Purification And Application Of Antibacterial Material Of Bacillus Amyloliquefaciens MG-3

Bacillus amyloliquefaciens MG-3 strain was isolated from the leaves of mango,and could inhibit a variety of fungal pathogens including Colletotrichum gloeosporioide.In this study,the antibacterial components from strain MG-3 were studied.The main results as follows:(1)Using Cgloesporioide as the indicator strain.First,The Plackett-Burman design was used to screen the key factors that influence inhibition rate.The steepest ascent method was used to access the optimal region of the key factors.And then the central composite design and the response surface methodology were used to optimize the optimum ratio of the medium.The results showed that the key factors and their ratio were soluble starch 13.32 g/L,beef extract 5.05 g/L,ammonium acetate 10.78 g/L.The predicted value of inhibition rate 71.70%was verified by validation experiments value 70%,increasing 1.12 times.(2)Firstly,the fresh-keeping effects of loquat fruit treated with fermentation broth of strain MG-3 was observed.The results showed that MG-3 strain could inhibit the incidence and browning,reduce respiratory intensity of loquat fruit caused by C.gloeosporioide invasion,and delayed senescence and rot of loquat fruit.Secondly,the effect of active oxygen metabolism in loquat fruit treated with fermentation broth of strain MG-3 was investigated.The results showed that compared with the control group,MG-3 fermentation broth can significantly enhanced the activity of superoxide dismutase(SOD),catalase(CAT)and ascorbated peroxidase(APX),the content of ascorbic acid(ASA)and reduced glutathione(GSH)content,while reduced the production rate of O2-,cell membrane permeability,malondialdehyde(MDA)content and lipoxygenase(LOX)activity.Finally,the effect of disease resistance in loquat fruit treated with fermentation broth of strain MG-3 was investigated.The results showed that compared with the control group,MG-3 fermentation broth can significantly enhanced phenylalanine ammonia-lyase(PAL),polyphenol oxidase(PPO)and peroxidase(POD)activity,increased total phenolic and lignin content,enhanced chitinase(CHI)and β-1,3-glucanase(GLU)activity,which showed that MG-3 could induced disease resistance and reduced anthracnose rot on loquat fruit.(3)The antifungal substance was purified from fermentation supernatant of strain MG-3 by using RP-C18 column chromatography,Sephadex LH-20 gel column chromatography,normal phase silica gel column chromatography and thin layer chromatography.Based on Q-TOF-MS analysis,the antifungal substance L-1 was indentified as the lipopeptide antibiotic IturinA(C14～C15).(4)The antagonistic test of IturinA on C.gloeosporioide was studied,the results showed that IturinA could inhibit the growth of mycelium and spore germination of C.gloeosporioide.The minimal inhibitory concentrations(MIC),half maximal inhibitory concentrations(IC50)and minimum fugicide concentration(MFC)of IturinA against C.gloeosporioide were 36.47,74.95 and 110 μg·mL-1 respectively.Mycelium morphology was observed by microscope,the results showed that pathogen mycelium became deformity and swelling.The physiological indicators of C.gloeosporioide was observed,IturinA could increase cell membrane permeability and the content of MDA,decreased the content of protein,but it had no effect on the chitin content.Further research showed that IturinA could reduce lesion diameter on loquat fruit compared with the control and inhibit anthracnose rot effectively.