The lab incubation study was conducted to measure the P mobilization capacity of the P. bilaiae from MBM as per the approach reported by Wakelin et al. (32) for tri-calcium phosphate. Finely ground (50–250 μm size) MBM (0.1 g) and Idaho PR (equivalent amount P) were mixed with 100 ml of sterilized culture medium (0.1 g L⁻¹ NaCl, 0.4 g L⁻¹ NH₄Cl, 0.78 g L⁻¹, KNO₃, 0.5 g L⁻¹ MgSO₄, 0.1 g L⁻¹ CaCl₂, and 10 g L⁻¹ sucrose) in 250 ml Erlenmeyer flasks. The P. bilaiae culture (200 μg) was inoculated into five replicate flasks and incubated at 25°C in dark with continuously gentle shaking. Six treatments were included in this experiment: Control, Control + P. bilaiae, MBM, MBM + P. bilaiae, Idaho PR, and Idaho PR + P. bilaiae in a completely randomized design. After 7 days of incubation, solution pH, soluble PO4- and ergosterol content were measured.

A greenhouse experiment was conducted to measure the effect of AMF, P. bilaiae, and AMF + P. bilaiae on the P mobilization and plant P uptake from MBM. The pots (12 L) were filled with a 12 kg sand-soil (9:1) mixture with a bulk density of 1.543 g cm⁻³. The sand and soil were steam autoclaved at 121°C for 1 h. The low proportion of soil in the substrate was to minimize the background P content and was based on preliminary greenhouse experiments. The nutrient free white sand with a bulk density of 1.72 g cm⁻³ and pH of 7.5 was used in the study. A total of 6 treatments (control, MBM, MBM + AMF, MBM + P. bilaiae, MBM + AMF + P. bilaiae, and KH₂PO₄) were tested in a greenhouse experiment and replicated five times in a completely randomized design. The MBM was applied at a rate of 800 μg P pot⁻¹. The AMF and P. bilaiae culture were applied at a rate of 25 g pot⁻¹ (132 spores g⁻¹) and 0.5 g pot⁻¹ (8.0 ^* 10⁸ spores g⁻¹), respectively. The pots were filled in three layers with the first 3 cm layer without any P fertilizers and P mobilizer inoculum, middle 15 cm layer mixed with P fertilizers and AMF and P. bilaiae inoculum, and top 3 cm layer again without any P fertilizers and P mobilizer inoculum. All the other essential macro and micro-nutrients were applied using the modified 1x Hoagland solution (P free; 300 ml after every 4 days). The maize (Zea mays) was used as a test plant species for this experiment. Three maize seeds were sown in each pot and after germination thinned to one plant per pot. Plants were harvested 40 days after planting. Plant shoot, root biomass, shoot, root P uptake, percent root mycorrhizae colonization, root morphology, plant photosynthesis rate, stomatal conductance, root acid phosphomonoesterase activity, soil P fractionation, and soil acid, alkaline phosphomonoesterase activity, were measured.

Ergosterol (lab incubation) was extracted as reported by (33). Thirty milliliters of the incubation solution was filtered through a 0.45 μm nitrocellulose filter paper. The filter paper along with filtrate was dissolved into 0.14 M methanolic KOH and incubated at 82°C for 50 min in dark. After the incubation, samples were placed on the ice for 5 min and acidified to a pH < 2 with 0.5 ml of 50% HCl. To the acidified samples, 2 ml of hexane was added and vortexed for 5 min under dark conditions. The top hexane phase (1.5 ml) was collected into the glass vials and dried under a vacuum. The dried contents were reconstituted in 20 μl of chloroform + 80 μl of methanol and transferred into glass vials with 200 μl inserts. The control samples were spiked with 0.5 μg/ml of ergosterol standard. Ergosterol was quantified using high-pressure liquid chromatography (HPLC). Separation of ergosterol compounds was performed on an Onyx C18 column (monolithic silica, 130 Å; 100 × 4.6 mm I.D.; Phenomenex, Torrance, CA) using a constant flow of methanol-water (99:1, v/v) solvent at a rate of 1 ml min⁻¹. Ergosterol was detected at 282 nm absorbance using LC-UV and quantified using ergosterol standards.

Soil P fractionation was carried out according to the method reported by Hedley et al. (34). The soil at the time of harvest was subjected to sequential chemical extraction with 1 M NH₄Cl, 0.5 M NaHCO₃, chloroform fumigation + 0.5 M NaHCO₃, 0.1 M NaOH, and 1 M HCl, in the specified order. Soil (4 g on a dry weight basis) was extracted with 40 ml of different extractants for 16 h. After extraction, the soil slurry was centrifuged at 1,509 g for 10 min and the supernatant was filtered through a Whatman No. 1 filter paper. The supernatant for 0.5 M NaHCO₃ and 0.1 M NaOH extractions was further filtered through 0.45 μm syringe filters to remove humic acid. The clear filtrate was stored at −20°C until further use. The soluble P from each fraction was determined by the molybdenum blue/ ascorbic acid method (35). The extraction procedure fractionates the P into loosely sorbed-P (NH₄Cl-P), olsen-P (NaHCO₃-P), microbial bound P (chloroform fumigation + NaHCO₃-P), Fe/Al-P (NaOH-P), and calcium bound-P [HCl-P (34)]. Soil pH was measured in water: soil solution with a 2:1 (v:w) ratio on the pH meter (Mettler Toledo, USA).

