温郁金内生拮抗细菌B-11的分离及其抑菌活性
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摘要
【背景】植物内生菌广泛分布在自然界中,具有巨大的潜在开发与应用价值。【目的】对温郁金内生细菌进行分离鉴定,并从中筛选出具有生防潜能的菌株。【方法】采用常规组织分离法对温郁金根茎内生细菌进行分离并利用16S rRNA基因序列分析进行初步鉴定;采用平板对峙法以铁皮石斛炭疽病菌(Colletotrichum gloeosporioides)为供试菌株对温郁金内生细菌进行拮抗菌的筛选;通过形态学鉴定、生理生化鉴定以及16S rRNA基因序列分析,确定拮抗菌株B-11的分类地位;以6个不同属的植物病原真菌为供试菌株对拮抗菌株B-11的抑菌谱进行测定,并研究其对病原菌菌丝的影响;利用特异性平板和MALDI-TOF-MS检测技术对拮抗菌株B-11产生的抑菌物质进行检测;采用离体叶片接种法研究拮抗菌株B-11对铁皮石斛炭疽病菌的防治效果。【结果】从温郁金根茎中分离得到25株内生细菌菌株,这些细菌菌株分属于12个属,其中芽孢杆菌属为优势菌属,占分离菌株的28%;通过初筛获得8株对C.gloeosporioides有抑菌活性的菌株,其中菌株B-11的抑菌活性最强,经鉴定该菌为贝莱斯芽孢杆菌(Baclliusvelezensis);抑菌谱测定发现菌株B-11能够对供试的属于不同属的6种植物病原真菌的菌丝生长具有抑制作用,显微观察发现经对峙培养6 d后这6种病原菌的菌丝体出现畸形膨大、分枝增多等现象;特异性平板检测结果表明拮抗菌株B-11能够产生蛋白酶、β-葡聚糖酶和嗜铁素,但不产生几丁质酶;MALDI-TOF-MS检测结果表明拮抗菌株B-11能够产生伊枯草素、丰源素和表面活性素3种脂肽类抗生素,其中伊枯草素的产量最高;离体叶片接种实验表明,拮抗菌B-11的离心去菌发酵液对铁皮石斛炭疽病的防治效率可达64%。【结论】温郁金根茎含有丰富的内生细菌资源,其内生细菌菌株B-11有潜力作为开发抗真菌代谢物和新药物的重要微生物资源。
[Background] Endophytes are widely distributed in nature, and have great potential for further exploration and application. [Objective] To isolate and identify endophytic bacteria of Curcuma wenyujin and evaluate their antimicrobial activity. [Methods] Endophytic bacteria from rhizomeof C. wenyujin were separated using routine tissue-separation method and identified by 16 S rRNA gene sequences analysis. Their antimicrobial activity against Colletotrichum gloeosporioides was tested by confront-culture method. Strain B-11. C. gloeosporioides was identified through morphological, physiological, biochemical characteristics and 16 S rRNA gene sequences analysis. Its influence on the hyphae of 6 pathogenic fungi, belonging to different families, was detected. Its antimicrobial components were determined by 4 specific culture mediums and MALDI-TOF-MS. The control efficacy against C. gloeosporioides on Dendrobium officinale was analyzed by inoculation of leaves in vitro. [Results] Twenty-five strains of endophytic bacteria,belonging to 12 families, were isolated and Bacillus spp. were the dominant species account for 28%. Eight strains showed antagonistic effect against C. gloeosporioides. Bacillus velezensis strain B-11 inhibited the growth of 6 different pathogenic fungi, with the hyphae inflated, deformed and branches increased. It can produce diverse antifungal substances including protease, β-gluconase and siderophore and lipopeptide.Inoculation experiment in vitro found that the cell-free culture of it had a control efficacy of 64% against C.gloeosporioides on D. officinale. [Conclusion] Endophytic bacterium strain B-11 in the rhizome of Curcuma wenyujin is an efficient biocontrol agent with potential for antibiotics development.
