内生解淀粉芽孢杆菌BEB17脂肽类和聚酮类化合物的抑菌活性分析
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摘要
BEB17是本实验室从健康香蕉植株根部中分离得到的一株具有较强抑制真菌活性的内生解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。在本研究中,我们通过PCR检测多个脂肽类和聚酮类化合物的合成相关基因,并结合基质协助激光解吸附离子化-飞行时间质谱(MALDI-TOF-MS)和液相色谱-质谱联用(LC-MS)分析,发现该菌株能产生表面活性素Surfactin、泛革素Fengycin和伊枯草菌素Iturin三类脂肽类化合物以及Bacillibactin、Difficidin和Bacillaene三类聚酮类化合物。通过比较两种发酵培养基(M9和Landy培养基)和两种提取方法(酸沉淀法和大孔树脂抽提法),结果发现酸沉淀法提取较为高效,Landy培养基更易于BEB17脂肽类和聚酮类化合物的发酵。活性检测表明,两种提取方法提取的粗提物对香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense race 4,Foc4)都具有较好的抑制作用,其中大孔树脂抽提法提取的物质抑制效果最好,当其浓度为300μg·m L-1时抑制率可达80.75%,说明该方法制备的粗提物的活性物质含量相对较高。进一步研究表明,该提取物能引起Foc4菌丝膨大和畸形,并抑制其孢子萌发。分析表明,该提取物对Foc4细胞膜具有破坏作用。
The strain BEB17 is an endophytic Bacillus amyloliquefaciens isolated from the roots of healthy banana plants in our laboratory,which can produce volatile gases with inhibitory activity against many pathogenic fungi. In this study,we found this strain contained the genes encoding the synthesis of multiple lipopeptide and polyketide compounds via PCR amplification. In agreement,this strain did produce several known bioactive compounds,including Surfactin,Sufengycin,Iturin,Bacillibactin,Difficidin and Bacillaene,identified by using MALDI-TOF-MS and LC-MS. By comparing the two fermentation medium( M9 and Landy medium) and two extraction methods( acid precipitation and macroporous resin extraction),we show that the acid precipitation method is more efficient,and Landy medium is more prone for fermentation of lipopeptide and polyketide by the strain BEB17. The activity tests showed that the crude extracts from two extraction methods described above had a good inhibitory effect on banana fusarium wilt( Fusarium oxysporum f. sp. cubense race4,Foc4). Furthermore,the crude extracts from macroporous resin extraction method display the best inhibitory effect compared to other treatments. When the concentration is 300 μg·mL-1,the inhibition rate can reach80.75%. This shows that this method of crude extract prepared by the active substance content is relatively higher. The further research shows that it can cause the mycelium of Foc4 swelling and deformity,and inhibit its spore germination. In addition,the Foc4 cell membrane were damaged by trypan blue staining.
The strain BEB17 is an endophytic Bacillus amyloliquefaciens isolated from the roots of healthy banana plants in our laboratory,which can produce volatile gases with inhibitory activity against many pathogenic fungi. In this study,we found this strain contained the genes encoding the synthesis of multiple lipopeptide and polyketide compounds via PCR amplification. In agreement,this strain did produce several known bioactive compounds,including Surfactin,Sufengycin,Iturin,Bacillibactin,Difficidin and Bacillaene,identified by using MALDI-TOF-MS and LC-MS. By comparing the two fermentation medium( M9 and Landy medium) and two extraction methods( acid precipitation and macroporous resin extraction),we show that the acid precipitation method is more efficient,and Landy medium is more prone for fermentation of lipopeptide and polyketide by the strain BEB17. The activity tests showed that the crude extracts from two extraction methods described above had a good inhibitory effect on banana fusarium wilt( Fusarium oxysporum f. sp. cubense race4,Foc4). Furthermore,the crude extracts from macroporous resin extraction method display the best inhibitory effect compared to other treatments. When the concentration is 300 μg·mL-1,the inhibition rate can reach80.75%. This shows that this method of crude extract prepared by the active substance content is relatively higher. The further research shows that it can cause the mycelium of Foc4 swelling and deformity,and inhibit its spore germination. In addition,the Foc4 cell membrane were damaged by trypan blue staining.
引文
[1]Bae H,Roberts D P,Lim H S,et al.Endophytic Trichoderma isolates from tropical environments delay disease onset and induce resistance against Phytophthora capsici in hot pepper using multiple mechanisms.[J].Molecular Plant Microbe Interactions,2011,24(3):336-351.
