炭样小单孢菌产抗生素对水稻细菌性条斑病菌的抗菌作用及其机理
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摘要
【背景】水稻细菌性条斑病菌为水稻细菌性条斑病的病原菌,土壤中分离到的一株具有广谱抗菌活性的炭样小单孢菌JXNU-1,其发酵产物(即抗生素JX)对植物病原菌具有较强的抑菌活性。【目的】研究抗生素JX对水稻细菌性条斑病菌的抗菌作用及其机理。【方法】采用杯碟法测定抑菌圈大小,二倍稀释法测定最低抑菌浓度、最低杀菌浓度,并且从菌体形态观察、电导率变化、培养液大分子漏出、蛋白质合成、核酸合成和膜电位变化6个方面探究其作用机理。【结果】抗生素JX对水稻细菌性条斑病菌的抑菌圈直径达18.84±0.28mm,最低抑菌浓度和最低杀菌浓度分别为1.39μg/m L和2.78μg/mL,且杀菌速度很快,作用12 h的杀菌率达100%。在抗生素JX作用下,水稻细菌性条斑病菌的细胞形态发生改变,培养液电导率、膜电位和大分子漏出量均随抗生素浓度增加而增大,但菌体蛋白质含量随着抗生素浓度增加而降低,同时,通过实时荧光定量PCR方法检测发现ef-p表达量下调。【结论】抗生素JX对水稻细菌性条斑病菌具有较强的抗菌作用,推测其抑菌机理是通过抑制菌体蛋白质的生物合成和影响细胞膜完整性而起作用。
[Background] Xanthomonas oryzae pv. oryzicola is a pathogen to cause rice bacterial leaf streak. Micromonospora carbonacea JXNU-1 with broad-spectrum antibacterial activities was isolated from soil previously, and its fermentation products(designated as "antibiotic JX") show strong antibacterial activity against plant pathogenic bacteria. [Objective] The antibacterial activity of antibiotic JX against Xanthomonas oryzae pv. oryzicola and its mechanism were studied. [Methods] The size of the inhibition zone was measured with cylinder plate method, and the determination of minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC) was performed by serial tube double dilution. Additionally, the mechanism was elaborated through the investigations of cell morphology,electrical conductivity of bacterial suspension, macromolecule leakage, biosynthesis level of protein and nucleic acid and bacterial membrane potential. [Results] The average diameter of inhibition zones was18.84±0.28 mm. The MIC and MBC was 1.39 μg/mL and 2.78 μg/mL, respectively. Complete sterilization was achieved within 12 hours, indicating a fast sterilization of antibiotic JX. In the presence of antibiotic JX, an obvious change was observed for the cell morphology of Xanthomonas oryzae pv. oryzicola.Moreover, the conductivity and macromolecule leakage of bacterial suspension as well as bacterial membrane potential increased with antibiotic concentration, but the protein content decreased. The down-regulated ef-p expression was detected by real-time fluorescence quantitative PCR. [Conclusion]The antibiotic JX exhibits a strong antibacterial ability against Xanthomonas oryzae pv. oryzicola by means of disturbing the integrity of bacterial membranes and inhibiting biosynthesis of proteins.
[Background] Xanthomonas oryzae pv. oryzicola is a pathogen to cause rice bacterial leaf streak. Micromonospora carbonacea JXNU-1 with broad-spectrum antibacterial activities was isolated from soil previously, and its fermentation products(designated as "antibiotic JX") show strong antibacterial activity against plant pathogenic bacteria. [Objective] The antibacterial activity of antibiotic JX against Xanthomonas oryzae pv. oryzicola and its mechanism were studied. [Methods] The size of the inhibition zone was measured with cylinder plate method, and the determination of minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC) was performed by serial tube double dilution. Additionally, the mechanism was elaborated through the investigations of cell morphology,electrical conductivity of bacterial suspension, macromolecule leakage, biosynthesis level of protein and nucleic acid and bacterial membrane potential. [Results] The average diameter of inhibition zones was18.84±0.28 mm. The MIC and MBC was 1.39 μg/mL and 2.78 μg/mL, respectively. Complete sterilization was achieved within 12 hours, indicating a fast sterilization of antibiotic JX. In the presence of antibiotic JX, an obvious change was observed for the cell morphology of Xanthomonas oryzae pv. oryzicola.Moreover, the conductivity and macromolecule leakage of bacterial suspension as well as bacterial membrane potential increased with antibiotic concentration, but the protein content decreased. The down-regulated ef-p expression was detected by real-time fluorescence quantitative PCR. [Conclusion]The antibiotic JX exhibits a strong antibacterial ability against Xanthomonas oryzae pv. oryzicola by means of disturbing the integrity of bacterial membranes and inhibiting biosynthesis of proteins.
