Studies On The Constructing Of Multifunctional Engineer Strain For Cabbage Disease Biocontrol

This experiment used Bacillus KR strains and Pseudomonas B13 strains tooperate as arch_strains to conduct protoplast fusion,established fusants.the mainresults were presented as follows:The highest formation rate of protoplast of KR was 31.6% under the condition ofthe concentration of sucrose 0.6 mol/L, pH 8.0,the concentration of lysozyme 8mg/mL, 37℃,the time of lysozyme treatmen 80 min; the highest formation rate ofprotoplast of B13 was 95.2% under the condition of the concentration of NaCl 0.6mol/L, pH 7.0,the concentration of lysozyme 6 mg/mL, 37℃,the time of lysozymetreatment 45 min.In order to improve the regeneration rate, KR regeneration rate under thecondition of the time of lysozyme treatment 20 min, interlayer, the concentration ofsucrose 0.6 mol/L, Ca²⁺ 0.03mol/L, Mg²⁺ 0.02 mol/L,the concentration of Sodiumsuccinate acid 0.3 tool/L, the concentration of L-tryptophan 0.2 g/L,culture mediumplaced 72 h under 37℃; B13 regeneration rate under the condition of the time oflysozyme treatment 15 min,interlayer, the concentration of NaCl 0.6 mol/L, Ca²⁺0.02mol/L, Mg²⁺ 0.01 mol/L, the concentration of Sodium succinate acid 0.3 mol/L,the concentration of L-tryptophan 0.1 g/L, culture medium placed 48 h under 37℃.Using drug resistance as the genetic markers of protoplast fusion between KRand B13. In this protoplast fusion experiment between two bacteria, the applyingdensity in selective culture medium of Tobramycin and Chloramphenicol are 2.0U/mL and 5×10^-3g/mL.Using PEG, we got 182 fusants,generating continuously 10 times,derived asteady fnsant KB1. Judging of characters of fusants on morphological andconfiguration,physics and chemical: The KB1 are rhabditiform,their averagemacro-axis are 2.99381μm, average brachy-axis are 0.45195μm, Gram negative,periichous flagellum, sporangium. The studies on biology characters of KB1: theantimicrobial zone on Soft Rot Disease are 1.70 cm under condition of A culturemedium ferment,35℃, pH 6.5, 60 h; Black Spot Disease are 1.61 cm after 8 d onPDA which contain 10% A culture medium ferment under 35℃, pH 8, 60,distinctness smaller than CK.