[2]Wu Y J,Lin Y F,Lin H T,et al.Advances in the researches of biocontrol bacteria Bacillus amyloliquefaciens(in Chinese)[J].Packaging and Food M achinery(包装与食品机械),2012,30(6):49-52.
[3]Chen X H,Vater J,Piel J,et al.Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB42[J].Journal of Bacteriology,2006,188(11):4024.
[4]Arguellesarias A,Ongena M,Halimi B,et al.Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens.[J].M icrobial Cell Factories,2009,8(1):1-12.
[5]Strieker M,Tanovic'A,Marahiel M A.Nonribosomal peptide synthetases:structures and dynamics.[J].Current Opinion in Structural Biology,2010,20(2):234-240.
[6]Koumoutsi A,Chen X H,Henne A,et al.Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in strain FZB42[J].Transactions of the Chinese Society of Agricultural Engineering,2013,29(12):177-184.
[7]Chen X H,Scholz R,Borriss M,et al.Difficidin and bacilysin produced by plant-associated Bacillus amyloliquefaciens are efficient in controlling fire blight disease[J].Journal of Biotechnology,2009,140(1-2):38-44.
[8]Zhao P,Quan C,Wang Y,et al.Bacillus amyloliquefaciens Q-426 as a potential biocontrol agent against Fusarium oxysporum f.sp.spinaciae[J].Journal of Basic M icrobiology,2014,54(5):448.
[9]Zhao Y,Selvaraj J N,Xing F,et al.Antagonistic action of Bacillus subtilis strain SG6 on Fusarium graminearum[J].Plos One,2014,9(3):e92486.
[10]Fang Z D.Research method for plant pathology(2nd Ed.)(in Chinese)[M].Beijing:China Agriculture Press(北京:中国农业出版社),1998:63.
[11]Wei Y H,Chu I M.Enhancement of surfactin production in iron-enriched media by Bacillus subtilis ATCC21332-Isolation,characterization,and its inhibition of fibrin clot formation[J].Enzyme&M icrobial Technology,1998(8):724-728.
[12]Sambrook J,Fritsch E F,Maniatis T.Molecular cloning:a laboratory manual(2nded)[M].New York:Cold Spring Harbor,1989.
[13]Arguelles-Arias A,Ongena M,Halimi B,et al.Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens.[J].M icrobial Cell Factories,2009,8(1):1-12.
[14]Alvarez F,Castro M,Príncipe A,et al.The plant-associated Bacillus amyloliquefaciens strains M EP218 and ARP23 capable of producing the cyclic lipopeptidesiturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease[J].Journal of Applied M icrobiology,2012,112(1):159-174.
[15]Yang R X,Ji J H,Wang Z H,et al.Isolation,indentification and inhibitory activity of lipopeptidesof endophytic bacteria from the root of Paeonia suffruticosa(in Chinese)[J].Microbiology China(微生物学通报),2015,42(6):1081-1088.
[16]Feng D X.Research on isolation and purification of antimicrobial peptides(in Chinese)[J].M odern Journal of Animal Husbandry and Veterinary M edicine(现代畜牧兽医),2015(11):9-16.
[17]Ongena M,Jacques P.Bacillus lipopeptides:versatile w eapons for plant disease biocontrol[J].Trends in M icrobiology,2008,16(3):115.
[18]Yang M,Cui Y,Wu W.Content determination of mexiletine hydrochloride tablets by HPLC-M S/M S(in Chinese)[J].China Pharmaceuticals(中国药业),2017,26(2):35-37.
[19]Chen X H,Koumoutsi A,Scholz R,et al.Comparative analysis of the complete genome sequence of the plant grow th promoting bacterium Bacillus amyloliquefaciens FZB42.[J].Nature Biotechnology,2007,25(9):1007-1014.
[20]Wang Y,Wang J,Quan C,et al.Purification and antifungal characterization of an antifungal substance from Bacillus amyloliquifaciens[J].China Biotechnology,2007,27(12):41-45.
[21]Maget-Dana R,Peypoux F.Iturins,a special class of pore-forming lipopeptides:biological and physicochemical properties.[J].Toxicology,1994,87(1-3):151.
[22]Stein T.Bacillus subtilis antibiotics:structures,syntheses and specific functions[J].M olecular M icrobiology,2005,56(4):845-857.
[23]Bais H P,Fall R,Vivanco J M.Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J].Plant Physiology,2004,134(1):307-319.
