解淀粉芽孢杆菌TF28抗菌脂肽芬芥素的分离鉴定及抑菌作用
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摘要
对一株解淀粉芽孢杆菌TF28产生的抗菌脂肽进行分离鉴定及抑菌活性研究,采用酸沉淀、乙酸乙酯和甲醇萃取技术制备了抗菌脂肽粗提物,经过2次HPLC分离纯化,在保留时间32～42min内获得8个抗菌脂肽纯品,经MALDI-TOF-MS鉴定为芬芥素(fengycins),对尖孢镰刀菌和禾谷镰刀菌显示出较强的抑菌活性,该研究为提高菌株TF28抗菌脂肽产量的定向遗传改造奠定基础。
The antifungal lipopeptides produced by Bacillus amyloliquefaciens TF28 have been isolated and identified and their antifungal activity have been studied.The extract of antifungal lipopeptides was prepared by acid precipitation,ethyl acetate and methanol extraction.After two rounds of HPLC separation,eight antifungal lipopeptides were obtained within 32~42 min of retention time,and they have been identified as fengycins by MALDI-TOF-MS,which show strong antifungal activity against Fusarium oxysporum and Fusarium graminearum.These results will contribute to the directional genetic manipulation of the strain TF28 to improve the yield of antifungal lipopeptides.
The antifungal lipopeptides produced by Bacillus amyloliquefaciens TF28 have been isolated and identified and their antifungal activity have been studied.The extract of antifungal lipopeptides was prepared by acid precipitation,ethyl acetate and methanol extraction.After two rounds of HPLC separation,eight antifungal lipopeptides were obtained within 32~42 min of retention time,and they have been identified as fengycins by MALDI-TOF-MS,which show strong antifungal activity against Fusarium oxysporum and Fusarium graminearum.These results will contribute to the directional genetic manipulation of the strain TF28 to improve the yield of antifungal lipopeptides.
引文
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[19]姬婧媛,杨洁,高小宁,等.植物内生枯草芽孢杆菌E1R-j脂肽类化合物的分离鉴定及抑菌作用.农药学学报,2015,17(02):172-178.Ji J Y,Yang J,Gao X N,et al.Isolation and identification of lipopeptides produced by endophytic bacteria Bacillus subtilis E1R-j and its anti-fungal mechanism studies,Chinese Journal of Pesticide Science,2015,17(02):172-178.
[20]Deleu M,Paquot M,Nylander T.Effect of fengycin,a lipopeptide produced by Bacillus subtilis,on model biomembranes.Biophys J,2008,94(7):2667-2679.
[21]Arrebola E,Jacobs R,Korsten L.Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens.J Appl Microbiol2010,108(2):386-395.
[22]Maget-Dana R,Thimon L,Peypoux F,et al.Surfactin/iturin Ainteractions may explain the synergistic effect of surfactin on the biological properties of iturin A.Biochimie,1992,74(12):1047-1051.
[23]Tao Y,Bie X M,Lv F X,et al.Antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer.J Microbiol,2011,49(1):146-150.
[24]Ongena M,Duby F,Jourdan E,et al.Bacillus subtilis M4decreases plant susceptibility towards fungal pathogens by increasing host resistance associated with differential gene expression.Appl Microbiol Biotechnol,2005,67(5):692-698.
[25]Ongena M,Jourdan E,Adam A,et al.Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants.Environ Microbiol,2007,9(4):1084-1090.
[2]郝建安,曹志辉,赵凤梅,等.解淀粉芽孢杆菌NK10.BAhjaWT抑真菌作用的研究.微生物学通报,2008,35(6):903-908.Hao J A,Cao Z H,Zhao F M,et al.Exploring the antifungal activity of Bacillus amyloliquefaciens NK10.BAhjaWT.Microbiology China,2008,35(6):903-908.
[3]Niazi A,Manzoor S,Bejai S,et al.Complete genome sequence of a plant associated bacterium Bacillus amyloliquefaciens subsp.plantarum UCMB5033.Stand Genomic Sci,2014,9(3):718-725.
[4]吴一晶,林艺芬,林河通,等.生防菌解淀粉芽孢杆菌研究进展.包装与食品机械,2012,30(6):49-52.Wu Y J,Lin Y F,Lin H T,et al.Advances in the researches of biocontrol bacteria Bacillus amyloliquefaciens.Packaging and Food Machinery,2012,30(6):49-52.
[5]张龙来,康向辉,魏孝义,等.1株解淀粉芽孢杆菌HN011抑菌次级代谢产物的分析.华南农业大学学报,2016,37(1):63-69.Zhang L L,Kang X H,Wei X Y,et al.Research on secondary metabolites from Bacillus amyloliquefaciens strain HN011.Journal of South China Agricultural University,2016,37(1):63-69.
[6]Stein T.Bacillus subtilis antibiotics:structures,syntheses and specific functions.Mol Microbiol,2005,56(4):845-857.
