ex (52.59 g) in presence of SSP (43.19g) followed by P. oxalicum (43.19 g) in presence of TCP and then by P. oxalicum + Ex (52.59 g) in presence of RP. The overall dry matter content of canola plants was also increased following root and shoot growth. Table 11 shows the results of the R:S ratio as a function of P. oxalicum inoculation treatments at 60 DAPs. The highest R:S ratio is noted in the control treatment in the combined inoculation treatment (P. oxalicum + Ex (0.27)). The extract of P. oxalicum recorded the lowest R:S ratio (0.08 and 0.08) with SSP and TCP, respectively. (Fig. 7).

Table 11

Effect of PSF on a shoot, roots dry weight(g), and R:S ratio of canola plants fertilizer with different sources of phosphate after 60 days of sowing
Treatments	CONTROL			SSP			TCP			RP
Treatments	A	B	C	A	B	C	A	B	C	A	B	C
Control	19.70^d	3.00^d	0.32^a	42.68	3.47	0.08	28.43	3.06	0.11	31.92	4.68	0.15
P. oxalicum	24.56^b	4.25^bc	0.17^d	38.53^b	5.47	0.14	43.19	6.41	0.15	35.76	6.72	0.19
EX P. oxalicum	14.90^f	3.20^d	0.22^c	27.54	2.78	0.10	27.11	2.20	0.08	23.58	3.13	0.13
P. oxalicum + EX	22.15^c	6.00^a	0.27^b	45.84^c	5.53	0.12	52.59	5.46	0.10	42.84	5.82	0.14

A): Shoot dry weight (g); (B): Root dry weight (g) and (C): R:S ratio dry weight (g), (A): Shoot length (cm); (B): Root Length (cm) and (C): Plants height (cm), Mean was done and there is not significantly different at P = 0.05 (Duncan's test) the same letter within columns.

3.5.4. Effect of RP and TCP on Canola P. Content and uptake.

Data of Canola P content and uptake illustrated in (Table 12) which showed that the single inoculation with P. expansum or with extract of P. expansum in presence of SSP or RP or TCP noted the highest important value of P content in the plant of canola compared to uninoculated control. Single inoculation treatment with P. oxalicum in the presence of RP or TCP fertilizer P uptake in comparison to all other treatments was substantially higher. The P-uptake, on the other hand, was lowest in the uninoculated control group.

Table 12

Effect of PSF on Canola P. content (%) and P uptake (mg plant^− 1) fertilized with different sources of phosphate
Treatments	CONTROL		SSP		TCP		RP
	P content	P uptake	P content	P uptake	P content	P uptake	P content	P uptake
Control	0.062^d	19.89^c	0.125^b	57.69^c	0.098^d	30.89	0.118^a	43.19^e
P. oxalicum	0.108^a	31.12^b	0.109^d	47.74^d	0.115^b	57.04	0.115^b	48.85^d
EX P. oxalicum	0.071^c	12.85^e	0.078^e	23.65^e	0.104^c	30.48	0.099^d	26.44^f
P. oxalicum + EX	0.109^a	30.68^b	0.113^c	58.05^c	0.114^b	66.18	0.119^a	57.91^c
Mean was done and there is no significant difference at P = 0.05 (Duncan's test) the same letter within columns.

3.5.5. Total phosphate solubilization microorganisms (PSM).

Table 13 shows that inoculation with P. oxalicum in the form of spores, supernatant, or both significantly raised total PSM counts for all mineral phosphate fertilizer sources. When comparing uninoculated control plants to inoculated control plants, the acquired data demonstrated an increase in counts in inoculated pots in the duration of planting from 28 to 45 days. These increases in the T.C. of P. oxalicum populations could be attributed to the isolated fungi's ability to survive under NaCl stress (Tables 13, 14, and 115) (the experimental soil is saline), as well as their potential effect on phosphorus solubilization (Tables 13, 14, and 15). The stimulation, on the other hand, varied depending on the source of P used. This was true both in the beginning and later stages of growth development. P. oxalicum, in particular, in the fertilization with RP or TCP compared to SSP fertilizer, enhanced the improvement in counts. After 60 days of planting time, the differences between those two treatments and the uninoculated control could be seen. The average rise was around 67.05 percent in TCP and 60.57 percent in the RP. In general, the increased population of PSF in all treatments, both inoculated and uninoculated, reached their peak almost on the 30 days then declined at 45 days (Table 14) and stability to 60 DAP (Table 15). The introduced PSF increased its population in treatments inoculated with singly or with superintend. However, the population of PSF in the inoculated with superintending of P. oxalicum treatments did not exceed that in the uninoculated treatments.