Soil acid and alkaline phosphomonoesterase activity were measured according to the method reported by Jatana et al. (7). Soil (1 g on a dry weight basis) immediately after harvest was mixed with 4 ml of modified universal buffer [MUB; 2.42 g tris(hydroxymethyl)aminomethane, 2.32 g maleic acid, 2.8 g citric acid, 1.26 g boric acid in 96.7 ml 1 N NaOH in total 1 L] of pH 6.5 (with 0.1 M HCl; acid phosphomonoesterase) or pH 11.0 (with 0.1 M NaOH; alkaline phosphomonoesterase). The soil buffer solution was incubated in the water bath for 1 h at 37°C after mixing with 1 ml of 0.05 M disodium p-nitrophenyl phosphate hexahydrate (prepared in the same MUB for acid and alkaline phosphomonoesterase). After the incubation, the reaction was immediately terminated by adding 1 ml of 0.5 M CaCl₂ and 4 ml of 0.5 M NaOH. The soil suspension was then centrifuged at 1509 g for 10 min. The clear solution was filtered through a Whatman No. 1 filter paper. The absorbance of the clear supernatant was measured at 410 nm on a spectrophotometer (DU® 640 spectrophotometer, Beckman Coulter, Fullerton, CA, USA). The substrate solution (disodium p-nitrophenyl phosphate hexahydrate) was added to the control after the addition of 1 ml 0.5 M CaCl₂ and 4 ml 0.5 M NaOH. Both enzyme activities were expressed as μg p-nitrophenol (PNP) g⁻¹ dry soil h⁻¹.

Root morphology of plants at harvest was evaluated by imaging the roots with Win-RHIZO version 3.9 (Regent Instruments Inc.). Specifically, roots were physically separated into different root types (primary, crown, and lateral roots) and the root images were taken in a plexiglass tray (0.2 × 0.3 m). The images were scanned through the Win-RHIZO system. After the root image analysis, the root samples were dried to calculate the specific root length (SRL; root length/dry weight of the roots).

Percent root mycorrhizal colonization was observed using the trypan blue staining procedure reported by Frater et al. (36). Roots were cleaned in a 10% KOH solution at 60°C for 24 h. After cleaning, the roots were washed with deionized water for 10 min followed by acidification in 1% HCl for 20 min. The acidified roots were soaked in trypan blue (0.05%, trypan blue powder) solution made in 1:1:1 (v:v:v) glycerol, lactic acid, water for 5 min on a hot plate set at 90°C. The roots were taken out of the trypan blue solution and washed gently under deionized water for 10 min. The washed roots were kept in deionized water at 4°C for a couple of days to remove the excess trypan dye present in the roots. We then assessed the percentage of root colonized by AMF using the gridline intercept technique (37). This technique determines the presence/absence of fungal organs (i.e., arbuscules/hyphae/vesicles) for each instance that a root intersected with a gridline.

Root phosphomonoesterase activity was measured as reported by Venterink (38). Roots tips were collected after thorough washing with deionized water. The washed root tips were immediately frozen in dry ice and stored at −80°C for later analysis. Frozen root tips were ground into a fine powder, weighed (250 mg), and homogenized at 6,000 rpm for 2 min in 500 μl (1:2, m/v) of MUB [100 mM tris(hydroxymethyl)aminomethane, 100 mM maleic acid, 5 mM citric acid, and 100 mM boric acid; pH 5.5 for acid phosphomonoesterase]. Homogenized root tissues were centrifuged at 14,000 g for 15 min at 4°C and the supernatant was collected. To the 300 μl of supernatant, 4 ml of MUB and 1 ml of 0.003 M p-nitrophenyl phosphate hexahydrate were added and vortexed for 30 s. The mixture was then incubated at 37°C for 10 min in a water bath. After the incubation, the reaction was terminated by adding 1 ml 0.5 M CaCl₂ and 4 ml 0.5 M NaOH. The precipitates were recovered by centrifugation at 12,000 g for 15 min. The PNP content of the supernatant was quantified spectrophotometrically (DU® 640 spectrophotometer, Beckman Coulter, Fullerton, CA, USA) at 410 nm by using an authentic standard (PNP). Enzyme activity was expressed as μg pPNP g⁻¹ root h⁻¹.

Net photosynthetic rate and stomatal conductance of individual leaves were measured between 09:00 a.m. and 12:00 p.m. at 18 and 36 days after planting. Two recently fully opened leaves were selected from each plant and analyzed using a portable photosynthesis system (model LI-6400, LI-COR, Lincoln, NE, USA). The leaf blade width was measured before inserting the maize leaf into the chamber to account for the leaf area. The LI-6400 was programmed with a constant leaf chamber block temperature at 25°C. A fixed substrate level of 400 mm³ m⁻¹ CO₂ (12-g cartridge) and an LED 6,400 R/B light at 1,500 μmol m⁻² s ⁻¹ were provided.

The laboratory and greenhouse experiments were conducted in a completely randomized design with five replicates. The one-way analysis of variance was performed on the data using JMP Pro 13.1.0 (SAS Institute Inc., Cary NC). Different treatment pairs were compared using the Tukey HSD test. The treatments were considered significantly different at the P < 0.05 level. The data for the percent mycorrhizae colonization were log-transformed to meet the assumption of normality before the analysis of variance.