[7]Montesinos E.Antimicrobial peptides and plant disease control.FEMS Microbiol Lett,2007,270(1):1-11.
[8]汪静杰,赵东洋,刘永贵,等.解淀粉芽孢杆菌SWB16菌株脂肽类代谢产物对球孢白僵菌的拮抗作用.微生物学报,2014,54(7):778-785.Wang J J,Zhao D Y,Liu Y G,et al.Antagonism against Beauveria bassiana by lipopeptide metabolites produced by entophyte Bacillus amyloliquefaciens strain SWB16.Acta Microbiologica Sinica,2014,54(7):778-785.
[9]Ongena M,Jacques P.Bacillus lipopeptides:versatile weapons for plant disease biocontrol.Trends Microbiol,2008,16(3):115-125.
[10]Alvarez F,Castro M,Principe A,et al.The plant-associated Bacillus amyloliquefaciens strains MEP2 18 and ARP2 3 capable of producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease.J Appl Microbiol,2012,112(1):159-174.
[11]张淑梅,沙长青,王玉霞,等.大豆内生细菌的分离及根腐病拮抗菌的筛选鉴定.微生物学通报,2008(10):1593-1599.Zhang S M,Sha C Q,Wang Y X,et al.Isolation and characterization of antifungal endophytic bacteria from soybean.Microbiology China,2008(10):1593-1599.
[12]Zhang S,Jiang W,Li J,et al.Whole genome shotgun sequence of Bacillus amyloliquefaciens TF28,a biocontrol entophytic bacterium.Stand Genomic Sci,2016,11:73.
[13]Zhang S,Wang Y,Meng L,et al.Isolation and characterization of antifungal lipopeptides produced by endophytic Bacillus amyloliquefaciens TF28.African Journal of Microbiology Research,2012,6(8):1747-1755.
[14]刘宇帅,孟利强,陈静宇,等.解淀粉芽胞杆菌TF28产抗菌脂肽培养基优化.微生物学杂志,2017,37(3):52-58.Liu Y S,Meng L Q,Chen J Y,et al.Optimization of medium for the production of antifungal lipopeptides by Bacillus amyloliquefaciens TF28.Journal of Microbiology,2017,37(3):52-58.
[15]Vater J,Kablitz B,Wilde C,et al.Matrix-assisted laser desorption ionization--time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge.Appl Environ Microbiol,2002,68(12):6210-6219.
[16]de Faria A F,Stefani D,Vaz B G,et al.Purification and structural characterization of fengycin homologues produced by Bacillus subtilis LSFM-05 grown on raw glycerol.J Ind Microbiol Biotechnol,2011,38(7):863-871.
[17]Wang J,Liu J,Wang X,et al.Application of electrospray ionization mass spectrometry in rapid typing of fengycin homologues produced by Bacillus subtilis.Lett Appl Microbiol,2004,39(1):98-102.
[18]李宝庆,鹿秀云,郭庆港,等.枯草芽孢杆菌BAB-1产脂肽类及挥发性物质的分离和鉴定.中国农业科学,2010,43(17):3547-3554.Li B Q,Lu X Y,Guo Q G,et al.Isolation and identification of lipopeptides and volatile compounds produced by Bacillus subtilis strain BAB-1,Scientia Agricultura Sinica,2010,43(17):3547-3554.
[19]姬婧媛,杨洁,高小宁,等.植物内生枯草芽孢杆菌E1R-j脂肽类化合物的分离鉴定及抑菌作用.农药学学报,2015,17(02):172-178.Ji J Y,Yang J,Gao X N,et al.Isolation and identification of lipopeptides produced by endophytic bacteria Bacillus subtilis E1R-j and its anti-fungal mechanism studies,Chinese Journal of Pesticide Science,2015,17(02):172-178.
[20]Deleu M,Paquot M,Nylander T.Effect of fengycin,a lipopeptide produced by Bacillus subtilis,on model biomembranes.Biophys J,2008,94(7):2667-2679.
[21]Arrebola E,Jacobs R,Korsten L.Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens.J Appl Microbiol2010,108(2):386-395.
[22]Maget-Dana R,Thimon L,Peypoux F,et al.Surfactin/iturin Ainteractions may explain the synergistic effect of surfactin on the biological properties of iturin A.Biochimie,1992,74(12):1047-1051.
[23]Tao Y,Bie X M,Lv F X,et al.Antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer.J Microbiol,2011,49(1):146-150.
[24]Ongena M,Duby F,Jourdan E,et al.Bacillus subtilis M4decreases plant susceptibility towards fungal pathogens by increasing host resistance associated with differential gene expression.Appl Microbiol Biotechnol,2005,67(5):692-698.
[25]Ongena M,Jourdan E,Adam A,et al.Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants.Environ Microbiol,2007,9(4):1084-1090.