茅尾海无瓣海桑根际土壤细菌多样性及抑菌活性分析
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摘要
【目的】研究茅尾海无瓣海桑根际土壤细菌多样性及抑菌活性,挖掘更多潜在新物种和高效活性物质,为进一步研究红树植物根际土壤中细菌的次生代谢产物及研发新型高效生物农药提供参考依据。【方法】从广西钦州茅尾海红树林保护区采集无瓣海桑根际土壤,采用6种分离培养基分离可培养细菌;通过16S r RNA基因序列的系统发育学分析其物种多样性,并采用牛津杯法考察根际细菌对甘蔗鞭黑粉菌的抑制活性。【结果】从无瓣海桑根际中分离得到可培养细菌97株,通过16S r RNA序列比对排重后,从中获得57株不同细菌,可归为4门5纲9目15科21属,有1株Cellvibrio sp.的潜在新种。其中29株细菌能够抑制甘蔗鞭黑粉菌活性,分别有12、14和14株细菌对甘蔗鞭黑粉菌菌丝生长、"+"担孢子增殖和"-"担孢子增殖表现出抑制活性,抑菌圈直径范围为7.54~21.81 mm,其中有9株细菌能够抑制2~3种甘蔗鞭黑粉菌的形态。【结论】广西茅尾海红树林保护区中无瓣海桑根际细菌具有较高的物种多样性,蕴藏着潜在新物种资源,且富含抑菌活性菌株。
【Objective】Diversity and inhibition activity of rhizospheric bacteria which came from Sonneratia apetala in Maowei Sea were studied to find out more potential new species and high-efficiency active materials,and provided reference for the further research on secondary metabolites of the rhizospheric bacteria of mangrove plant and the further development of new and efficient biological pesticides.【Method】The rhizosphere soil of S. apetala were collected from Maowei Sea mangrove reserve in Qinzhou,Guangxi. Six different media were employed to isolate the cultured bacteria.The species diversity was revealed through phylogenetic analysis of the 16 S r RNA gene sequence,and Oxford cup method was used to investigate the inhibitory activity of the rhizosphere bacteria against Sporisorium scitamineum.【Result】The97 strains of culturable bacteria were isolated from the rhizosphere of S. apetala.After comparing the 16 S r RNA sequence alignments,57 different strains of bacteria were obtained,which could be classified into 21 genera,15 families,9 orders,5 classes and 4 phyla. There was also a potential new species of Cellvibrio sp. The 29 strains could inhibit the activity of S. scitamineum. That there were 12,14 and 14 strains of bacteria exhibited inhibitory activity against mycelium growth,proliferation of"+"spores,and proliferation of"-"spores of S. scitamineum respectively. The diameter of inhibition zone was between 7.54 and 21.81,and 9 strains of which could inhibit the morphology of 2 to 3 species of S. scitamineum.【Conclusion】The rhizospheric bacteria of S. apetala which come from Maowei Sea mangrove reserve in Qinzhou,Guangxi have high species diversity,contain potential new species resources,and are rich in antibacterial activity strains.
【Objective】Diversity and inhibition activity of rhizospheric bacteria which came from Sonneratia apetala in Maowei Sea were studied to find out more potential new species and high-efficiency active materials,and provided reference for the further research on secondary metabolites of the rhizospheric bacteria of mangrove plant and the further development of new and efficient biological pesticides.【Method】The rhizosphere soil of S. apetala were collected from Maowei Sea mangrove reserve in Qinzhou,Guangxi. Six different media were employed to isolate the cultured bacteria.The species diversity was revealed through phylogenetic analysis of the 16 S r RNA gene sequence,and Oxford cup method was used to investigate the inhibitory activity of the rhizosphere bacteria against Sporisorium scitamineum.【Result】The97 strains of culturable bacteria were isolated from the rhizosphere of S. apetala.After comparing the 16 S r RNA sequence alignments,57 different strains of bacteria were obtained,which could be classified into 21 genera,15 families,9 orders,5 classes and 4 phyla. There was also a potential new species of Cellvibrio sp. The 29 strains could inhibit the activity of S. scitamineum. That there were 12,14 and 14 strains of bacteria exhibited inhibitory activity against mycelium growth,proliferation of"+"spores,and proliferation of"-"spores of S. scitamineum respectively. The diameter of inhibition zone was between 7.54 and 21.81,and 9 strains of which could inhibit the morphology of 2 to 3 species of S. scitamineum.【Conclusion】The rhizospheric bacteria of S. apetala which come from Maowei Sea mangrove reserve in Qinzhou,Guangxi have high species diversity,contain potential new species resources,and are rich in antibacterial activity strains.