引文
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[7]Huang YH,Gao XQ,Long ZE.The antibacterial activities of antibiotics produced by Micromonospora carbonacea to plant pathogenic fungi[J].Agrochemicals,2009,48(11):843-845(in Chinese)黄运红,高兴强,龙中儿.炭样小单孢菌抗生素抗植物病原真菌作用[J].农药,2009,48(11):843-845
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[9]Shen P,Fan XR,Li GW.Experiment of Microbiology[M].Beijing:Higher Education Press,2003:111-113(in Chinese)沈萍,范秀容,李广武.微生物学实验[M].北京:高等教育出版社,2003:111-113
[10]Xu SY,Bian RL,Chen X.Methodology of Pharmacological Experiment[M].Beijing:People’s Medical Publishing House,2006:1647-1664(in Chinese)徐叔云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社,2006:1647-1664
[11]Lou ZX,Wang HX,Zhu S,et al.Antibacterial activity and mechanism of action of chlorogenic acid[J].Journal of Food Science,2011,76(6):M398-M403
[12]Wang WL.Study on the antibacterial mechanism of herbal extracts and drug resistance of E.coli[D].Baoding:Master’s Thesis of Agricultural University of Hebei,2015(in Chinese)王唯霖.中草药提取物的抑菌机理及对大肠杆菌的耐药性研究[D].保定:河北农业大学硕士学位论文,2015
[13]Novo D,Perlmutter NG,Hunt RH,et al.Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique[J].Cytometry,1999,35(1):55-63
[14]Peng WM,Huang YH,Li J,et al.Studies on the antimicrobial activity and its mechanism of an antibiotic,from Micromonospora carbonacea JXNU-1,against gram-negative bacteria in vitro[J].Journal of Jiangxi Normal University(Natural Science),2013,37(2):162-165(in Chinese)彭伟梦,黄运红,李瑾,等.炭样小单孢菌JXNU-1产抗生素对G-的体外抗菌作用及其机理的初步研究[J].江西师范大学学报:自然科学版,2013,37(2):162-165
[15]Kim BR,Nam HY,Kim SU,et al.Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice[J].Biotechnology Letters,2003,25(21):1869-1872
[16]Wang Q,Xie MJ.Antibacterial activity and mechanism of Luteolin on Staphylococcus aureus[J].Acta Microbiologica Sinica,2010,50(9):1180-1184(in Chinese)王倩,谢明杰.木犀草素对金黄色葡萄球菌的抑菌活性及其机制[J].微生物学报,2010,50(9):1180-1184
[17]Zhang YB,Liu XY,Jiang PP,et al.Mechanism and antibacterial activity of cinnamaldehyde against Escherichia coli and Staphylococcus aureus[J].Modern Food Science&Technology,2015,31(5):31-35(in Chinese)张赟彬,刘笑宇,姜萍萍,等.肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌作用及抑菌机理研究[J].现代食品科技,2015,31(5):31-35
[18]Cox SD,Mann CM,Markham JL,et al.Determining the antimicrobial actions of tea tree oil[J].Molecules,2001,6(2):87-91
[19]Chen CZ,Cooper SL.Interactions between dendrimer biocides and bacterial membranes[J].Biomaterials,2002,23(16):3359-3368
[20]Li W.Study about membrane potential in physiology hyperpolarization and depolarization[J].World Health Digest,2012(48):57-58(in Chinese)李薇.浅谈生理学中的膜电位--超极化和去极化[J].中外健康文摘,2012(48):57-58
