Isolation of phosphate solubilizing fungi from phosphate mine and its molecular identification based on the rule of Calmodulin gene

Phosphate is one of the macronutrients and involved in many essential plant processes such as respiration, cell division, root development, photosynthesis, sugar decomposition, nutrient transfer inside the plant, the transfer of genetic characteristics from one generation to the next, and the regulation of metabolic pathways. Deficiency occurs when phosphorus content in the soil reaches less than 0.1%. Today, in order to achieve sustainable development, the use of low-cost natural nutrients such as rock phosphate is necessary. In recent years, the use of phosphate solubilizing microorganisms has been studied for dissolution of rock phosphate. The aim of this study was to isolate indigenous phosphate solubilizing fungi and identify the most effective isolate at the species level.
Samples were taken from the soil around the Dalir phosphate mine located in Chalous, (Mazandaran). After preparation of the dilution series from soil suspension and culture in NBRIP-BPB and PVK, phosphate-solublilizing fungal colonies were isolated. The ability of phosphate solubilization of fungal isolates was also studied in NBRIP liquid medium. The phosphate solubilized was measured in the liquid medium and the pH of the samples was measured using pH meter. An isolate that significantly reduced the pH of the environment, released more phosphorus and had a higher solubility index, was selected. The selected isolate was identified by ITS polymerase chain reaction and then calmodulin-based PCR methods.
The results indicate that the use of the NBRIP-BPB medium is more efficient than PVK medium for P solubilizing fungi isolation. Screening was carried out in solid and liquid media for each isolate, and SANRU isolate was more prominent phosphate solubilizers compare to the other isolates. The results of comparing the sequence of the ITS gene with the sequences in the gene bank showed that SANRU strain had a similarity of 100% with A. niger and A.tubingensis species. In order to overcome this uncertainty, a calmodulin-based PCR method was carried out. The replication of a part of the Calmodulin gene sequence revealed that the SANRU strain belongs to the tubingensis section, and the sequencing sample was registered with the access number KT222864 at the World Gene Bank.
According to the results of this study, simultaneous screening in a solid and liquid medium is recommended to determine phosphate-solubilizing ability. According to the results of this study, the method of sequencing of calmodulin in comparison with ITS has a much higher accuracy for differentiation of Aspergillus species, therefore, it is recommended for accurate identification of Aspergillus species.