Table 13

Effect of inoculation canola plants with PSF on the total count of bacteria, fungi, and phosphate solubilizing fungi (x10⁴ g^− 1 dry soil) after 28 days from sowing
Treatments	Control			SSP			TCP			RP
	T.B	T.F	T. PSF	T.B	T.F	T.PSF	T.B	T.F	T. PSF	T.B	T.F	T. PSF
Control	130^c	43^c	28^c	118^a	15^d	44^f	90^d	20^bc	10^f	210^a	17^de	23^de
P. oxalicum	163^a	89^a	47^b	80^c	39^a	22^a	153^a	17^cd	12^d	106^e	30^b	41^b
EX P. oxalicum	100^d	25^e	21^d	63^e	32^b	90^e	87^d	32^a	22^b	120^d	14^f	26^d
P. oxalicum + EX	150^b	82^ab	157^a	76^d	9^e	107^c	127^b	30^a	25^a	153^b	20c^d	52^a
Mean was done and there is not significantly different at P = 0.05 (Duncan's test) the same letter within columns

T. B = total bacteria, T.F.= total fungi, PSF = Phosphorous solubilizing fungi

Table 14

Effect of inoculation canola plants with PSF on the total count of bacteria, fungi, and phosphate solubilizing fungi (x10⁴ g^− 1 dry soil) after 45 days from sowing
Treatments	Control			SSP			TCP			RP
	T.B	T. F	T. PSF	T. B	T. F	T.PSF	T.B	T. F	T. PSF	T.B	T. F	T.PSF
Control	3	20	1	8	15	2	15	10	2	3	10	1
P. oxalicum	1l	26	2	10	70	13	52	29	5	23	4	4
EX P.oxalicum	2	11	3	12	16	2	53	5	1	15	3	2
P. oxalicum + EX	4	16	4	19	57	16	12	6	4	74	7	4
T. B = total bacteria, T.F.= total fungi, PSF = Phosphorous solubilizing fungi

Table 15

Effect of inoculation canola plants with PSF on the total count of bacteria, fungi, and phosphate solubilizing fungi (x10⁴ g^− 1 dry soil) after 60 days from sowing
Treatments	Control			SSP			TCP			RP
	T.B	T.B	T.PSF	T.B	T. F	T. PSF	T.B	T.F	T.PSF	T.B	T.F	T. PSF
Control	3^c	23^c	18^c	118^a	15^d	4^f	9^d	4b^c	3^f	2^a	4^de	2^de
P. oxalicum	10^a	89^a	14^b	8^c	39^a	12^a	33^a	7^cd	5^d	16^e	3^b	4^b
EX P. oxalicum	2^d	25^e	16^d	13^e	32^b	4^e	27^d	5^a	2^b	20^d	4^f	2^d
P. oxalicum + EX	5^b	82^ab	17^a	16^d	9^e	17^c	12^b	7^a	5^a	53^b	6c^d	5^a
Mean was done and there is not significantly different at P = 0.05 (Duncan's test) the same letter within columns

T. B = total bacteria, T.F.= total fungi, PSF = Phosphorous solubilizing fungi

3.5.6. Acidic phosphatase activity, available P, and pH in the rhizosphere of canola plants.

Data listed in Tables 16, 17, and 18 illustrated that a slight reduction in pH was noticed after 60 days of the final harvest. Furthermore, in all P sources, the final pH did not drop to extremely acidic values. In contrast, accessible P. rose in soils with fungal inoculums as compared to soils that were not inoculated. (Tables 16, 17, and 18). Plants inoculated with PSF gave a significant amount of available P in soil rhizosphere fertilizer with TCP or RP treatments compared to control. Higher values of soil available P were obtained at 45 days after planting (Table 17) and decreased to lower values at 60 days after planting (Table 18). Acidic phosphatase activity was detected in all PSF, but activities varied among the isolates (Tables16, 17, and 18). The activity of acid phosphatase in all inoculated treatment soils was higher than the uninoculated soil.

