Molecular Mechanism Of Phosphate Solubilization To Phosphate Deficient Stress In Penicillium Oxalicum BK

As one of the major limiting nutritive elements in plants growth, phosphorus (P) are abundant in soil, but most of them are in the form of insoluble inorganic phosphorus and the bioavailable for plant is inefficient. For a long time, microorganisms that are capable of mobilizing P into available forms were used in fields as biofertilizers. It is generally accepted that the production of organic acids is the main mechanism of inorganic phosphate-solubilizing in these organism. But most studies about phosphate-solubilizing mechanisms especially for fungi were concentrated on the analysis of organic acids production, and the mainly research techniques were biochemical detection and qualitative and quantitative analysis. Unfortunately, there is no further progress about the molecular regulating mechanism on phosphorus solubilization in phosphate-solubilizing microorganisms (PSMs), which restricted the application of PSMs as biofertilizer in agroecological system.In this study, we began with screening high effective phosphate-solubilizing fungus (PSF) from soil, then analyzed its metabolic mechanism of organic acids in the process of phosphate-solubilizing by the means of qualitative and quantitative. Two key genes which maybe related to the process of inorganic phosphate solubilizing under phosphate deficient stress are also studied. Therefore, these results will illuminated the molecular mechanism of phosphate-solubilizing response to phosphate deficient stress in fungus.The results are as followed:An insoluble inorganic phosphate-solubilizing fungus named BK was isolated from tomato rhizosphere based on the medium that Ca3(PO4)2was the sole P source. The initial capacity of phosphate-solubilizing based on liquid culture medium was685mg/1. Cluster of ITS rDNA sequence of BK showing that the microbe belong to Penicillium oxalicum. After optimization of carbon sources and nitrogen sources, the maximum of phosphate-solubilizing is853mg/1.It was defined that the capacity of phosphate-solubilizing was gradually growing up while the pH value of broth declined. When the maximum concentration of soluble phosphorus appeared on the fifth day, the pH value dropped to2.5. It suggested that acidification probably involved in the mechanism of phosphate-solubilizing by P. oxalicum BK. The optimal condition of high performance liquid chromatography (HPLC) for analysis of metabolite during the progress of phosphate-solubilizing was developed. The mobile phase was a mixture of methanol and0.02mol/L KH2PO4buffer (pH2.6)(1%:99%, v/v) at a flow rate of0.5ml/min. The injection volume was10μl and UV detector was at210run. The column temperature was25℃. With above conditions, the metabolites were analysised on qualitative and quantitative under both the conditions of insoluble phosphate and soluble phosphate. It is significantly indicated that there were much more than4-fold malic acid was produced by P. oxalicum BK under tricalcium phosphate stress than that of soluble phosphate, and the secretion of oxalic acid was restrained by insoluble phosphate which could further verify the fact that the accumulation of malic acid played a major role in the process of phosphate-solubilizing by P. oxalicum BK.The expression patterns of mitochondrial malate dehydrogenase (mMDH) gene and fumarase gene, which are key associated with phosphate-solubilizing, were studied under the conditions of phosphate deficient stress and soluble phosphate. It is indicated that the transcription of mMDH gene under phosphate deficient stress was overexpressed from the third day and the highest expression was8.7folds higher than in soluble phosphorus culture condition by the method of Real-time quantitative PCR (q-PCR). The fumarase gene was overexpressed from the fifth day and nearly1.9folds than the soluble phosphorus culture condition. In addition, mMDH activity and fumarase activity were also consistent with the changing trend of two gene’s transcription. Hence, for the first time, this research illuminated that P. oxalicum BK could increase the secretion and accumulation of malic acid under phosphate deficient stress by the overexpression of related gene to adapt the stress of phosphate deficient in the environment.