Screening Of The Specific Solubilizing Phosphate And Poassium CX-7Strain And Research On Its Applying Experiment

A specific solubilizing phosphate and potassium strain has been separated from297bacillus strains screened from different soil samples of8provinces. Effect of solubilizingphosphate and potassium, fermentation condition and its applying experiment has beenvalued. The main research results are list as below.1Screening and separation of bacillus strains with multifunction297bacillus strains have been separated from the soil samples of8provinces. Thereare89strains of thesewith the ability of solubilizing phosphate and potassium. Highability of solubilizing phosphate and potassium strains should be separated from these.1.1Determination ability of solubilizing phosphate and potassium in liquid flaskBy solubilizing phosphate and potassium test using the liquid flask, BY-3, BY-4,CX-7, CX-14, NX-42, NX-43and SS-12strains has been separated from89strains. Inthis experiment, the soluble phosphorus and potassium were determine in the liquid flaskand K-7strain（ACCC10013） is as contrast. CX-7strain is good compared with thesestrains. The solubilizing rock phosphate and potassium feldspar activity is1.84times and2.53times as that as K-7strain.1.2Pathogenic microorganisms antagonistic testsWheat, cotton and other pathogenic microorganisms antagonistic test have beendone using BY-3, BY-4, CX-7, CX-14, NX-42, NX-43and SS-12strains and its culture.By plate confrontation, it is proved that CX-7strain has wide antimicrobial pectrum,which is Verticillium dahliae, Rhizoctonia solani, Fusarium culmorum, Fusariumoxysporum, Fusarium graminearum and the antagonistic diameter are17.3mm,18.3mm,16.7mm,17.7mm and16.9mm respectively.1.3Pot experimentsThe maize pot experiments are performed by BY-3, BY-4, CX-7, CX-14, NX-42,NX-43and SS-12strains. As a result, compared with control the plant height of corninoculated with CX-7strains increases7.8%. Corn leaf area increases by18.0%. Themaize height and leaf area highly significant difference compared with that of thecontrast, and it reached significant difference compared with that of other strains using3414statistics software.2Identification of CX-7strain and analysis of metabolites2.1Classification and identification of CX-7strainBy colony morphology, cell characteristics, determination of physiological and biochemical reaction and the phylogenetic tree and the analysis of sequence of CX-7strain16S-rDNA according to Berger’s Manual of Systematic Bacteriology, the straintested was preliminarily identified as Paenibacillus kribbensis.2.2Metabolites analysis of CX-7strainThe contents of oxalic acid, tartaric acid, citric acid and malic acid in the CX-7fermentation broth measured by high-performance liquid chromatograph （HPLC） are4.57mg/L,12.63mg/L,10.48mg/L and10.12mg/L after fermenting for48h respectively.After precipitation treatment with ethanol and drying, the highest content ofpolysaccharide in the fermentation broth was measured to8.5mg/L in24h.It is confirmed that CX-7strains may secrete the antimicrobial protein substancesby NH4SO4sedimentation methods, organic reagent extraction and thermal denaturationtreatment. The protein was purified by DEAE-Sephadex chromatography, singleantibacterial polypeptide is obtained. The molecular weight was detected by SDS-PAGE,which is approximately50KDa, the suitable reaction is pH7.0and the optimumtemperate is25°C. Mn2+and Mg2+can activate it, while Cu2+, Zn2+and Na+can inhibit it.3Production techniques research of CX-7strainThe best sporulation medium and optimal culture conditions of CX-7strains aredetermined by single factor and orthogonal test. The best media is: starch5%, soybeanmeal1.5%, MgSO40.07%, NaCl0.05%. Optimal culture conditions are as follows: theage of seed20h, inoculation amount4%, initial pH8.0, filling amount100mL/250mL.By many batches of20m3fermentation tank experiment, the fermentation period ofPaenibacillus kribbensis CX-7strains is36h, of which the biomass reaches8.9×108cfu/mL using culture flask seed. During the fermentation process, the best conditions areventilation ratio1:0.8-1.0, inoculum amount8%, culture temperature32-34°C.Through DHY320disc centrifuge Paenibacillus kribbensis CX-7strain fermentationbroth is concentrated by centrifugation, with the centrifugal rotation speed of7300r/min,flow of0.5T/h, concentration ratio of8times, the yield of95%. The optimumtechnological parameters of the spin flash dryer are: air inlet temperature of135°C, outlettemperature50-60°C, the feed rate of80kg/h. The bacterial content of Paenibacilluskribbensis CX-7strain powder got under this condition is2.89×1010cfu/g, with watercontent not more than5%. This agent can be used in the agriculture production directly.4Application effect study of CX-7strain4.1Effect of soil enzyme activity and soil nutrition by CX-7strainResearch on soil phosphorus, potassium nutrient and enzyme activity of maize potexperiment demonstrate that sucrase can be increased by5.27mg/g·24h, the catalase by0.47(0.1mol/L K2MnO4mL/g), the phosphatase by0.29PhOH mg/g, the urease activityby NH3-N0.18mg/g; as well as potassium content is increased by20%, phosphoruscontent can be increased by57.4%, so it can improve the environment of maize rhizosphere soil.5.2Application effect inoculated by CX-7strain preparation in the fieldThe results of tobacco inoculation experiments shows that the application of theCX-7strain preparation (50×108cfu/g) of0.5kg per mu can promote plant growth anddevelopment, improve tobacco production, production value, as well as superior andmiddle-superior leaf rate, and improve tobacco inherent chemical quality. Also it cangreatly improve the quality grade of tobacco. The result of wheat coating experimentshows that the application of the CX-7strain preparation (50×108cfu/g) of0.1kg per mucan promote plant growth and increasing the yield.