[21]Zhou JC,Zhou GY,Liu JA,et al.Antifungal mechanisms of the ethyl acetate extracts from Phytolacca americana and Ageratum conyzoides against Colletotrichum gloeosporioides of Dalbergia odorifera[J].Plant Protection,2017,43(4):46-50(in Chinese)周洁尘,周国英,刘君昂,等.垂序商陆及胜红蓟提取物对降香黄檀炭疽病菌的抑菌机理研究[J].植物保护,2017,43(4):46-50
[22]Chen P.Study on antagonism mechanisms of the antibiotics JXfrom Microspore anthracis JXNU-1 against Kocuria rhizophila[D].Nanchang:Master’s Thesis of Jiangxi Normal University,2016(in Chinese)陈鹏.炭样小单孢菌JXNU-1产抗生素JX对嗜根考克氏菌的拮抗作用机理研究[D].南昌:江西师范大学硕士学位论文,2016
[23]Jiang XL,Xie DL.The mechanism of action of agricultural antibiotics[J].Chinese Journal of Biological Control,1994,10(2):76-81(in Chinese)蒋细良,谢德龄.农用抗生素的作用机理[J].中国生物防治,1994,10(2):76-81
[24]Isono K.Nucleoside antibiotics:structure,biological activity,and biosynthesis[J].The Journal of Antibiotics,1988,41(12):1711-1739
[25]Vara J,Perez-Gonzalez JA,Jimenez A.Biosynthesis of puromycin by Streptomyces alboniger:characterization of puromycin N-acetyltransferase[J].Biochemistry,1985,24(27):8074-8081
[26]Wolf H,Z?hner H.Metabolic products of microorganisms.99.Kirromycin[J].Archiv Für Mikrobiologie,1972,83(2):147-154
[27]Zhang HR,Meng SX,Cao J,et al.Antibacterial mechanisms of endophytic fungi from Gynostemma pentaphyllum on Escherichia coli[J].Microbiology China,2015,42(1):157-162(in Chinese)张慧茹,孟素香,曹健,等.绞股蓝内生真菌抗大肠杆菌抗菌机制的研究[J].微生物学通报,2015,42(1):157-162
[28]Wang YG,Yang GR,Ma XQ,et al.Antibacterial mechanisms of endophytic fungi Alternaria alternata of ethyl acetate extract on Escherichia coli[J].Chinese Traditional and Herbal Drugs,2018,49(2):374-381(in Chinese)王永刚,杨光瑞,马雪青,等.内生真菌链格孢菌醋酸乙酯提取物对大肠杆菌抑菌机制的研究[J].中草药,2018,49(2):374-381
[29]Zhang XM,Liu Q,Liu X,et al.Research progress on the Micromonosporaceae family[J].Journal of Northwest A&FUniversity(Natural Science Edition),2013,41(9):175-185(in Chinese)张晓敏,刘秋,刘限,等.小单孢菌科研究进展[J].西北农林科技大学学报:自然科学版,2013,41(9):175-185
[2]Zhang RS,Wang XY,Luo CP,et al.Identification of the lipopeptides from Bacillus amyloliquefaciens Lx-11 and biocontrol efficacy of surfactin against bacterial leaf streak[J].Scientia Agricultura Sinica,2013,46(10):2014-2021(in Chinese)张荣胜,王晓宇,罗楚平,等.解淀粉芽孢杆菌Lx-11产脂肽类物质鉴定及表面活性素对水稻细菌性条斑病的防治作用[J].中国农业科学,2013,46(10):2014-2021
[3]Shen SW,Liu YF,Zhou SJ,et al.The study of synergistic control of bacterial leaf streak with 4 antagonistic bacteria,streptomycin and leaf Kyzalone[J].Jiangsu Agricultural Sciences,2010(4):98-100(in Chinese)沈素文,刘永锋,周苏津,等.4株拮抗细菌与链霉素、叶枯唑协同控制水稻细菌性条斑病研究[J].江苏农业科学,2010(4):98-100
[4]Long ZE,Zhu YJ,Huang YH,et al.Isolation and identification of a strain of Micromonospora with broad-spectrum antimicrobial activity[J].Microbiology China,2008,35(3):378-383(in Chinese)龙中儿,朱跃进,黄运红,等.一株具有广谱抗菌活性小单孢菌的分离和鉴定[J].微生物学通报,2008,35(3):378-383