引文
常涛,吴志强,黄亮亮,耿俊杰,朱召军,武文丽.2015.茅尾海红树林水域仔稚鱼群落结构及与主要环境因子关系[J].应用海洋学学报,34(2):219-226.[Chang T,Wu Z Q,Huang L L,Geng J J,Zhu Z J,Wu W L.2015.Community structures of fish larvae and its relation with environmental factors in the mangrove of Maoweihai Bay[J].Journal of Applied Oceanography,34(2):219-226.]
郭凯旋.2012.红树植物内生细菌的种群数量周年变化、盐度适应性及抗菌活性研究[D].湛江:广东海洋大学.[Guo K X.2012.Annual variation in population densities,adaptability to salinity and antimicrobial activity of endophytic bacteria from mangrove plants[D].Zhanjiang:Guangdong Ocean University.]
李菲,高程海,竺利波,余炼,覃媚,颜栋美.2016.茅尾海无瓣海桑内生细菌多样性及其细胞毒活性[J].微生物学报,56(4):689-697.[Li F,Gao C H,Zhu L B,Yu L,Qin M,Yan D M.2016.Diversity and cytotoxic activity of endophytic bacteria isolated from Sonneratia apetala of Maowei Sea[J].Acta Microbiologica Sinica,56(4):689-697.]
李菲,黄庶识,王伟,江蕾,米顺利,余炼.2018.海绵Pseudoceratina sp.共栖细菌多样性及其抑制甘蔗鞭黑粉菌活性研究[J].广西科学,25(1):87-93.[Li F,Huang S S,Wang W,Jiang L,Mi S L,Yu L.2018.Diversity of sponge-derived bacteria isolated from Pseudoceratina sp.against Sporisorium scitamineum in vitro[J].Guangxi Sciences,25(1):87-93.]
李倩茹,符夏梨.2009.红树林土壤微生物与土壤酶活性分析[J].广东农业科学,36(7):93-96.[Li Q R,Fu X L.2009.Soil microorganisms and activity of soil enzymes in mangrove[J].Guangdong Agricultural Sciences,36(7):93-96.]
李小群,李菲,李家怡,颜栋美,苏志维,高程海.2017.海芒果可培养细菌的分离鉴定及其抑制海洋鱼类致病菌活性研究[J].广西科学院学报,33(2):108-113.[Li X Q,Li F,Li J Y,Yan D M,Su Z W,Gao C H.2017.Identification and inhibition activity inhibit fish pathogens of cultivated bacteria isolated from Cerbera manghas[J].Journal of Guangxi Academy of Sciences,33(2):108-113.]
李逾.2012.三种红树植物共附生放线菌的分离及其抗病毒活性菌株的筛选[D].海口:海南大学.[Li Y.2012.Isolation and screening anti-viral actinomycetes from three mangrove[D].Haikou:Hainan University.]
梁碧怡,陆羽,王军,周翔,刘岚,林永成.2012.广东沿海原生态红树林环境放线菌分离及抗分枝杆菌活性评价[J].海洋通报,31(3):314-319.[Liang B Y,Lu Y,Wang J,Zhou X,Liu L,Lin Y C.2012.Isolation and screening of actinomycetes aginst Mycobacterium in Guangdong intact mangrove[J].Marine Science Bulletin,31(3):314-319.]
林巧玲,严玉宁,何红,孙兴涛,杨定祥.2017.红树植物老鼠簕内生细菌数量的时空动态变化[J].微生物学通报,44(9):2037-2042.[Lin Q L,Yan Y N,He H,Sun X T,Yang D X.2017.Spatiotemporal dynamic of endophytic bacteria numbers in Acanthus illicifolius[J].Microbiology China,44(9):2037-2042.]
卢乃会,严玉宁,何红,卢仕严,黄勤知.2013.红树植物秋茄内生细菌数量时空变化动态初步研究[J].广东海洋大学学报,33(4):7-10.[Lu N H,Yan Y N,He H,Lu S Y,Huang Q Z.2013.Spatiotemporal dynamic of endophytic bacteria in Kandelia candel[J].Journal of Guangdong Ocean University,33(4):7-10.]