[Background] Endophytes are widely distributed in nature, and have great potential for further exploration and application. [Objective] To isolate and identify endophytic bacteria of Curcuma wenyujin and evaluate their antimicrobial activity. [Methods] Endophytic bacteria from rhizomeof C. wenyujin were separated using routine tissue-separation method and identified by 16 S rRNA gene sequences analysis. Their antimicrobial activity against Colletotrichum gloeosporioides was tested by confront-culture method. Strain B-11. C. gloeosporioides was identified through morphological, physiological, biochemical characteristics and 16 S rRNA gene sequences analysis. Its influence on the hyphae of 6 pathogenic fungi, belonging to different families, was detected. Its antimicrobial components were determined by 4 specific culture mediums and MALDI-TOF-MS. The control efficacy against C. gloeosporioides on Dendrobium officinale was analyzed by inoculation of leaves in vitro. [Results] Twenty-five strains of endophytic bacteria,belonging to 12 families, were isolated and Bacillus spp. were the dominant species account for 28%. Eight strains showed antagonistic effect against C. gloeosporioides. Bacillus velezensis strain B-11 inhibited the growth of 6 different pathogenic fungi, with the hyphae inflated, deformed and branches increased. It can produce diverse antifungal substances including protease, β-gluconase and siderophore and lipopeptide.Inoculation experiment in vitro found that the cell-free culture of it had a control efficacy of 64% against C.gloeosporioides on D. officinale. [Conclusion] Endophytic bacterium strain B-11 in the rhizome of Curcuma wenyujin is an efficient biocontrol agent with potential for antibiotics development.
引文
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[9]You JM,Xiong K,Mu S,et al.Identification of endophytic bacteria BZJN1 and research on biological control of root rot of Atractylodes macrocephala[J].China Journal of Chinese Materia Medica,2018,43(3):478-483(in Chinese)游景茂,熊坤,穆森,等.内生细菌BZJN1的鉴定及对白术根腐病的生物防治研究[J].中国中药杂志,2018,43(3):478-483
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[15]Yan J,Qi NB,Wang SP,et al.Characterization of secondary metabolites of an endophytic fungus from Curcuma wenyujin[J].Current Microbiology,2014,69(5):740-744
[16]Chen L,Liang ZN,Zhu H.Research advances in the studies of plant entophytic[J].Biotechnology Bulletin,2015,31(8):30-34(in Chinese)陈龙,梁子宁,朱华.植物内生菌研究进展[J].生物技术通报,2015,31(8):30-34
[17]Huang HF,Zheng CJ,Mo ZR,et al.Antibacterial sesquiterpenoids from the petroleum ether extract of Curcuma wenyujin Dreg[J].Chemistry of Natural Compounds,2016,52(3):527-530
[18]Dong XZ,Cai MY.Common Bacterial System Identification Manual[M].Beijing:Science Press,2001(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001
[19]Matarese F,Sarrocco S,Gruber S,et al.Biocontrol of Fusarium head blight:interactions between Trichoderma and mycotoxigenic Fusarium[J].Microbiology,2012,158(1):98-106
