大兴安岭地区粘细菌资源的多样性及其生物活性
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摘要
【背景】粘细菌是一类具有社会性行为的高等原核生物,其代谢产物具有丰富、多样、新颖的生物活性,是筛选天然药物的良好资源,具有很大的研究开发应用价值。【目的】分析大兴安岭地区粘细菌资源的多样性;分离纯化可培养的粘细菌,分析其抗菌活性并从中筛选对马铃薯晚疫病菌具有拮抗作用的菌株。【方法】通过变性梯度凝胶电泳(DGGE)技术对从大兴安岭地区采集的15个土样中粘细菌资源的多样性进行分析,利用兔粪诱导、大肠杆菌划线诱导和滤纸诱导3种方法从土样中分离可培养的粘细菌,结合形态观察、生理生化特征和16S rRNA基因序列分析确定这些菌株的分类地位,通过平板对峙法对其进行抗菌活性分析。【结果】DGGE的分析结果显示,15个土样中共鉴定到13个属的粘细菌,基本覆盖了大部分已知的粘细菌种属以及部分未分类的粘细菌,表现出了丰富的多样性。共分离得到88株菌,从中得到22株纯菌,经鉴定属于2个属(粘球菌属与珊瑚球菌属)8个种(黄色粘球菌、橙色粘球菌、变绿粘球菌、珊瑚粘球菌、具枝粘球菌、大孢珊瑚球菌、弱小珊瑚球菌和珊瑚状珊瑚球菌)。抗菌结果显示:22株纯菌均表现出可以对一种甚至多种指示菌产生抗性,其中19株抑制大肠杆菌的生长,14株抑制马铃薯晚疫病菌的生长,8株抑制金黄色葡萄球菌的生长,13株抑制酿酒酵母菌的生长,7株抑制枯草芽孢杆菌的生长。【结论】内蒙古大兴安岭地区蕴藏着丰富的粘细菌资源,粘球菌属及珊瑚球菌属可能为该地区粘细菌菌群中的优势菌。分离纯化出的粘细菌菌株均表现出抑制一种甚至多种指示菌生长的活性,其中64%的纯菌对马铃薯晚疫病菌产生抗性,具有进一步研究的潜在价值。
[Background] Myxobacteria are a group of higher prokaryotes with social behavior. They have great research value in screening natural drugs because their metabolites have rich, diverse and novel biological activities. [Objective] We studied the diversity of myxobacteria in Daxing'an Mountains of Inner Mongolia, and isolated and purified the culturable myxobacteria, and studied their antimicrobial activities and screened strains against Phytophthora infestans. [Methods] The diversity of myxobacteria was studied by PCR-denaturing gradient gel electrophoresis(DGGE). Culturable myxobacteria were isolated from 15 soil samples collected from Daxing'an Mountains by rabbit dung pellets inducing method, Escherichia coli inducing method, and filter paper inducing method. The purified strains were identified by morphological observation, physiological and biochemical characteristics, and the 16 S rRNA gene sequence analysis. Then, the antimicrobial activities of the strains were determined by plate confrontation method. [Results] The results of DGGE showed that 13 genera of myxobacteria were identified from 15 soil samples, covering most of the known genera of myxobacteria and some of the unclassified myxobacteria. 88 strains were isolated in this study and 22 of them were purified. The purified strains belonged to two genera, Myxococcus and Corallococcus, and 8 species, Myxococcus xanthus, Myxococcus flavescens, Myxococcus virescens, Myxococcus coralloides, Myxococcus stipitatus, Corallococcus macrosporus, Corallococcus exiguous, Corallococcus coralloides. All of the purified myxobacterial strains exhibited one or more kinds of antimicrobial activities. 19 strains had the activity against E. coli. 14 strains had the activity against Phytophthora infestans. Eight strains showed the activity against Staphylococcus aureus. 13 strains had the activity against Saccharomyces cerevisiae. Seven strains showed the activity against Bacillus subtilis. [Conclusion] Myxobacterial resource in Daxing'an Mountains of Inner Mongolia is rich. Myxococcus and Corallococcus could be the dominant populations of myxobacterial community in this region. The purified myxobacterial strains exhibited one or more kinds of antimicrobial activities and 64% of them have the activity against Phytophthora infestans, which has the potential value for further research in the future.