缪承杜,庄令,林海鹏,洪葵.2008.海南文昌清澜港红树林真菌抗B16肿瘤细胞活性菌株的筛选[J].生物工程学报,24(6):975-979.[Miao C D,Zhuang L,Lin H P,Hong K.2008.Screening of cytotoxic activity against B16 tumor cell of mangrove fungi isolate from Qinglan Harbor in Hainan[J].Chinese Journal of Biotechnology,24(6):975-979.]
孙静,王素英,张德超.2014.海南红树林根系土壤中可培养细菌的多样性分析[J].海洋科学,38(7):27-33.[Sun J,Wang S Y,Zhang D C.2014.Diversity of culturable bacteria from the soil of root system of mangrove forest of Beigang Island in Hainan Province[J].Marine Sciences,38(7):27-33.]
吴家法,吴思婷,李智鸣,肖尚荣,吴霜,张政,杨立芳,龙寒,禤金彩,姜明国.2017.茅尾海红树林土壤可培养放线菌多样性及其抗尖孢镰刀菌活性分析[J].中国抗生素杂志,42(4):294-301.[Wu J F,Wu S T,Li Z M,Xiao SR,Wu S,Zhang Z,Yang L F,Long H,Xuan J C,Jiang M G.2017.Biodiversity and screening of culturable actinobacteria against Fusarium oxysporum isolated from mangrove soil in Maowei sea[J].Chinese Journal of Antibiotics,42(4):294-301.]
夏丽娟,张焜,黄华容,张占生.2014.5种红树根际土壤真菌和内生真菌的分离及抑菌活性的研究[J].中国农学通报,30(4):259-263.[Xia L J,Zhang K,Huang H R,Zhang Z S.2014.The antimicrobial activity of 5 kinds of mangrove fungi in Rhizosphere soil and endophytic fungi[J].Chinese Agricultural Science Bulletin,30(4):259-263.]
殷萌清,冯建祥,黄小芳,蔡中华,林光辉,周进.2017.天然及人工红树林土壤微生物群落结构分析[J].生态科学,36(5):1-10.[Yin M Q,Feng J X,Huang X F,Cai Z H,Lin G H,Zhou J.2017.Soil microbial community structure in natural and transplanted mangrove(Kandelia obovata)forests[J].Ecological Science,36(5):1-10.]
张偲,张长生,田新朋,王发左,李杰.2010.中国海洋微生物多样性研究[J].中国科学院院刊,25(6):651-658.[Zhang S,Zhang C S,Tian X P,Wang F Z,Li J.2010.The study of diversities of marine microbes in China[J].Bulletin of the Chinese Academy of Sciences,25(6):651-658.]
张永夏,王艳枝,王伟,李乐兴,陈红锋.2017.东江虎门入海口红树林的群落特征及生态恢复[J].江西农业学报,29(2):40-44.[Zhang Y X,Wang Y Z,Wang W,Li L X,Chen H F.2017.Community characteristics and ecological restoration of mangrove at estuary of Dongjiang River in Humen[J].Acta Agriculturae Jiangxi,29(2):40-44.]
周双清,黄小龙,黄东益,胡新文,陈吉良.2010.Chelex-100快速提取放线菌DNA作为PCR扩增模板[J].生物技术通报,26(2):123-125.[Zhou S Q,Huang X L,Huang DY,Hu X W,Chen J L.2010.A rapid method for extracting DNA from actinomycetes by Chelex-100[J].Biotechnology Bulletin,26(2):123-125.]
Keswani J,Whitman W B.2001.Relationship of 16S r RNAsequence similarity to DNA hybridization in prokaryotes[J].International Journal of Systematic&Evolutionary Microbiology,51(2):667-678.
Santoferrara L F,Grattepanche J D,Katz L A,Mcmanus G B.2014.Pyrosequencing for assessing diversity of eukaryotic microbes:Analysis of data on marine planktonic ciliates and comparison with traditional methods[J].Environmental Microbiology,16(9):2752-2763.
Takahashi Y,Omura S.2003.Isolation of new actinomycete strains for the screening of new bioactive compounds[J].Journal of General and Applied Microbiology,49(3):141.