[20]Zhao WJ,Fu B,Xu SY,et al.Antagonism of biocontrol bacteria SF1103 and SF1104 on wilt pathogen in Cucumber[J].Acta Agriculturae Boreali-occidentalis Sinica,2017,26(10):1537-1543(in Chinese)赵文娟,付博,徐升运,等.生防菌SF1103、SF1104对黄瓜枯萎病菌的拮抗作用[J].西北农业学报,2017,26(10):1537-1543
[21]Li XY,Mao ZC,Wu YX,et al.Rapid detection of cyclic lipopeptide metabolites from Bacillus[J].Acta Phytopathologica Sinica,2014,44(6):718-722(in Chinese)李兴玉,毛自朝,吴毅歆,等.芽孢杆菌环脂肽类次生代谢产物的快速检测[J].植物病理学报,2014,44(6):718-722
[22]Fang XM,Bai JL,You XF,et al.Endophytic bacteria diversity of Aspidistra Ker-Gawl.and their antimicrobial activities[J].Microbiology China,2018,45(4):836-847(in Chinese)方晓梅,柏菁璘,游雪甫,等.蜘蛛抱蛋属植物内生细菌的多样性及其抗菌活性筛选[J].微生物学通报,2018,45(4):836-847
[23]Wang YH,Wang H,Xu L,et al.Isolation and identification of endophytic fungi from Curcuma aromatica Salisb[J].Lishizhen Medicine and Materia Medica Research,2009,20(11):2803-2806(in Chinese)王艳红,汪洪,徐磊,等.温莪术内生真菌的分离及其分类鉴定[J].时珍国医国药,2009,20(11):2803-2806
[24]Ruíz-Garcia C,Béjar V,Martínez-Checa FI,et al.Bacillus velezensis sp.nov.,a surfactant-producing bacterium isolated from the river Velez in Malaga,southern Spain[J].International Journal of Systematic and Evolutionary Microbiology,2005,55(1):191-195
[25]Cai XC,Liu CH,Wang BT,et al.Genomic and metabolic traits endow Bacillus velezensis CC09 with a potential biocontrol agent in control of wheat powdery mildew disease[J].Microbiological Research,2017,196:89-94
[26]Li S,Du ST,Li CY.Screening and Identification of the antagonistic strain DL-59 of B.velezensis against A.brassicae and biocontrol efficiency[J].Frontiers of Agriculture in China,2011,5(4):581-587
[27]Nakamura N,Nakano K,Sugiura N,et al.A novel cyanobacteriolytic bacterium,Bacillus cereus,isolated from a Eutrophic Lake[J].Journal of Bioscience and Bioengineering,2003,95(2):179-184
[28]Wang J,Zhang L,Teng KL,et al.Cerecidins,novel lantibiotics from Bacillus cereus with potent antimicrobial activity[J].Applied and Environmental Microbiology,2014,80(8):2633-2643
[29]Xu T,Zhu TH,Li SJ.β-1,3-1,4-glucanase gene from Bacillus velezensis ZJ20 exerts antifungal effect on plant pathogenic fungi[J].World Journal of Microbiology and Biotechnology,2016,32(2):26
[30]Liu XY,Ren B,Chen M,et al.Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3[J].Applied Microbiology and Biotechnology,2010,87(5):1881-1893
[31]Zhang YG,Ni H,Zhou YY,et al.Effects of meteorological conditions on strawberry anthrax and drug control tests[J].Chinese Horticulture Abstracts,2016,32(8):51-54(in Chinese)张叶根,倪航,周洋洋,等.气象条件对草莓炭疽病的影响及药剂防治试验[J].中国园艺文摘,2016,32(8):51-54
[32]Huang L,Li QC,Hou Y,et al.Bacillus velezensis strain HYEB5-6 as a potential biocontrol agent against anthracnose on Euonymus japonicus[J].Biocontrol Science and Technology,2017,27(5):636-653
[2]Redecker D,Kodner R,Graham LE.Glomalean fungi from the Ordovician[J].Science,2000,289(5486):1920-1921
[3]Stierle A,Strobel G,Stierle D.Taxol and taxane production by Taxomyces andreanae,an endophytic fungus of Pacific yew[J].Science,1993,260(5105):214-216