[24]Deleu M,Lorent J,Lins L,et al.Effects of surfactin on membrane models displaying lipid phase separation[J].Biochimica et biophysica acta,2013,1828(2):801-815.
[25]Zhang J,Ren P F,Tian L,et al.Identication of a anti-M DRB compound from the marine-derived Bacillus sp.B-9987(in Chinese)[J].Chinese Journal of M arine Drugs(中国海洋药物),2016,35(5):45-49.
[26]Wu L,Wu H,Chen L,et al.Difficidin and bacilysin from Bacillus amyloliquefaciens FZB42 have antibacterial activity against Xanthomonas oryzae rice pathogens[J].Scientific Reports,2015,5:12975.
[27]Xiang Y P,Zhou H F,Liu Y F,et al.Isolation and identification of lipopeptide antibiotics produced by Bacillus amyloliquefaciens B1619 and the inhibition of the lipopeptide antibiotics to Fusarium oxysporum f.sp.Lycopersici(in Chinese)[J].Scientia Agricultura Sinica(中国农业科学),2016,49(15):2935-2944.
[28]Wang Y.Purification and characterization of antimicrobial substances produced by Bacillus subtilis strain Bv10(in Chinese)[D].Nanning:Guangxi University(南宁:广西大学),2013.
[29]Luo C P,Liu Y Z,Wu H F,et al.Antifungal activity and rice disease biocontrol performance of lipopeptide antibiotic bacillomycin L(in Chinese)[J].Chinese Journal of Biological Control(中国生物防治学报),2011,27(1):76-81.
[30]Landy M,Rosenman S B,Warren G H.An antibiotic from Bacillus subtilis active against pathogenic fungi[J].Journal of Bacteriology,1947,54(4):24.
[31]Ran J J,Xu J H,Hu X D,et al.Identication of a Bacillus strain producing lipopeptide and cloning of genes related to lipopeptide(in Chinese)[J].Food Science(食品科学),2016,37(17):127-132.
[32]Rao Y K,Lin H Y,Wu W S,et al.Evaluation of HPLC and M EKC methods for the analysis of lipopeptide antibiotic iturin A produced by Bacillus amyloliquefaciens[J].International Journal of Applied Science&Engineering,2008,6(2):85-96.
[2]Wu Y J,Lin Y F,Lin H T,et al.Advances in the researches of biocontrol bacteria Bacillus amyloliquefaciens(in Chinese)[J].Packaging and Food M achinery(包装与食品机械),2012,30(6):49-52.
[3]Chen X H,Vater J,Piel J,et al.Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB42[J].Journal of Bacteriology,2006,188(11):4024.
[4]Arguellesarias A,Ongena M,Halimi B,et al.Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens.[J].M icrobial Cell Factories,2009,8(1):1-12.
[5]Strieker M,Tanovic'A,Marahiel M A.Nonribosomal peptide synthetases:structures and dynamics.[J].Current Opinion in Structural Biology,2010,20(2):234-240.
[6]Koumoutsi A,Chen X H,Henne A,et al.Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in strain FZB42[J].Transactions of the Chinese Society of Agricultural Engineering,2013,29(12):177-184.
[7]Chen X H,Scholz R,Borriss M,et al.Difficidin and bacilysin produced by plant-associated Bacillus amyloliquefaciens are efficient in controlling fire blight disease[J].Journal of Biotechnology,2009,140(1-2):38-44.
[8]Zhao P,Quan C,Wang Y,et al.Bacillus amyloliquefaciens Q-426 as a potential biocontrol agent against Fusarium oxysporum f.sp.spinaciae[J].Journal of Basic M icrobiology,2014,54(5):448.
[9]Zhao Y,Selvaraj J N,Xing F,et al.Antagonistic action of Bacillus subtilis strain SG6 on Fusarium graminearum[J].Plos One,2014,9(3):e92486.
[10]Fang Z D.Research method for plant pathology(2nd Ed.)(in Chinese)[M].Beijing:China Agriculture Press(北京:中国农业出版社),1998:63.
[11]Wei Y H,Chu I M.Enhancement of surfactin production in iron-enriched media by Bacillus subtilis ATCC21332-Isolation,characterization,and its inhibition of fibrin clot formation[J].Enzyme&M icrobial Technology,1998(8):724-728.
[12]Sambrook J,Fritsch E F,Maniatis T.Molecular cloning:a laboratory manual(2nded)[M].New York:Cold Spring Harbor,1989.