[Background] Myxobacteria are a group of higher prokaryotes with social behavior. They have great research value in screening natural drugs because their metabolites have rich, diverse and novel biological activities. [Objective] We studied the diversity of myxobacteria in Daxing'an Mountains of Inner Mongolia, and isolated and purified the culturable myxobacteria, and studied their antimicrobial activities and screened strains against Phytophthora infestans. [Methods] The diversity of myxobacteria was studied by PCR-denaturing gradient gel electrophoresis(DGGE). Culturable myxobacteria were isolated from 15 soil samples collected from Daxing'an Mountains by rabbit dung pellets inducing method, Escherichia coli inducing method, and filter paper inducing method. The purified strains were identified by morphological observation, physiological and biochemical characteristics, and the 16 S rRNA gene sequence analysis. Then, the antimicrobial activities of the strains were determined by plate confrontation method. [Results] The results of DGGE showed that 13 genera of myxobacteria were identified from 15 soil samples, covering most of the known genera of myxobacteria and some of the unclassified myxobacteria. 88 strains were isolated in this study and 22 of them were purified. The purified strains belonged to two genera, Myxococcus and Corallococcus, and 8 species, Myxococcus xanthus, Myxococcus flavescens, Myxococcus virescens, Myxococcus coralloides, Myxococcus stipitatus, Corallococcus macrosporus, Corallococcus exiguous, Corallococcus coralloides. All of the purified myxobacterial strains exhibited one or more kinds of antimicrobial activities. 19 strains had the activity against E. coli. 14 strains had the activity against Phytophthora infestans. Eight strains showed the activity against Staphylococcus aureus. 13 strains had the activity against Saccharomyces cerevisiae. Seven strains showed the activity against Bacillus subtilis. [Conclusion] Myxobacterial resource in Daxing'an Mountains of Inner Mongolia is rich. Myxococcus and Corallococcus could be the dominant populations of myxobacterial community in this region. The purified myxobacterial strains exhibited one or more kinds of antimicrobial activities and 64% of them have the activity against Phytophthora infestans, which has the potential value for further research in the future.
引文
[1]Zhang Q.Isolation,identification and activity of myxobacteria[D].Shanghai:Master’s Thesis of Shanghai Normal University,2005(in Chinese)张琦.粘细菌的分离、鉴定及活性研究[D].上海:上海师范大学硕士学位论文,2005
[2]Li YZ,Li J,Zhou L,et al.Isolation and identification of myxobacteria[J].Acta Microbiologica Sinica,2000,40(6):652-656(in Chinese)李越中,李健,周璐,等.我国粘细菌(Myxobacteria)资源的分离与鉴定[J].微生物学报,2000,40(6):652-656
[3]Zhu YL.A study on myxobacteria[J].Xuzhou Institute of Technology,2005,20(5):25-27(in Chinese)朱蕴兰.粘细菌研究[J].徐州工程学院学报,2005,20(5):25-27
[4]Nan BY,Zusman DR.Uncovering the mystery of gliding motility in the myxobacteria[J].Annual Review of Genetics,2011,45(1):21-39