Tamura K,Peterson D,Peterson N,Stecher G,Nei M,Kumar S.2011.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,28(10):2731-2739.
Zengler K,Toledo G,Rappe M,Elkins J,Mathur E J,Short JM,Keller M.2002.Cultivating the uncultured[J].Proceedings of the National Academy of Sciences of the United States of America,99(24):15681-15686.
郭凯旋.2012.红树植物内生细菌的种群数量周年变化、盐度适应性及抗菌活性研究[D].湛江:广东海洋大学.[Guo K X.2012.Annual variation in population densities,adaptability to salinity and antimicrobial activity of endophytic bacteria from mangrove plants[D].Zhanjiang:Guangdong Ocean University.]
李菲,高程海,竺利波,余炼,覃媚,颜栋美.2016.茅尾海无瓣海桑内生细菌多样性及其细胞毒活性[J].微生物学报,56(4):689-697.[Li F,Gao C H,Zhu L B,Yu L,Qin M,Yan D M.2016.Diversity and cytotoxic activity of endophytic bacteria isolated from Sonneratia apetala of Maowei Sea[J].Acta Microbiologica Sinica,56(4):689-697.]
李菲,黄庶识,王伟,江蕾,米顺利,余炼.2018.海绵Pseudoceratina sp.共栖细菌多样性及其抑制甘蔗鞭黑粉菌活性研究[J].广西科学,25(1):87-93.[Li F,Huang S S,Wang W,Jiang L,Mi S L,Yu L.2018.Diversity of sponge-derived bacteria isolated from Pseudoceratina sp.against Sporisorium scitamineum in vitro[J].Guangxi Sciences,25(1):87-93.]
李倩茹,符夏梨.2009.红树林土壤微生物与土壤酶活性分析[J].广东农业科学,36(7):93-96.[Li Q R,Fu X L.2009.Soil microorganisms and activity of soil enzymes in mangrove[J].Guangdong Agricultural Sciences,36(7):93-96.]
李小群,李菲,李家怡,颜栋美,苏志维,高程海.2017.海芒果可培养细菌的分离鉴定及其抑制海洋鱼类致病菌活性研究[J].广西科学院学报,33(2):108-113.[Li X Q,Li F,Li J Y,Yan D M,Su Z W,Gao C H.2017.Identification and inhibition activity inhibit fish pathogens of cultivated bacteria isolated from Cerbera manghas[J].Journal of Guangxi Academy of Sciences,33(2):108-113.]
李逾.2012.三种红树植物共附生放线菌的分离及其抗病毒活性菌株的筛选[D].海口:海南大学.[Li Y.2012.Isolation and screening anti-viral actinomycetes from three mangrove[D].Haikou:Hainan University.]
梁碧怡,陆羽,王军,周翔,刘岚,林永成.2012.广东沿海原生态红树林环境放线菌分离及抗分枝杆菌活性评价[J].海洋通报,31(3):314-319.[Liang B Y,Lu Y,Wang J,Zhou X,Liu L,Lin Y C.2012.Isolation and screening of actinomycetes aginst Mycobacterium in Guangdong intact mangrove[J].Marine Science Bulletin,31(3):314-319.]
林巧玲,严玉宁,何红,孙兴涛,杨定祥.2017.红树植物老鼠簕内生细菌数量的时空动态变化[J].微生物学通报,44(9):2037-2042.[Lin Q L,Yan Y N,He H,Sun X T,Yang D X.2017.Spatiotemporal dynamic of endophytic bacteria numbers in Acanthus illicifolius[J].Microbiology China,44(9):2037-2042.]
卢乃会,严玉宁,何红,卢仕严,黄勤知.2013.红树植物秋茄内生细菌数量时空变化动态初步研究[J].广东海洋大学学报,33(4):7-10.[Lu N H,Yan Y N,He H,Lu S Y,Huang Q Z.2013.Spatiotemporal dynamic of endophytic bacteria in Kandelia candel[J].Journal of Guangdong Ocean University,33(4):7-10.]
