Research Of Phosphate Solubilizing Microorganism From Caragana Rhizosphere Root And Soil Respectively Then Analysis Of Its Bacteria Community Structure

Caragana Fabr. are shrubs have been cultivated to improve saline-alkaline soils, which could enhace the availability of nitrogen, phosphorus and potassium in soil. Firstly we used the soil as the research object, which from the rhizosphere of Caragana plants grown on the saline-alkaline wasteland in Datong basin. We collected the soil and curtured to screen the phosphate solubilizing fungus. And detected that the colonies of fungus had the capability of solubilizing tricalcium phosphate. Secondly we collected and grinded the roots of Caragana to screen the phosphate solubilizing bacterias. We isolated four strains of colonies with clear halos were considered to be phosphate solubilizing bacterias. And took one of the four strains as the target DNA for real time PCR to detect the amounts of target DNA in samples of Caragana soil, Caragana rhizosphere soil and roots of Caragana. At last the tricalcium phosphate agar medium was used to isolate the phosphate solubilizing fungus and using molecular born sequence technology analysis of two soil bacteria diversity, compared with alkali soil after grow microbial communities change. This research contents for the development of biological phosphorus fertilizer can provide effective strains of resources, and to further shows the alkali soil environment with the improvement in the process to have crucial role. This research mainly have the following conclusion:1.In our study, the soil sample from rhizospheres of Caragana plants grown on saline-alkaline wasteland was collected and used for culture to screen the phosphate solubilizing fungus and investigate its phosphate solubilizing proprties. The tricalcium phosphate agar medium was used. The colonies with clear halos were considered to be phosphate solubilizing fungus in the screening. One strain of fungus with phosphate-solubilizing ability was isolated in the current paper. The ITS sequence of the strain determined by PCR was of100%homology with that of Aspergillus niger when compared with the known sequence in NCBI database using pairwise BLAST. The isolated strain was identified as Aspergillus niger on the basis of morphological characteristics and ITS genetic sequence analyses. To study the phosphate-solubilizing ability of the strain, the pH value and available phosphorus content of inorganic phosphorus culture medium, the weight of mycelium and the absorbed amount of phosphorous by the fungus were measured during a culture period of168hours. The pH value dropped sharply from7.0to2.0within the first36hours of culture, and then generally leveled off during the rest of the culture. This process provides an acidic condition for the solubilizing of insoluble phosphates and is probably one of the main reasons for the changes in the rhizosphere of Caragana plants. At the same time, the available phosphorus content in culture increased to4.7mg at the36th hour of culture and remained at this level for another96hours, then underwent a slight increase in the last24hours of culture.This indicates a high phosphate solubilizing ability of the strain. The weight of mycelium reached its peak (0.32g) at the60th hour of culture and stayed at around0.20g for60hours before reached a second peak (0.27g) at144th hour and then decrease to0.20g at the end of culture. On the other hand, the absorbed amount of phosphorous by the fungus decreased dramatically from5.4mg dropped to0.5mg during the first48hours of culture, then stayed leveled off until the end of culture, indicating that the absorbance and the release of phosphorous later on by Aspergillus niger may be one of the mechanisms for the transformation of insoluble phosphates into available phosphorus by the fungus. These data evidence that Aspergillus niger isolated from the rhizosphere of Caragana plants is able to utilize the insoluble phosphates in soil and transform it into available phosphorus for the plants, thus help with the remediation of soil biology, growth of plants and improvement of soils. The results also provided scientific evidence for the improvement of saline-alkaline soil environment by Caragana plants and the development of biologicalfertilizer made from Aspergillus niger.2.Using inorganic phosphorus solid medium as a screening matrix too, add sterile H2O to grind roots of Caragana, cultured on medium,choosed four strains of bacteria phosphate-solubilizing with larger solubilizing circle then purified and preservated, using molecular biology experiment technology for their separation identified, the four strains of bacteria are belong to Pseudomonas sp. and Pantoea vagans, use one of plants C9as the research object, by Real Time PCR to detect amounts of the srain in root and soil,we found that the gene in three samples of the number for:soil with Caragana<soil with Caragana rhizosphere root<endophytes with root. The results shows the substance with Caragana, it can improve salinity-alkalinity ecological environment gradually.3. Use RFLP to analysis diversity of soil bacteria in saline-alkali soil and Caragana soil, the research result indicates that:bacteria in the community structure of Caragana soil keep basic stability, but there has appeared Bradyrhizobium sp. and nitrogen fixation spiral bacteria (Azospirillum sp.), the two kinds of bacteria can improve the soil environment,play an important role.So it determine that Caragana can improve salinity environment.