Extract Of The Active Compositions Of Brevibacillus Brevs HAB-5and Its Mutants Screening

HAB-5is a Brevibacillus brevis separated from rhizosphere soil by our laboratory, the study found that it has strong inhibitory effect on against a variety of plant pathogenic fungi. This text did preliminary study on inhibitory spectrum of HAB-5strains, fermentation conditions, extraction of active substance and its characteristics, to clear the main active ingredient of HAB-5in inhibiting plant pathogens. By insert Xanthomonas hrp gene into mutation, we also improved the HAB-5, in order to raise its biocontrol effect. The results are as follows:1. HAB-5shows stronger antagonism against the three types of Colletotrichum、 Fusarium oxysporum f. sp Vasinfectu and that contains18kinds of pathogens and it showed the best antimicrobial effects against C7-2from WenChang and the inhibitory rate was70.89%;2. We made improvements to the LB medium, according to the shake flask fermentation filter HAB-5fermented active substances, the best condition is ZS medium,28℃-37℃,2d, the best medium volume:50ml/250ml.3. Lysate and total protein of HAB-5bacterial cells do not have antagonistic activity, it shows that HAB-5active substances of antagonism does not exist in the bacteria protein or its antagonism activity lost bacteriostatic activity in the process, but when separate cultivation and pathogen confrontation with0.5cm bear take1cm away from bacteria in culture medium (MB), it produced obvious bacteriostasis, while HAB-5bacterium supernatant liquid can also produce strong bacteriostatic effect to pathogenic bacteria. So HAB-5’s main antimicrobial active substances can secrete to culture medium easily.3.HAB-5supernatant treated by ammonium sulfate did not have antifungal active. However, the supernatant or MB crude was further extracted by Low-temperature ethanol precipitation method, and the crude had strong antifungal effect, and produced3same electrophoresis bands, its relative molecular mass were60.0kDa,59.0kDa and43.5kDa respectively. Separating by Sephadex G-50gel column, one of the extraction samples with stronger inhibition zone against the pathogen had single band at14.4kDa, which it same as the band of MB induced by the pathogen with SDS-PAGE analysis. Therefore,the key antifungal composition of HAB-5strain is mainly peptide with14.4kDa.5. We made the precipitations gained by the means of the chilled alcohnol precipitation as it’s antimicrobial crude extraction and test its stability, indicating that the antibacterial crude extract have certain stability to temperature, after40℃and60℃processing for30min does not affect its bacteriostatic activity, and after80℃and90℃processing for30min its bacteriostatic activity also remained above80%, after100℃and121℃processing it have no antibacterial activity after20min; HAB-5crude extractings under the condition of acidic pH3.0, its bacteriostatic activity declined slightly; Under pH5.0,5.0and7.0, bacteriostatic activity did not change significantly; Under the condition of alkaline pH11antibacterial crude extract bacteriostatic activity loss; Not sensitive to light or ultraviolet irradiation and have certain tolerance of protease.6.1n this paper, we used the chemical method to improved Brevibacillus brevis HAB-5, Turn into hpalxoo and hpaxm genes, screening get581transformants that transformed hpalxoo gene; Screening get650transformants that transformed hpaxm gene. Using PCR detect each of50transformants, found that amplified specific420bp hpal gene. By preliminary validate to sub-functions of transformants, we found colony morphology changed, while about the antagonistic activity, only transformed Blxoo genes compared with the original strains HAB-5, the antagonism activity has not changed, other transformants antagonistic activity diminished or disappeared. The detected transformants Blxoo, Blxm and its total protein, after heating and non-heating treatment, did not have produced an allergic reaction on tobacco.7.Evaluated by indoor germination test method determine promoting effect on tomato seeds of the bacterial suspension of bacillus HAB-5, supernatant, ethanol crude extract bacterial suspension of Blxoo transformants and Blxm bacterial suspension, Blxm bacterial suspension has the best growth-promoting effects to tomato seeds, germ grows improving effect of1×10cfu/mL bacteria suspension compared with CK improve112.6%, radicle grows improving effect of1×108cfu/mL bacteria suspension compared with CK improve120.8%; Supernatant fluid and crude ethanol extract in high concentrations has inhibitory effect on tomato seeds, stems and root, but when diluted100times, has certain role in the growth. We also use acupuncture method to determine disease prevention effects to detached mango anthrax leaves of the bacterial suspension of bacillus HAB-5, supernatant, ethanol crude extract, bacterial suspension of Blxoo transformants and Blxm bacterial suspension, through1×10cfu/ml bacterial suspension and Blxm bacterial suspension processing,69.4%higher than the sterile water of inhibition rate, and the inhibition to mango anthrax as same as prochloraz.