缪承杜,庄令,林海鹏,洪葵.2008.海南文昌清澜港红树林真菌抗B16肿瘤细胞活性菌株的筛选[J].生物工程学报,24(6):975-979.[Miao C D,Zhuang L,Lin H P,Hong K.2008.Screening of cytotoxic activity against B16 tumor cell of mangrove fungi isolate from Qinglan Harbor in Hainan[J].Chinese Journal of Biotechnology,24(6):975-979.]
孙静,王素英,张德超.2014.海南红树林根系土壤中可培养细菌的多样性分析[J].海洋科学,38(7):27-33.[Sun J,Wang S Y,Zhang D C.2014.Diversity of culturable bacteria from the soil of root system of mangrove forest of Beigang Island in Hainan Province[J].Marine Sciences,38(7):27-33.]
吴家法,吴思婷,李智鸣,肖尚荣,吴霜,张政,杨立芳,龙寒,禤金彩,姜明国.2017.茅尾海红树林土壤可培养放线菌多样性及其抗尖孢镰刀菌活性分析[J].中国抗生素杂志,42(4):294-301.[Wu J F,Wu S T,Li Z M,Xiao SR,Wu S,Zhang Z,Yang L F,Long H,Xuan J C,Jiang M G.2017.Biodiversity and screening of culturable actinobacteria against Fusarium oxysporum isolated from mangrove soil in Maowei sea[J].Chinese Journal of Antibiotics,42(4):294-301.]
夏丽娟,张焜,黄华容,张占生.2014.5种红树根际土壤真菌和内生真菌的分离及抑菌活性的研究[J].中国农学通报,30(4):259-263.[Xia L J,Zhang K,Huang H R,Zhang Z S.2014.The antimicrobial activity of 5 kinds of mangrove fungi in Rhizosphere soil and endophytic fungi[J].Chinese Agricultural Science Bulletin,30(4):259-263.]
殷萌清,冯建祥,黄小芳,蔡中华,林光辉,周进.2017.天然及人工红树林土壤微生物群落结构分析[J].生态科学,36(5):1-10.[Yin M Q,Feng J X,Huang X F,Cai Z H,Lin G H,Zhou J.2017.Soil microbial community structure in natural and transplanted mangrove(Kandelia obovata)forests[J].Ecological Science,36(5):1-10.]
张偲,张长生,田新朋,王发左,李杰.2010.中国海洋微生物多样性研究[J].中国科学院院刊,25(6):651-658.[Zhang S,Zhang C S,Tian X P,Wang F Z,Li J.2010.The study of diversities of marine microbes in China[J].Bulletin of the Chinese Academy of Sciences,25(6):651-658.]
张永夏,王艳枝,王伟,李乐兴,陈红锋.2017.东江虎门入海口红树林的群落特征及生态恢复[J].江西农业学报,29(2):40-44.[Zhang Y X,Wang Y Z,Wang W,Li L X,Chen H F.2017.Community characteristics and ecological restoration of mangrove at estuary of Dongjiang River in Humen[J].Acta Agriculturae Jiangxi,29(2):40-44.]
周双清,黄小龙,黄东益,胡新文,陈吉良.2010.Chelex-100快速提取放线菌DNA作为PCR扩增模板[J].生物技术通报,26(2):123-125.[Zhou S Q,Huang X L,Huang DY,Hu X W,Chen J L.2010.A rapid method for extracting DNA from actinomycetes by Chelex-100[J].Biotechnology Bulletin,26(2):123-125.]
Keswani J,Whitman W B.2001.Relationship of 16S r RNAsequence similarity to DNA hybridization in prokaryotes[J].International Journal of Systematic&Evolutionary Microbiology,51(2):667-678.
Santoferrara L F,Grattepanche J D,Katz L A,Mcmanus G B.2014.Pyrosequencing for assessing diversity of eukaryotic microbes:Analysis of data on marine planktonic ciliates and comparison with traditional methods[J].Environmental Microbiology,16(9):2752-2763.
Takahashi Y,Omura S.2003.Isolation of new actinomycete strains for the screening of new bioactive compounds[J].Journal of General and Applied Microbiology,49(3):141.
Tamura K,Peterson D,Peterson N,Stecher G,Nei M,Kumar S.2011.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,28(10):2731-2739.
Zengler K,Toledo G,Rappe M,Elkins J,Mathur E J,Short JM,Keller M.2002.Cultivating the uncultured[J].Proceedings of the National Academy of Sciences of the United States of America,99(24):15681-15686.