Isolation and functional evaluation of phosphate-solubilizing fungi with salt-tolerant characteristics

Objectives To supply effective microbial strains for biofertilizer production, we isolated phosphate-solubilizing fungi from the saline-alkaline soils planting sunflower in Inner Mongolia.

Methods Petri dishes method was used to isolate phosphate-solubilizing fungus and the isolated fungus was identified by ITS rDNA sequence homology analysis. Broth medium culture was used to measure the dissolving capacity of insoluble phosphate of the isolated M2. Insoluble phosphates: Ca₃(PO₄)₂, AlPO₄, and five phosphorous rock powders as sole P source, respectively, were used to make PVK broth medium containing 5 g/L of phosphate separately, and 1 mL of sterilized cultural media, strain P83 and strain M2 were added into 100 mL of the broth medium and incubated at 28℃ for 3, 6 and 9 days at 160 r/min. Cultures were centrifuged at 12000 r/min for 5 min at 4℃ and the supernatant immediately analyzed for available phosphorus. To determinate the salt tolerance of isolate M2 and strain P83 in petridishes, the PDA solid medium containing 0%, 5%, 7.5%, 10% and 12.5% of NaCl were employed and the growth diameters of P-solubilizing isolates P83 and M2 were recorded, respectively, after 5 days of incubation. A pot experiment was carried out to investigate solubilizing phosphate effect of strain M2. Maize seeds (Zea mays L. ‘Zhendan 958’) were planted in the four types of soils (paddy soil, viscous fluvo-aquic soil, salinized fluvo-aquic soil and calcareous fluvo-aquic soil) in the presence of Ca₃(PO₄)₂, AlPO₄ and Kunyang RP. The fresh and dry biomass and soil available phosphorus in treatments of P83 and M2 inoculations were measured at 40 days after sowing. A field trial was conducted to investigate the plant growth promotion effect of the produced bio-fertilizer with M2 strain. Peanut seeds were coated onto the mixture of fungal spores suspension (strain ATCC20851, P83 and M2) with sterilized peat. The yields of peanut straw and seeds were recorded and the soil available phosphorus was analyzed after harvest. Accumulation profiles of organic acid and phytohormone produced by the isolated M2 were analyzed using LC-MS method.

Results The isolated M2 strain was identified as Penicillium oxalicum and had a strong ability to solubilize insoluble phosphates. The phosphate release rate from the solubilized Ca₃(PO₄)₂ by M2 strain was 59.2% and the concentration of available P was 972 mg/L under shaking incubation after 6 days’ culture. The soluble P in AlPO₄ liquid culture was lower than that in the medium with Ca₃(PO₄)₂, the available P content was 988 mg/L and the solubilized rate of AlPO₄ reached up to 48.2%. The contents of solubilized P by M2 from Jinping RP, Kaiyang RP, Jinning RP, Fanshan RP, Kunyang RP were ranged from 21.0 to 556 mg/L after 6 days’ shaking incubation. The isolated M2 strain grew well in the presence of 7.5% NaCl, exhibiting satisfactory salt resistance property. The pot experiment showed that the M2 strain had significant growth promoting effects in all the four types of soils. Compared with the no-inoculation control, the fresh and dry biomass of corn in the M2 inoculated treatments were increased by 26.36%–99.18% and 20.05%–262.94%, respectively, and the soil available P contents were enhanced by 19.24–25.28 mg/kg. The adaptability of isolated M2 to four types of soils was better than that of strain P83. Field experiment showed that the M2 inoculation exhibited a greater enhancement in peanut yield, and the yield was averaged at 4.50 t/hm² with an increase of 23.29% over the control. Seven organic acids and two phytohormones were all detectable in the liquid cultures of Ca₃(PO₄)₂, AlPO₄ and Kunyang RP, the concentrations of oxalic acid and citric acid in the three liquid cultures were as high as 653.46 mg/L and 269.61 mg/L in the presence of M2 stain, respectively. The concentrations of indole acetic acid (IAA) were ranged from 32.38 to 66.17 mg/L, and zeatin concentrations were ranged from 0.05 to 0.07 mg/L.

Conclusions A novel phosphate-solubilizing M2 strain was isolated and identified to be Penicillium oxalicum. The isolated M2 strain behaves well in solubilizing insoluble phosphates under culture with petri dishes, broth medium and pot experiments, and could significantly increase soil available phosphate and corn biomass, suggesting that Penicillium oxalicum M2 strain is prospective for biofertilizer production in the future.