Table 16

Effect of different treatments of fungi on phosphate activity, available P, and pH value of soil after 28 days from sowing fertilizer with different sources of phosphate (28 days from sowing).
Treatments	Control			SSP			TCP			RP
	P. ase	AP	pH	P. ase	Av P	pH	P. ase	AP	pH	P. ase	AP	pH
Control	6.14^e	2.53^d	7.81^a	9.49^c	4.70^c	7.46^a	11.96^e	4.00^f	7.20^c	17.16^c	4.18^e	7.22^f
P. oxalicum	14.27^c	5.88^b	7.55^bc	24.53^f	3.40^cd	7.14^ab	30.62^a	10.57^c	7.20^a	33.83^d	11.40^c	7.41^f
EX P. oxalicum	6.79^d	2.00^a	7.59^cd	10.13^f	7.30^ab	7.21^c	16.31^a	5.67^b	7.20^a	17.00^f	5.95^cd	7.30^ab
P.oxalicum + EX	15.45^b	6.00^a	7.44^d	25.66^c	7.90^d	7.0^ab	33.20^f	11.44^c	7.30^a	36.45^cd	11.66^c	7.30^cd

Table 17

Effect of inoculation with PSF on rhizosphere soil phosphatases activity, available P. and pH of canola(45 days from sowing).
Treatments	CONTROL			SSP			TCP			RP
	P. ase	AP	pH	P. ase	Av P	pH	P. ase	AP	pH	P. ase	AP	pH
Control	3.53^e	1.43^d	7.61^a	7.49^c	4.70^c	7.30^a	8.11^e	2.00^f	7.10^c	11.15^c	2.18^e	7.12^c
P. oxalicum	10.47^c	3.35^c	7.39^c	20.70^a	3.40^d	7.12^c	20.74^d	7.70^ab	7.15^b	29.90^ab	7.90^c	7.11^c
EX P. oxalicum	4.62^d	1.85^d	7.41^bc	6.05^cd	7.30^ab	7.11^c	8.61^e	3.27^d	7.20^a	7.80^e	4.00^d	7.30^a
P. oxalicum + EX	12.75^b	4.50^a	7.35^d	21.69^a	7.90^a	7.00^d	27.60^a	8.00^a	7.10^c	30.15^a	9.00^b	7.20^b

* Mean was done and there is no significantly different at P = 0.05 (Duncan's test) the same letter within columns

SSP = superphosphate, TCP = tricalcium phosphate, RP = Rock phosphate, AV = available phosphate, P.ase = phosphatase

Table 17

Effect of inoculation with PSF on rhizosphere soil phosphatases activity, available P. and pH of canola
Treatments	CONTROL			SSP			TCP			RP
	P. ase	AP	pH	P. ase	Av P	pH	P. ase	AP	pH	P. ase	AP	pH
Control	3.53^e	1.43^d	7.61^a	7.49^c	4.70^c	7.30^a	8.11^e	2.00^f	7.10^c	11.15^c	2.18^e	7.12^c
P. oxalicum	10.47^c	3.35^c	7.39^c	20.70^a	3.40^d	7.12^c	20.74^d	7.70^ab	7.15^b	29.90^ab	7.90^c	7.11^c
EX P. oxalicum	4.62^d	1.85^d	7.41^bc	6.05^cd	7.30^ab	7.11^c	8.61^e	3.27^d	7.20^a	7.80^e	4.00^d	7.30^a
P. oxalicum + EX	12.75^b	4.50^a	7.35^d	21.69^a	7.90^a	7.00^d	27.60^a	8.00^a	7.10^c	30.15^a	9.00^b	7.20^b

(45 days from sowing).

SSP = superphosphate, TCP = tricalcium phosphate, RP = Rock phosphate, AV = available phosphate, P.ase = = Acidic phosphatase. Mean was done and there is not significantly different at P = 0.05 (Duncan's test) the same letter within columns

Table 18

Effect of inoculation with PSF on rhizosphere soil phosphatases activity, available P (mg g-1 soil), and pH of the rhizosphere of canola plants fertilizer with different sources of phosphate (60 DAP)
Treatments	CONTROL			SSP			TCP			RP
	P. ase	AP	pH	P. ase	Av P	pH	P. ase	AP	pH	P. ase	AP	pH
Control	2.75^e	1.01^f	7.60^a	5.31^c	3.55^c	7.30^a	5.11^f	2.00^e	7.10^c	9.65^c	2.18^e	7.12^c
P. oxalicum	8.52^c	2.79^c	7.38^c	18.35^a	2.89^d	7.11^c	18.88^d	5.79^b	7.15^b	27.51^a	8.00^b	7.11^c
EX P. oxalicum	3.66^d	1.21^d	7.40^bc	4.51^cd	6.45^ab	7.11^c	6.96^e	3.01^d	7.20^a	5.99^d	3.51^c	7.30^a
P. oxalicum + EX	10.95^b	3.89^a	7.33^d	18.90^a	6.10^a	7.00^d	26.11^a	7.11^a	7.11^c	27.19^a	9.30^a	7.19^b
SSP = superphosphate, TCP = tricalcium phosphate, RP = Rock phosphate, AV = available phosphate, P.ase = = Acidic phosphatase * Mean was done and there is no significantly different at P = 0.05 (Duncan's test) the same letter within columns