[4]Gamboa MA,Laureano S,Bayman P.Measuring diversity of endophytic fungi in leaf fragments:does size matter?[J].Mycopathologia,2003,156(1):41-45
[5]W?li PR,Ahlholm JU,Helander M,et al.Occurrence and genetic structure of the systemic grass endophyte Epichloёfestucae in fine Fescue populations[J].Microbial Ecology,2007,53(1):20-29
[6]Zhang WR,Guo XC,Li Q,et al.Antimicrobial activity of endophytic fungi isolated from leaf and fruit of Eucommia ulmoides[J].Chinese Traditional and Herbal Drugs,2016,47(16):2921-2926(in Chinese)张维瑞,郭秀春,李钦,等.杜仲叶和果实中内生真菌的分离及抑菌活性[J].中草药,2016,47(16):2921-2926
[7]Liu L,Pan F,Yang YB,et al.Isolation and identification of fungal endophytes from Ophiopogon japonicus and their antimicrobial activities[J].Chinese Traditional and Herbal Drugs,2016,47(8):1382-1391(in Chinese)刘雷,潘峰,杨远兵,等.川麦冬内生真菌分离和鉴定及抑菌活性初步研究[J].中草药,2016,47(8):1382-1391
[8]Zhang ZJ,Han JX,Liu XZ,et al.Optimization of B16endophytic strain culture for its control of ginseng Cylindrocarpon destructans[J].Microbiology China,2017,44(11):2698-2707(in Chinese)张正杰,韩金秀,刘学周,等.内生菌株B16发酵条件优化及其对人参锈腐病的防效[J].微生物学通报,2017,44(11):2698-2707
[9]You JM,Xiong K,Mu S,et al.Identification of endophytic bacteria BZJN1 and research on biological control of root rot of Atractylodes macrocephala[J].China Journal of Chinese Materia Medica,2018,43(3):478-483(in Chinese)游景茂,熊坤,穆森,等.内生细菌BZJN1的鉴定及对白术根腐病的生物防治研究[J].中国中药杂志,2018,43(3):478-483
[10]Chinese Pharmacopoeia Commission.Chinese Pharmacopoeia[M].Beijing:China Medical Science Press,2015(in Chinese)国家药典委员会.中华人民共和国药典[M].北京:中国医药科技出版社,2015
[11]Lou Y,Zhao F,He H,et al.Guaiane-type sesquiterpenes from Curcuma wenyujin and their inhibitory effects on nitric oxide production[J].Journal of Asian Natural Products Research,2009,11(8):737-747
[12]Yang L,Wei DD,Chen Z,et al.Reversal of multidrug resistance in human breast cancer cells by Curcuma wenyujin and Chrysanthemum indicum[J].Phytomedicine,2011,18(8/9):710-718
[13]Zeng X,Lian ML,Mao BZ.The research progress in chemical components,pharmacological activity and main pathogenic diseases in Curcuma wenyujin[J].Pharmaceutical Biotechnology,2017,24(6):554-560(in Chinese)曾欣,练美林,毛碧增.温郁金化学成分、药理作用及病害研究进展[J].药物生物技术,2017,24(6):554-560
[14]Yan J,Qi N,Wang S,et al.Diversity and antimicrobial activity of endophytic fungi from traditional medicinal plant Curcuma wenyujin[J].Journal of Pure and Applied Microbiology,2014,8(2):1701-1705
[15]Yan J,Qi NB,Wang SP,et al.Characterization of secondary metabolites of an endophytic fungus from Curcuma wenyujin[J].Current Microbiology,2014,69(5):740-744
[16]Chen L,Liang ZN,Zhu H.Research advances in the studies of plant entophytic[J].Biotechnology Bulletin,2015,31(8):30-34(in Chinese)陈龙,梁子宁,朱华.植物内生菌研究进展[J].生物技术通报,2015,31(8):30-34
[17]Huang HF,Zheng CJ,Mo ZR,et al.Antibacterial sesquiterpenoids from the petroleum ether extract of Curcuma wenyujin Dreg[J].Chemistry of Natural Compounds,2016,52(3):527-530
[18]Dong XZ,Cai MY.Common Bacterial System Identification Manual[M].Beijing:Science Press,2001(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001