[5]Wang H,Zhang QL.Research progress of myxobacteria metabolites[J].Journal of International Pharmaceutical Research,2005,32(4):246-250(in Chinese)王赫,张庆林.粘细菌代谢产物的研究进展[J].国际药学研究杂志,2005,32(4):246-250
[6]Tao LX.Isolation and identification of myxobacteria and study on its antibiotic activity[D].Hohhot:Master’s Thesis of Inner Mongolia Agricultural University,2012(in Chinese)陶龙鑫.粘细菌的分离鉴定及活性分析[D].呼和浩特:内蒙古农业大学硕士学位论文,2012
[7]Zhou L,Li YZ,Li J.Multicellular morphogenesis in myxobacteria and its molecular regulation[J].Progress in Biochemistry and Biophysics,1999,26(6):544-547(in Chinese)周璐,李越中,李健.粘细菌的多细胞形态发生及其分子调控[J].生物化学与生物物理进展,1999,26(6):544-547
[8]Ren XB,Wu ZH,Cui HC,et al.Isolation and identification of the strain YR-7 against Phytophthora infestans and study on its antibiotic substances[J].Microbiology China,2016,43(7):1513-1523(in Chinese)任兴波,武志华,崔海辰,等.致病疫霉拮抗菌株YR-7的分离鉴定及其活性物质[J].微生物学通报,2016,43(7):1513-1523
[9]Plaza A,Garcia R,Bifulco G,et al.Aetheramides A and B,potent HIV-inhibitory depsipeptides from a myxobacterium of the new genus“Aetherobacter”[J].Organic Letters,2012,14(11):2854-2857
[10]Okanya PW,Mohr KI,Gerth K,et al.Hyaladione,an S-methyl cyclohexadiene-dione from Hyalangium minutum[J].Journal of Natural Products,2012,75(4):768-770
[11]Wenzel Silke C,Müller R.Myxobacteria-microbial factories for the production of bioactive secondary metabolites[J].Molecular Biosystems,2009,5(6):567-574
[12]Dawid W.Biology and global distribution of myxobacteria in soils[J].FEMS Microbiology Reviews,2000,24(4):403-427
[13]Jiang DM.Phylogenetic diversity of cultured and uncultured myxobacteria in soil and marine sediments[D].Jinan:Doctoral Dissertation of Shandong University,2008(in Chinese)蒋德明.土壤及海底沉积物中可培养与未可培养粘细菌系统发育多样性研究[D].济南:山东大学博士学位论文,2008
[14]Li XJ.Microbial community dynamics of biological soil crusts in desert induced by addition of cellulose[D].Hohhot:Master’s Thesis of Inner Mongolia Agricultural University,2012(in Chinese)李晓军.外源纤维素诱导对荒漠生物土壤结皮细菌群落结构的影响[D].呼和浩特:内蒙古农业大学硕士学位论文,2012
[15]Jiang HY.Soil microorganism and activities of soil enzyme in Larix gmelinii forest of Daxingan Mountains[D].Hohhot:Doctoral Dissertation of Inner Mongolia Agricultural University,2010(in Chinese)姜海燕.大兴安岭兴安落叶松林土壤微生物与土壤酶活性研究[D].呼和浩特:内蒙古农业大学博士学位论文,2010
[16]Wu ZH.Isolation and identification of myxobacteria from the soil samples in Liangcheng,Inner Mongolia and study on their antibiotic activity[D].Hohhot:Master’s Thesis of Inner Mongolia Agricultural University,2014(in Chinese)武志华.内蒙古凉城地区粘细菌的分离鉴定及活性分析[D].呼和浩特:内蒙古农业大学硕士学位论文,2014
[17]Ye HY,Zhang JC.A measuring method of soil moisture[J].Journal of West China Forestry Science,1984(1):63-66(in Chinese)叶厚源,张金畅.介绍一种土壤湿度速测法[J].云南林业科技,1984(1):63-66
[18]Zhang JE.Ecology Common Experimental Research Methods and Techniques[M].Beijing:Chemical Industry Press,2007(in Chinese)章家恩.生态学常用实验研究方法与技术[M].北京:化学工业出版社,2007
[19]Xing XL,Yue ZH,Chen RG,et al.Determination and precautions of soil available phosphorus[J].Agriculture of Henan,2011(2):48-49(in Chinese)邢晓丽,岳志宏,陈瑞鸽,等.土壤有效磷测定方法及注意事项[J].河南农业,2011(2):48-49