P. oxalicum is the best possible inoculation tool for phosphate solubilization and is recommended for deficient P soils, greenhouse experiments as well as field trials in the presence of RP, TCP more than commercial SSP. From insoluble rock phosphate and tricalcium phosphate sources, P. oxalicum was able to solubilize and fix a significant amount of phosphorus into the agricultural soil systems and there is a strong relation between pH and the amount of the solubilized P. The experimental treatments tested in the current study significantly enhanced canola growth, indicating that the right combinations of treatments have been suggested. Data of the P content and P uptake of canola plants show that the single inoculation with P. oxalicum in fertilizers with SSP or RP or TCP recorded the highest significant increment of P content in the plant of canola, compared to uninoculated control. In case of P uptake, single inoculation treatment P. oxalicum) compared to all other treatments. However, the P uptake was lowest in uninoculated control. The plant height, shoot, and root length of canola plants increased significantly with dual inoculation with superintending and fungal spores compared with single inoculation and control at 60 days after planting. The biohybrid nanocomposite formed by mixing the composite from RP and TCP showed an absorbance of 0.26 and 2.23 at the wavelength (l) 378 nm and 465 nm. Also, significant changes in the absorption index and indirect/direct bandgap of the biohybrid nanocomposite have been detected.

DFT	Density Functional Theory	BHC	Biological hybrid composite
		EU	Enzyme units
TDDFT	Time-Dependent Density Functional Theory	rpm	Rotation per minute
UV-Vis	Ultraviolet–Visible Spectroscopy	HOMO	Highest occupied Molecular Orbital
FMOs	fragment molecular orbitals theory	LUMO	Lowest Unoccupied Molecular Orbital
CASTEP	C Ambridge serial total energy package	PKV	Pikovskaya’s agar
OD	Optical Density	PDA	Potato Dextrose agar
BNC	Biosynthesized nanocomposite	PDB	Potato Dextrose broth
DNP	Dynamic nuclear polarization	DMB	Dimer Molecule Bonds
Df	Degrees of freedom	PNP	p-nitrophenol
r²	R-squared	RNA	Ribonucleic acid
DNP	Double Numerical plus polarization	EC	Electronic conductivity
PS	Phosphate Solubilization	MEP	Molecular Electrostatic Potential
PBE	Perdew–Burke–Ernzerh	PSM	phosphate solubilization microorganisms
SSP	Single Superphosphate	T.C.	Total count
DAP	Days after planting	PSF	Phosphate solubilization Fungi
PSM	phosphate solubilization microorganisms	T.F.	Total Fungi
P.ase	Acidic phosphatase activity	EX	Extract of P. oxalicum
NRC	National research center	P	phosphrous

Conflict of Interest

There are no conflicts of interest as declared by the authors.

Authors’ Contributions

Maysa G. Shalaby and M. Attia, original draft, Methodology, Data preparation and curation, deep analysis, and Writing Ahmed F. Al-Hossainy and Mai G.Shalaby original draft, Methodology, Data preparation and curation, deep analysis, and Writing, Ahmed F. Al-Hossainy and Maysa G. Shalaby, Data preparation Magdy Attia Methodology - original draft.

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A.A.J.J.o.E.M. Al-Hossainy, Combined Experimental and TDDFT-DFT Computation, Characterization, and Optical Properties for Synthesis of Keto-Bromothymol Blue Dye Thin Film as Optoelectronic Devices, 50 (2021) 3800-3813.
S.A. Mahmoud, A.A. Al-Dumiri, A.F. Al-Hossainy, Combined experimental and DFT-TDDFT computational studies of doped [PoDA+ PpT/ZrO2] C nanofiber composites and its applications, Vacuum, 182 (2020) 109777.
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No competing interests reported.