[5]Zhu YJ,Long ZE,Huang YH,et al.Studies on fermentation technology for a strain of rare actinomycete producing antibiotics[J].Chemistry&Bioengineering,2006,23(12):39-42(in Chinese)朱跃进,龙中儿,黄运红,等.一株稀有放线菌发酵产抗生素的工艺研究[J].化学与生物工程,2006,23(12):39-42
[6]Long ZE,Zhu YJ,Huang YH,et al.Isolation and purification of antibiotic from the fermentation broth of Micromonospora carbonacea JXNU-1 and its physical-chemical properties[J].Microbiology China,2008,35(9):1450-1454(in Chinese)龙中儿,朱跃进,黄运红,等.炭样小单孢菌JXNU-1广谱抗生素产物的分离及其理化性质[J].微生物学通报,2008,35(9):1450-1454
[7]Huang YH,Gao XQ,Long ZE.The antibacterial activities of antibiotics produced by Micromonospora carbonacea to plant pathogenic fungi[J].Agrochemicals,2009,48(11):843-845(in Chinese)黄运红,高兴强,龙中儿.炭样小单孢菌抗生素抗植物病原真菌作用[J].农药,2009,48(11):843-845
[8]Dai F,Huang YH,Ning X,et al.Antibacterial activities of antibiotics produced by Micromonospora carbonacea to Xanthomonas oryzae pv.oryzae[J].Agrochemicals,2011,50(4):302-304(in Chinese)戴菲,黄运红,宁茜,等.炭样小单孢菌抗生素对水稻白叶枯病菌的抑菌作用[J].农药,2011,50(4):302-304
[9]Shen P,Fan XR,Li GW.Experiment of Microbiology[M].Beijing:Higher Education Press,2003:111-113(in Chinese)沈萍,范秀容,李广武.微生物学实验[M].北京:高等教育出版社,2003:111-113
[10]Xu SY,Bian RL,Chen X.Methodology of Pharmacological Experiment[M].Beijing:People’s Medical Publishing House,2006:1647-1664(in Chinese)徐叔云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社,2006:1647-1664
[11]Lou ZX,Wang HX,Zhu S,et al.Antibacterial activity and mechanism of action of chlorogenic acid[J].Journal of Food Science,2011,76(6):M398-M403
[12]Wang WL.Study on the antibacterial mechanism of herbal extracts and drug resistance of E.coli[D].Baoding:Master’s Thesis of Agricultural University of Hebei,2015(in Chinese)王唯霖.中草药提取物的抑菌机理及对大肠杆菌的耐药性研究[D].保定:河北农业大学硕士学位论文,2015
[13]Novo D,Perlmutter NG,Hunt RH,et al.Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique[J].Cytometry,1999,35(1):55-63
[14]Peng WM,Huang YH,Li J,et al.Studies on the antimicrobial activity and its mechanism of an antibiotic,from Micromonospora carbonacea JXNU-1,against gram-negative bacteria in vitro[J].Journal of Jiangxi Normal University(Natural Science),2013,37(2):162-165(in Chinese)彭伟梦,黄运红,李瑾,等.炭样小单孢菌JXNU-1产抗生素对G-的体外抗菌作用及其机理的初步研究[J].江西师范大学学报:自然科学版,2013,37(2):162-165
[15]Kim BR,Nam HY,Kim SU,et al.Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice[J].Biotechnology Letters,2003,25(21):1869-1872
[16]Wang Q,Xie MJ.Antibacterial activity and mechanism of Luteolin on Staphylococcus aureus[J].Acta Microbiologica Sinica,2010,50(9):1180-1184(in Chinese)王倩,谢明杰.木犀草素对金黄色葡萄球菌的抑菌活性及其机制[J].微生物学报,2010,50(9):1180-1184
