| In China, over 90% of arable land is under phosphate (P) deficiency. The use of phosphate fertilizer is an important measure to increase crop yield. However, the excessive use of P fertilizer in the long term and the immobilization of P in soil have caused serious waste of resources and environmental pollution. Therefore, to develop and utilize the P-solubilizing bio fertilizer and to study the mechanism of the P-solubilization and related gene has important practical value and theoretical significanceIn this study,7 fungi with high efficient P-solubilization were isolated from north China. Of the 7 strains,3 strains, P4 (Penicillium oxalicum), P93 (Aspergillus aculeatus) and P85 (Aspergillus niger), were further studied on their P-solubilizing ability, optimum conditions, the secretion of organic acids, soil colonization ability, the plant growth-promoting ability. The results showed that P-solubilizing fungi inoculation significantly increased available phosphorus content and reduced the pH value of the solution. Strain P93, P4 and P85 showed the most significant efficiency in P-solubilizing when calcium phosphate was used as P resources. The optimum carbon types for them to dissolve rock phosphate were fructose, glucose and maltose, respectively, and the optimum nitrogen source types were urea, ammonium and nitrate, respectively. The optimum temperature was 25-35℃ and the optimum concentration of rock phosphate was 2 mg/mL. Malic and acetic acid was mainly produced by the strain P93; tartaric and citric acid was mainly produced by P4 and P85 mainly produced malic and citric acid. Under pot experiments, P-solubilizing strain P93, P4 and P85 inoculations significantly affected corn biomass and showed the best growth-promoting effects when soil was treated with calcium phosphate, calcium phosphate and Kunyang rock phosphate, respectively. The colonization ability of these three fungi showed obviously difference and the decreasing order is P93, P85 and P4. Rock phosphate application was in favor of fungal colonization. Inoculant P93 showed the most efficiency in increasing soil available phosphorus and organic acids content. Adding phosphorus source and cultivation plants could increase the organic acids content of the soil. Under field experiments, inoculations significantly increased corn yield, the highest yield was reached to 1.22 t/hm2. Compared with the control, three fungal inoculations increased corn yield by 15.6% (P93),15.3% (P4) and 12.6% (P85), respectively. Based on the above results, strain P93 was selected for the next research. The effects of inoculation P93 on soil microbial diversity was measured using PCR-DGGE method. The results showed that inoculation of P93 can significantly increase microbial diversity in corn rhizosphere.A P-solubilizing related gene named as psf-Y was isolated from the cDNA library of strain P93 which was identified as glutamate dehydrogenase gene and had an apparent open reading frame that encoding a protein which contains 460 amino acid residues and included two main domains, namely glu/α-ketoglutarate binding sites motif and NADP coenzyme binding site motif. The transformants carrying gene psf-Y could significantly reduce the medium pH at the same time improve the available P concentration. Nine target organic acids could also be detected. The psf-Y gene silencing of strain P93 was conducted by using hpRNA expression vector. P-solubilization efficiency of transformation fungus was significantly affected by the nitrogen concentration on the plate medium. Significant reduction of P-solubilization capacity of transformantion fungus was also be verified in liquid. Semi-quantitative RT-PCR results showed that the psf-Y gene expression level of wild-type strain was significantly higher than that in transformed strain. Plant expression vector pC-pyk-psf was constructed by using root specific expression promoter gene pyk10 and psf-Y gene and transformation into Arabidopsis thaliana through Agrobacterium-mediated method. Results of GUS gene expression analysis, PCR and RT-PCR detection validated that the exogenous gene was integrated into the Arabidopsis genome, and expressed at the transcriptional level. Under P-deficiency conditions, the root length, stem length and averaged fresh weight of transgenic plants was higher, but not significantly, than the wild type while total phosphorus content of transgenic Arabidopsis thaliana was significantly higher than the wild type. Under normal P level cultivation, the biomass of transgenic Arabidopsis thaliana was significantly higher than that of wild type. However, there was no significant difference in total P content could be finding between them. Quantitative RT-PCR detection showed that the psf-Y gene expression level in transgenic plants under P deficiency conditions was significantly higher than that under normal P culture conditions. |
PDF Full Text Request