[19]Matarese F,Sarrocco S,Gruber S,et al.Biocontrol of Fusarium head blight:interactions between Trichoderma and mycotoxigenic Fusarium[J].Microbiology,2012,158(1):98-106
[20]Zhao WJ,Fu B,Xu SY,et al.Antagonism of biocontrol bacteria SF1103 and SF1104 on wilt pathogen in Cucumber[J].Acta Agriculturae Boreali-occidentalis Sinica,2017,26(10):1537-1543(in Chinese)赵文娟,付博,徐升运,等.生防菌SF1103、SF1104对黄瓜枯萎病菌的拮抗作用[J].西北农业学报,2017,26(10):1537-1543
[21]Li XY,Mao ZC,Wu YX,et al.Rapid detection of cyclic lipopeptide metabolites from Bacillus[J].Acta Phytopathologica Sinica,2014,44(6):718-722(in Chinese)李兴玉,毛自朝,吴毅歆,等.芽孢杆菌环脂肽类次生代谢产物的快速检测[J].植物病理学报,2014,44(6):718-722
[22]Fang XM,Bai JL,You XF,et al.Endophytic bacteria diversity of Aspidistra Ker-Gawl.and their antimicrobial activities[J].Microbiology China,2018,45(4):836-847(in Chinese)方晓梅,柏菁璘,游雪甫,等.蜘蛛抱蛋属植物内生细菌的多样性及其抗菌活性筛选[J].微生物学通报,2018,45(4):836-847
[23]Wang YH,Wang H,Xu L,et al.Isolation and identification of endophytic fungi from Curcuma aromatica Salisb[J].Lishizhen Medicine and Materia Medica Research,2009,20(11):2803-2806(in Chinese)王艳红,汪洪,徐磊,等.温莪术内生真菌的分离及其分类鉴定[J].时珍国医国药,2009,20(11):2803-2806
[24]Ruíz-Garcia C,Béjar V,Martínez-Checa FI,et al.Bacillus velezensis sp.nov.,a surfactant-producing bacterium isolated from the river Velez in Malaga,southern Spain[J].International Journal of Systematic and Evolutionary Microbiology,2005,55(1):191-195
[25]Cai XC,Liu CH,Wang BT,et al.Genomic and metabolic traits endow Bacillus velezensis CC09 with a potential biocontrol agent in control of wheat powdery mildew disease[J].Microbiological Research,2017,196:89-94
[26]Li S,Du ST,Li CY.Screening and Identification of the antagonistic strain DL-59 of B.velezensis against A.brassicae and biocontrol efficiency[J].Frontiers of Agriculture in China,2011,5(4):581-587
[27]Nakamura N,Nakano K,Sugiura N,et al.A novel cyanobacteriolytic bacterium,Bacillus cereus,isolated from a Eutrophic Lake[J].Journal of Bioscience and Bioengineering,2003,95(2):179-184
[28]Wang J,Zhang L,Teng KL,et al.Cerecidins,novel lantibiotics from Bacillus cereus with potent antimicrobial activity[J].Applied and Environmental Microbiology,2014,80(8):2633-2643
[29]Xu T,Zhu TH,Li SJ.β-1,3-1,4-glucanase gene from Bacillus velezensis ZJ20 exerts antifungal effect on plant pathogenic fungi[J].World Journal of Microbiology and Biotechnology,2016,32(2):26
[30]Liu XY,Ren B,Chen M,et al.Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3[J].Applied Microbiology and Biotechnology,2010,87(5):1881-1893
[31]Zhang YG,Ni H,Zhou YY,et al.Effects of meteorological conditions on strawberry anthrax and drug control tests[J].Chinese Horticulture Abstracts,2016,32(8):51-54(in Chinese)张叶根,倪航,周洋洋,等.气象条件对草莓炭疽病的影响及药剂防治试验[J].中国园艺文摘,2016,32(8):51-54
[32]Huang L,Li QC,Hou Y,et al.Bacillus velezensis strain HYEB5-6 as a potential biocontrol agent against anthracnose on Euonymus japonicus[J].Biocontrol Science and Technology,2017,27(5):636-653