[20]Li YK,Zhang SM,Zhou FD.Determination of soil available potassium by tetraphenylborate nephelometry[J].Chinese Journal of Soil Science,1982(10):39-42(in Chinese)李酉开,张淑民,周斐德.四苯硼比浊法测定土壤速效钾[J].土壤通报,1982(10):39-42
[21]Li JY.Determination of soil hydrolyzable nitrogen(alkaline hydrolysis diffusion method)[J].Agriculture Science-Technology and Information,2010(10):15(in Chinese)李金彦.土壤水解性氮的测定(碱解扩散法)[J].农业科技与信息,2010(10):15
[22]Ji TW.Comparison on determining the organic matter contents in the soils by different heating methods in the potassium dichromate-volumetric method[J].Acta Agriculture Zhejiangensis,2005,17(5):311-313(in Chinese)季天委.重铬酸钾容量法中不同加热方式测定土壤有机质的比较研究[J].浙江农业学报,2005,17(5):311-313
[23]Yeates C,Gillings MR.Rapid purification of DNA from soil for molecular biodiversity analysis[J].Letters in Applied Microbiology,1998,27(1):49-53
[24]Wu ZH,Jiang DM,Li P,et al.Exploring the diversity of myxobacteria in a soil niche by myxobacteria-specific primers and probes[J].Environmental Microbiology,2005,7(10):1602-1610
[25]Guo WJ,Tao GJ,Tao WY,et al.A Myxobacterium strain Sorangium cellulosum AHB125 producing epothilone B and other anticancer substances[J].Natural Product Research,2007,21(14):1256-1265
[26]Zhou XL,Shen W,Rao ZM,et al.A rapid method for preparation of fungal chromosome DNA[J].Microbiology China,2004,31(4):89-92(in Chinese)周小玲,沈微,饶志明,等.一种快速提取真菌染色体DNA的方法[J].微生物学通报,2004,31(4):89-92
[27]Zhang ZD,Xie YQ,Chu M,et al.Study on isolation and identification of endophytes in yam rhizome[J].Xinjiang Agricultural Sciences,2010,47(1):126-129(in Chinese)张志东,谢玉清,楚敏,等.山药内生菌的分离及菌种鉴定研究[J].新疆农业科学,2010,47(1):126-129
[28]Li BY,Yao Q,Zhu HH.Approach to analyze the diversity of myxobacteria in soil by Semi-Nested PCR-denaturing gradient gel electrophoresis(DGGE)based on taxon-specific gene[J].PLo S One,2014,9(10):e108877
[29]Zhou XW.Sub-population census for Myxobacteria in soil and molecular strategy research for their territoriality[D].Jinan:Doctoral Dissertation of Shandong University,2013(in Chinese)周秀文.土壤中粘细菌群落的调查及领地性行为的分子机制的研究[D].济南:山东大学博士学位论文,2013
[30]Menne B,Rückert G.Myxobakterien(Myxobacterales)in H?hlensedimenten des Hagengebirges(N?rdliche Kalkalpen)[J].Die Hohle Z Karst Hohlenkd,1988,39:120-131
[31]Reichenbach H.Nannocystis exedens gen.nov.,spec.nov.,a new myxobacterium of the family Sorangiaceae[J].Archiv für Mikrobiologie,1970,70(2):119-138
[32]Wang C,Feng LY,Cao Y,et al.An effective methidathion-degrading bacteria strain and its degrading condition optimization with the response surface method[J].Chinese Journal of Applied and Environmental Biology,2016,22(2):224-229(in Chinese)王川,冯灵玉,曹瑜,等.一株杀扑磷高效降解菌的分离、鉴定及降解条件的响应面法优化[J].应用与环境生物学报,2016,22(2):224-229
[2]Li YZ,Li J,Zhou L,et al.Isolation and identification of myxobacteria[J].Acta Microbiologica Sinica,2000,40(6):652-656(in Chinese)李越中,李健,周璐,等.我国粘细菌(Myxobacteria)资源的分离与鉴定[J].微生物学报,2000,40(6):652-656