[17]Zhang YB,Liu XY,Jiang PP,et al.Mechanism and antibacterial activity of cinnamaldehyde against Escherichia coli and Staphylococcus aureus[J].Modern Food Science&Technology,2015,31(5):31-35(in Chinese)张赟彬,刘笑宇,姜萍萍,等.肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌作用及抑菌机理研究[J].现代食品科技,2015,31(5):31-35
[18]Cox SD,Mann CM,Markham JL,et al.Determining the antimicrobial actions of tea tree oil[J].Molecules,2001,6(2):87-91
[19]Chen CZ,Cooper SL.Interactions between dendrimer biocides and bacterial membranes[J].Biomaterials,2002,23(16):3359-3368
[20]Li W.Study about membrane potential in physiology hyperpolarization and depolarization[J].World Health Digest,2012(48):57-58(in Chinese)李薇.浅谈生理学中的膜电位--超极化和去极化[J].中外健康文摘,2012(48):57-58
[21]Zhou JC,Zhou GY,Liu JA,et al.Antifungal mechanisms of the ethyl acetate extracts from Phytolacca americana and Ageratum conyzoides against Colletotrichum gloeosporioides of Dalbergia odorifera[J].Plant Protection,2017,43(4):46-50(in Chinese)周洁尘,周国英,刘君昂,等.垂序商陆及胜红蓟提取物对降香黄檀炭疽病菌的抑菌机理研究[J].植物保护,2017,43(4):46-50
[22]Chen P.Study on antagonism mechanisms of the antibiotics JXfrom Microspore anthracis JXNU-1 against Kocuria rhizophila[D].Nanchang:Master’s Thesis of Jiangxi Normal University,2016(in Chinese)陈鹏.炭样小单孢菌JXNU-1产抗生素JX对嗜根考克氏菌的拮抗作用机理研究[D].南昌:江西师范大学硕士学位论文,2016
[23]Jiang XL,Xie DL.The mechanism of action of agricultural antibiotics[J].Chinese Journal of Biological Control,1994,10(2):76-81(in Chinese)蒋细良,谢德龄.农用抗生素的作用机理[J].中国生物防治,1994,10(2):76-81
[24]Isono K.Nucleoside antibiotics:structure,biological activity,and biosynthesis[J].The Journal of Antibiotics,1988,41(12):1711-1739
[25]Vara J,Perez-Gonzalez JA,Jimenez A.Biosynthesis of puromycin by Streptomyces alboniger:characterization of puromycin N-acetyltransferase[J].Biochemistry,1985,24(27):8074-8081
[26]Wolf H,Z?hner H.Metabolic products of microorganisms.99.Kirromycin[J].Archiv Für Mikrobiologie,1972,83(2):147-154
[27]Zhang HR,Meng SX,Cao J,et al.Antibacterial mechanisms of endophytic fungi from Gynostemma pentaphyllum on Escherichia coli[J].Microbiology China,2015,42(1):157-162(in Chinese)张慧茹,孟素香,曹健,等.绞股蓝内生真菌抗大肠杆菌抗菌机制的研究[J].微生物学通报,2015,42(1):157-162
[28]Wang YG,Yang GR,Ma XQ,et al.Antibacterial mechanisms of endophytic fungi Alternaria alternata of ethyl acetate extract on Escherichia coli[J].Chinese Traditional and Herbal Drugs,2018,49(2):374-381(in Chinese)王永刚,杨光瑞,马雪青,等.内生真菌链格孢菌醋酸乙酯提取物对大肠杆菌抑菌机制的研究[J].中草药,2018,49(2):374-381
[29]Zhang XM,Liu Q,Liu X,et al.Research progress on the Micromonosporaceae family[J].Journal of Northwest A&FUniversity(Natural Science Edition),2013,41(9):175-185(in Chinese)张晓敏,刘秋,刘限,等.小单孢菌科研究进展[J].西北农林科技大学学报:自然科学版,2013,41(9):175-185