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Formation of bio-hybrid nanocomposite [P. oxalicum/P 2 O 5 +Ca 3 (PO 4 ) 2 ] BHC by Penicillium oxalicum and its application on the growth of canola

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental Setup

2.1. Materials and reagents

2.2. Synthesis of biohybrid nanocomposite of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC.

2.2.1. Fungal isolate

2.2.2. Testing the ability of P. oxalicum for phosphate solubilization.

2.2.3. The solubilization of tricalcium and rock phosphates (TCP and RP) in liquid medium

2.2.4. Phosphate solubilization in liquid medium under salt stress.

2.3. Computational study of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC as an isolated molecule in the cell unit

2.4. Evaluation of efficient strains under Pot Experiment conditions

2.5. Tricalcium phosphate (TCP) and Rock Phosphate (RP)

2.6. Data analysis

3. Results And Discussion

3.1. The ability of P. oxalicum for phosphate solubilization.

3.2. The solubilization of tricalcium and rock phosphates (TCP and RP) in liquid medium

3.3. Phosphate solubilization in liquid medium under salt stress

3.4. Characterization [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC.

3.4.1. Raman spectrum of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC thin films.

3.4.2. Geometry study for [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC as an isolated molecule

3.4.3. Optical properties of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC.

3.5 Pot experiment:

3.5.1. Soil sample physiochemical analyses.

3.5.2. Effect of RP and TCP on canola height.

3.5.3. Effect of RP and TCP on canola dry weight.

3.5.4. Effect of RP and TCP on Canola P. Content and uptake.

3.5.5. Total phosphate solubilization microorganisms (PSM).

3.5.6. Acidic phosphatase activity, available P, and pH in the rhizosphere of canola plants.

Conclusion

Abbreviations

Declarations

References

Competing Interests

Supplementary Files

Status:

Version 1

Formation of bio-hybrid nanocomposite [P. oxalicum/P 2 O 5 +Ca 3 (PO 4 ) 2 ] BHC by Penicillium oxalicum and its application on the growth of canola

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental Setup

2.1. Materials and reagents

2.2. Synthesis of biohybrid nanocomposite of [P. oxalicum/P2O5 + Ca3(PO4)2]BHC.

2.2.1. Fungal isolate

2.2.2. Testing the ability of P. oxalicum for phosphate solubilization.

2.2.3. The solubilization of tricalcium and rock phosphates (TCP and RP) in liquid medium

2.2.4. Phosphate solubilization in liquid medium under salt stress.

2.3. Computational study of [P. oxalicum/P2O5 + Ca3(PO4)2]BHC as an isolated molecule in the cell unit

2.4. Evaluation of efficient strains under Pot Experiment conditions

2.5. Tricalcium phosphate (TCP) and Rock Phosphate (RP)

2.6. Data analysis

3. Results And Discussion

3.1. The ability of P. oxalicum for phosphate solubilization.

3.2. The solubilization of tricalcium and rock phosphates (TCP and RP) in liquid medium

3.3. Phosphate solubilization in liquid medium under salt stress

3.4. Characterization [P. oxalicum/P2O5 + Ca3(PO4)2]BHC.

3.4.1. Raman spectrum of [P. oxalicum/P2O5 + Ca3(PO4)2]BHC thin films.

3.4.2. Geometry study for [P. oxalicum/P2O5 + Ca3(PO4)2]BHC as an isolated molecule

3.4.3. Optical properties of [P. oxalicum/P2O5 + Ca3(PO4)2]BHC.

3.5 Pot experiment:

3.5.1. Soil sample physiochemical analyses.

3.5.2. Effect of RP and TCP on canola height.

3.5.3. Effect of RP and TCP on canola dry weight.

3.5.4. Effect of RP and TCP on Canola P. Content and uptake.

3.5.5. Total phosphate solubilization microorganisms (PSM).

3.5.6. Acidic phosphatase activity, available P, and pH in the rhizosphere of canola plants.

Conclusion

Abbreviations

Declarations

References

Competing Interests

Supplementary Files

Status:

Version 1

2.2. Synthesis of biohybrid nanocomposite of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC.

2.3. Computational study of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC as an isolated molecule in the cell unit

3.4. Characterization [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC.

3.4.1. Raman spectrum of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC thin films.

3.4.2. Geometry study for [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC as an isolated molecule

3.4.3. Optical properties of [P. oxalicum/P₂O₅ + Ca₃(PO₄)₂]^BHC.