[3]Zhu YL.A study on myxobacteria[J].Xuzhou Institute of Technology,2005,20(5):25-27(in Chinese)朱蕴兰.粘细菌研究[J].徐州工程学院学报,2005,20(5):25-27
[4]Nan BY,Zusman DR.Uncovering the mystery of gliding motility in the myxobacteria[J].Annual Review of Genetics,2011,45(1):21-39
[5]Wang H,Zhang QL.Research progress of myxobacteria metabolites[J].Journal of International Pharmaceutical Research,2005,32(4):246-250(in Chinese)王赫,张庆林.粘细菌代谢产物的研究进展[J].国际药学研究杂志,2005,32(4):246-250
[6]Tao LX.Isolation and identification of myxobacteria and study on its antibiotic activity[D].Hohhot:Master’s Thesis of Inner Mongolia Agricultural University,2012(in Chinese)陶龙鑫.粘细菌的分离鉴定及活性分析[D].呼和浩特:内蒙古农业大学硕士学位论文,2012
[7]Zhou L,Li YZ,Li J.Multicellular morphogenesis in myxobacteria and its molecular regulation[J].Progress in Biochemistry and Biophysics,1999,26(6):544-547(in Chinese)周璐,李越中,李健.粘细菌的多细胞形态发生及其分子调控[J].生物化学与生物物理进展,1999,26(6):544-547
[8]Ren XB,Wu ZH,Cui HC,et al.Isolation and identification of the strain YR-7 against Phytophthora infestans and study on its antibiotic substances[J].Microbiology China,2016,43(7):1513-1523(in Chinese)任兴波,武志华,崔海辰,等.致病疫霉拮抗菌株YR-7的分离鉴定及其活性物质[J].微生物学通报,2016,43(7):1513-1523
[9]Plaza A,Garcia R,Bifulco G,et al.Aetheramides A and B,potent HIV-inhibitory depsipeptides from a myxobacterium of the new genus“Aetherobacter”[J].Organic Letters,2012,14(11):2854-2857
[10]Okanya PW,Mohr KI,Gerth K,et al.Hyaladione,an S-methyl cyclohexadiene-dione from Hyalangium minutum[J].Journal of Natural Products,2012,75(4):768-770
[11]Wenzel Silke C,Müller R.Myxobacteria-microbial factories for the production of bioactive secondary metabolites[J].Molecular Biosystems,2009,5(6):567-574
[12]Dawid W.Biology and global distribution of myxobacteria in soils[J].FEMS Microbiology Reviews,2000,24(4):403-427
[13]Jiang DM.Phylogenetic diversity of cultured and uncultured myxobacteria in soil and marine sediments[D].Jinan:Doctoral Dissertation of Shandong University,2008(in Chinese)蒋德明.土壤及海底沉积物中可培养与未可培养粘细菌系统发育多样性研究[D].济南:山东大学博士学位论文,2008
[14]Li XJ.Microbial community dynamics of biological soil crusts in desert induced by addition of cellulose[D].Hohhot:Master’s Thesis of Inner Mongolia Agricultural University,2012(in Chinese)李晓军.外源纤维素诱导对荒漠生物土壤结皮细菌群落结构的影响[D].呼和浩特:内蒙古农业大学硕士学位论文,2012
[15]Jiang HY.Soil microorganism and activities of soil enzyme in Larix gmelinii forest of Daxingan Mountains[D].Hohhot:Doctoral Dissertation of Inner Mongolia Agricultural University,2010(in Chinese)姜海燕.大兴安岭兴安落叶松林土壤微生物与土壤酶活性研究[D].呼和浩特:内蒙古农业大学博士学位论文,2010
[16]Wu ZH.Isolation and identification of myxobacteria from the soil samples in Liangcheng,Inner Mongolia and study on their antibiotic activity[D].Hohhot:Master’s Thesis of Inner Mongolia Agricultural University,2014(in Chinese)武志华.内蒙古凉城地区粘细菌的分离鉴定及活性分析[D].呼和浩特:内蒙古农业大学硕士学位论文,2014
[17]Ye HY,Zhang JC.A measuring method of soil moisture[J].Journal of West China Forestry Science,1984(1):63-66(in Chinese)叶厚源,张金畅.介绍一种土壤湿度速测法[J].云南林业科技,1984(1):63-66
