Purification,Identification And Inhibitory Mechanism Of Antimicrobial Lipopeptides IturinA Produced By Biocontrol Bacterium Bacillus,Amyloliquefaciens Strain LY-1

Bacillus subtilis was considered to be a useful biocontrol bacteria,which can suppress the growth of plant pathogenic fungus and promote the growth of plant,thereby it has high research values and potential for development in biological control of postharvest diseases of fruits and vegetables.Bacillus amyloliquefaciens LY-1 was isolated from longan leaves and showed strong inhibition against pathogenic fungus.Based on previous achievements,the purification,identification,biological and physical characteristics,antifungal activity and mechanism of lipopeptide produced by Bacillus amyloliquefaciens LY-1 were investigated in this study.The main results were as followed:(1)The purification and identification methods of antimicrobial lipopeptide were established.The active crude extracts were obtained from fermented supernatant of Bacillus amyloliquefaciens LY-1 by hydrochloric acid precipitation and methanol extraction.Thereafter,an antifungal compound J was purified from RP-C18 column,Sephadex LH-20 and positive phase silica gel column.The antifungal compound J was identified and analyzed through Q-TOF-MS and 1H-NMR.The mass spectrum showed that its molecular weight was 1042.5 Da and 1056.5 Da.Thus,it can be inferred that the antifungal compound J was a mixture of Ituirn A2 and IturinA4,which was identified as two homologous compound of IturinA.(2)The biological and physical characteristics of IturinA produced by Bacillus amyloliquefaciens LY-1 were investigated,the results showed that IturinA has a broad-antifungal spectrum,especially against Colletotrichum gloeosporioides,Pestalotiopsis eriobotryfolia,Fusarium?Botryosphaeria,Lasiodiplodia theobromae,Phomopsis longanae Chi,Pestalotiopsis microspora,Botryosphaeria.Besides,IturinA has high stability to heat,acid and alkali,anti-ultraviolet radiation and resistance to protease hydrolysis,which indicated it has a potentail application in biological control.(3)The antimicrobial mechanism of IturinA on Pestalotiopsis microspora was studied.The minimal inhibitory concentration(MIC)of IturinA against Pestalotiopsis microspora was 15.63?g/mL,minimum lethal concentration(MLC)was 62.5 ?g/mL.IturinA could inhibit the hypha growth,spore germination and spore productivity of Pestalotiopsis microspora.The study of inhibitory mechanism from cellular level demonstrated that IturinA changed cell membrane permeability,induced cellular protein and nucleic acid leakage.Meanwhile,MDA content of Pestalotiopsis microspora increased.All these results indicated that IturinA could increase lipid peroxidation in membrane,damage the integrity of cell membrane,change cell membrane permeability,which might trigger cell death.In vitro bio-control on Chinese olive,it was showed that iturinA could resist the intrusion of outside pathogenic fungus and enhance plants disease resistance.