[13]Arguelles-Arias A,Ongena M,Halimi B,et al.Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens.[J].M icrobial Cell Factories,2009,8(1):1-12.
[14]Alvarez F,Castro M,Príncipe A,et al.The plant-associated Bacillus amyloliquefaciens strains M EP218 and ARP23 capable of producing the cyclic lipopeptidesiturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease[J].Journal of Applied M icrobiology,2012,112(1):159-174.
[15]Yang R X,Ji J H,Wang Z H,et al.Isolation,indentification and inhibitory activity of lipopeptidesof endophytic bacteria from the root of Paeonia suffruticosa(in Chinese)[J].Microbiology China(微生物学通报),2015,42(6):1081-1088.
[16]Feng D X.Research on isolation and purification of antimicrobial peptides(in Chinese)[J].M odern Journal of Animal Husbandry and Veterinary M edicine(现代畜牧兽医),2015(11):9-16.
[17]Ongena M,Jacques P.Bacillus lipopeptides:versatile w eapons for plant disease biocontrol[J].Trends in M icrobiology,2008,16(3):115.
[18]Yang M,Cui Y,Wu W.Content determination of mexiletine hydrochloride tablets by HPLC-M S/M S(in Chinese)[J].China Pharmaceuticals(中国药业),2017,26(2):35-37.
[19]Chen X H,Koumoutsi A,Scholz R,et al.Comparative analysis of the complete genome sequence of the plant grow th promoting bacterium Bacillus amyloliquefaciens FZB42.[J].Nature Biotechnology,2007,25(9):1007-1014.
[20]Wang Y,Wang J,Quan C,et al.Purification and antifungal characterization of an antifungal substance from Bacillus amyloliquifaciens[J].China Biotechnology,2007,27(12):41-45.
[21]Maget-Dana R,Peypoux F.Iturins,a special class of pore-forming lipopeptides:biological and physicochemical properties.[J].Toxicology,1994,87(1-3):151.
[22]Stein T.Bacillus subtilis antibiotics:structures,syntheses and specific functions[J].M olecular M icrobiology,2005,56(4):845-857.
[23]Bais H P,Fall R,Vivanco J M.Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J].Plant Physiology,2004,134(1):307-319.
[24]Deleu M,Lorent J,Lins L,et al.Effects of surfactin on membrane models displaying lipid phase separation[J].Biochimica et biophysica acta,2013,1828(2):801-815.
[25]Zhang J,Ren P F,Tian L,et al.Identication of a anti-M DRB compound from the marine-derived Bacillus sp.B-9987(in Chinese)[J].Chinese Journal of M arine Drugs(中国海洋药物),2016,35(5):45-49.
[26]Wu L,Wu H,Chen L,et al.Difficidin and bacilysin from Bacillus amyloliquefaciens FZB42 have antibacterial activity against Xanthomonas oryzae rice pathogens[J].Scientific Reports,2015,5:12975.
[27]Xiang Y P,Zhou H F,Liu Y F,et al.Isolation and identification of lipopeptide antibiotics produced by Bacillus amyloliquefaciens B1619 and the inhibition of the lipopeptide antibiotics to Fusarium oxysporum f.sp.Lycopersici(in Chinese)[J].Scientia Agricultura Sinica(中国农业科学),2016,49(15):2935-2944.
[28]Wang Y.Purification and characterization of antimicrobial substances produced by Bacillus subtilis strain Bv10(in Chinese)[D].Nanning:Guangxi University(南宁:广西大学),2013.
[29]Luo C P,Liu Y Z,Wu H F,et al.Antifungal activity and rice disease biocontrol performance of lipopeptide antibiotic bacillomycin L(in Chinese)[J].Chinese Journal of Biological Control(中国生物防治学报),2011,27(1):76-81.
[30]Landy M,Rosenman S B,Warren G H.An antibiotic from Bacillus subtilis active against pathogenic fungi[J].Journal of Bacteriology,1947,54(4):24.
[31]Ran J J,Xu J H,Hu X D,et al.Identication of a Bacillus strain producing lipopeptide and cloning of genes related to lipopeptide(in Chinese)[J].Food Science(食品科学),2016,37(17):127-132.
[32]Rao Y K,Lin H Y,Wu W S,et al.Evaluation of HPLC and M EKC methods for the analysis of lipopeptide antibiotic iturin A produced by Bacillus amyloliquefaciens[J].International Journal of Applied Science&Engineering,2008,6(2):85-96.