[18]Zhang JE.Ecology Common Experimental Research Methods and Techniques[M].Beijing:Chemical Industry Press,2007(in Chinese)章家恩.生态学常用实验研究方法与技术[M].北京:化学工业出版社,2007
[19]Xing XL,Yue ZH,Chen RG,et al.Determination and precautions of soil available phosphorus[J].Agriculture of Henan,2011(2):48-49(in Chinese)邢晓丽,岳志宏,陈瑞鸽,等.土壤有效磷测定方法及注意事项[J].河南农业,2011(2):48-49
[20]Li YK,Zhang SM,Zhou FD.Determination of soil available potassium by tetraphenylborate nephelometry[J].Chinese Journal of Soil Science,1982(10):39-42(in Chinese)李酉开,张淑民,周斐德.四苯硼比浊法测定土壤速效钾[J].土壤通报,1982(10):39-42
[21]Li JY.Determination of soil hydrolyzable nitrogen(alkaline hydrolysis diffusion method)[J].Agriculture Science-Technology and Information,2010(10):15(in Chinese)李金彦.土壤水解性氮的测定(碱解扩散法)[J].农业科技与信息,2010(10):15
[22]Ji TW.Comparison on determining the organic matter contents in the soils by different heating methods in the potassium dichromate-volumetric method[J].Acta Agriculture Zhejiangensis,2005,17(5):311-313(in Chinese)季天委.重铬酸钾容量法中不同加热方式测定土壤有机质的比较研究[J].浙江农业学报,2005,17(5):311-313
[23]Yeates C,Gillings MR.Rapid purification of DNA from soil for molecular biodiversity analysis[J].Letters in Applied Microbiology,1998,27(1):49-53
[24]Wu ZH,Jiang DM,Li P,et al.Exploring the diversity of myxobacteria in a soil niche by myxobacteria-specific primers and probes[J].Environmental Microbiology,2005,7(10):1602-1610
[25]Guo WJ,Tao GJ,Tao WY,et al.A Myxobacterium strain Sorangium cellulosum AHB125 producing epothilone B and other anticancer substances[J].Natural Product Research,2007,21(14):1256-1265
[26]Zhou XL,Shen W,Rao ZM,et al.A rapid method for preparation of fungal chromosome DNA[J].Microbiology China,2004,31(4):89-92(in Chinese)周小玲,沈微,饶志明,等.一种快速提取真菌染色体DNA的方法[J].微生物学通报,2004,31(4):89-92
[27]Zhang ZD,Xie YQ,Chu M,et al.Study on isolation and identification of endophytes in yam rhizome[J].Xinjiang Agricultural Sciences,2010,47(1):126-129(in Chinese)张志东,谢玉清,楚敏,等.山药内生菌的分离及菌种鉴定研究[J].新疆农业科学,2010,47(1):126-129
[28]Li BY,Yao Q,Zhu HH.Approach to analyze the diversity of myxobacteria in soil by Semi-Nested PCR-denaturing gradient gel electrophoresis(DGGE)based on taxon-specific gene[J].PLo S One,2014,9(10):e108877
[29]Zhou XW.Sub-population census for Myxobacteria in soil and molecular strategy research for their territoriality[D].Jinan:Doctoral Dissertation of Shandong University,2013(in Chinese)周秀文.土壤中粘细菌群落的调查及领地性行为的分子机制的研究[D].济南:山东大学博士学位论文,2013
[30]Menne B,Rückert G.Myxobakterien(Myxobacterales)in H?hlensedimenten des Hagengebirges(N?rdliche Kalkalpen)[J].Die Hohle Z Karst Hohlenkd,1988,39:120-131
[31]Reichenbach H.Nannocystis exedens gen.nov.,spec.nov.,a new myxobacterium of the family Sorangiaceae[J].Archiv für Mikrobiologie,1970,70(2):119-138
[32]Wang C,Feng LY,Cao Y,et al.An effective methidathion-degrading bacteria strain and its degrading condition optimization with the response surface method[J].Chinese Journal of Applied and Environmental Biology,2016,22(2):224-229(in Chinese)王川,冯灵玉,曹瑜,等.一株杀扑磷高效降解菌的分离、鉴定及降解条件的响应面法优化[J].应用与环境生物学报,2016,22(2):224-229