明党参内生菌的分离鉴定及其对金黄色葡萄球菌的抑制作用
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摘要
研究郑州市栽培明党参内生菌资源状况,筛选出对金黄色葡萄球菌具有拮抗作用的内生菌。采用平板培养法从明党参中分离内生细菌,通过混菌法和牛津杯双层平板扩散法筛选出对金黄色葡萄球菌具有明显抑制作用的菌株,并通过培养特征、形态特征、生化特征、全自动微生物质谱检测、16S rDNA基因序列分析和系统进化树分析对菌株进行了分类学鉴定。在明党参中共分离到29株内生细菌,通过初筛和复筛后,筛选出对金黄色葡萄球菌具有明显抑制作用的4株菌株NMY1、NMJ10、NMG10、PMG3,其中NMJ10对金黄色葡萄球菌的抑菌圈直径达20.3 mm,经鉴定菌株NMJ10和菌株PMG3为芽胞杆菌属新菌种(Bacillus spp.),菌株NMY1为枯草芽胞杆菌(B.subtilis),菌株NMG10为假蕈状芽胞杆菌(B.pseudomycoides)。明党参内生菌具有多样性,其中芽胞杆菌属为优势菌株,有拮抗金黄色葡萄球菌活性高的菌株存在,其代谢产物对金黄色葡萄球菌有较高抑制作用,为抗菌药源成分的开发提供了新的来源。
Resource conditions of endophytes in cultivated radix changii(Changium smyrnioides Wolff) in Zhengzhou City were investigated and screened endophytes that possessing antagonistic effect on Staphylococcus aureus. The endogenous bacteria were isolated from C. smyrnioides by plate culture method, strains with significant antagonistic effect on S. aureus were selected by mixed strains method and Oxford cup double plate diffusion method, and combined with culture characteristics, morphological characteristics, biochemical characteristics, automatic microbial mass spectrometry detection, 16 S rDNA gene sequence analysis and phylogenetic tree analysis, the strains were identified by taxonomic identification. Twenty-nine endophytic bacteria were isolated from C. smyrnioides. After initial screening and re-screening, four strains NMY1, NMJ10, NMG10, and PMG3 with significant antagonistic effect on S. aureus were selected. Among them the diameter of antibacterial ring of strain NMJ10 against S. aureus reaches 20.3 mm. According to the identification, strain NMJ10 and PMG3 were new strains of Bacillus(Bacillus spp.), strain NMY1 was initially identified as Bacillus subtilis, and strain NMG10 was B. pseudomycoides. Endophytes in C. smyrnioides had diversity and Bacillus were the dominant strains. There also exist strains with antagonism against S. aureus, and their metabolites have a high inhibitory effect against S. aureus, which can provide a new source to develop antibacterial medicines.
Resource conditions of endophytes in cultivated radix changii(Changium smyrnioides Wolff) in Zhengzhou City were investigated and screened endophytes that possessing antagonistic effect on Staphylococcus aureus. The endogenous bacteria were isolated from C. smyrnioides by plate culture method, strains with significant antagonistic effect on S. aureus were selected by mixed strains method and Oxford cup double plate diffusion method, and combined with culture characteristics, morphological characteristics, biochemical characteristics, automatic microbial mass spectrometry detection, 16 S rDNA gene sequence analysis and phylogenetic tree analysis, the strains were identified by taxonomic identification. Twenty-nine endophytic bacteria were isolated from C. smyrnioides. After initial screening and re-screening, four strains NMY1, NMJ10, NMG10, and PMG3 with significant antagonistic effect on S. aureus were selected. Among them the diameter of antibacterial ring of strain NMJ10 against S. aureus reaches 20.3 mm. According to the identification, strain NMJ10 and PMG3 were new strains of Bacillus(Bacillus spp.), strain NMY1 was initially identified as Bacillus subtilis, and strain NMG10 was B. pseudomycoides. Endophytes in C. smyrnioides had diversity and Bacillus were the dominant strains. There also exist strains with antagonism against S. aureus, and their metabolites have a high inhibitory effect against S. aureus, which can provide a new source to develop antibacterial medicines.
引文
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[2] 任安芝,高玉葆.植物内生真菌一类应用前景广阔的资源微生物[J].微生物学通报,2001,28(6):90-93.
[3] 陈向东.植物内生菌是有待深入开发的资源宝库[J].微生物学通报,2012,39(2):282-282.
[4] Saikkonen,Kari,W■li,et al.Evolution of endophyte-plant symbioses[J].Trends in Plant Science,2004,9(6):275-280.
[5] Hyde KD,Soytong K.The fungal endophyte dilemma[J].Fungal Diversity,2008,33(33):163-173.
[6] 刘守炉,叶锦生,陈重明,等.中国明党参属植物综合研究[J].植物研究,1991,11(2):75-83.
[7] 李松,肖玲.温肺化痰的明党参[J].首都食品与医药,2017,(3):55-55.
[8] 黄宝康,郑汉臣,张朝晖.明党参的研究概况[J].中药材,1998,(8):425-428.
[9] 邱英雄,傅承新.珍稀特有植物明党参的比较解剖学研究[J].浙江大学学报(农业与生命科学版),2000,26(4):427-432.
[10] 刘晓宁.珍稀药用植物明党参种质资源保护及快速繁殖研究[D].南京:南京中医药大学,2009.
[11] 盛海燕,常杰,殷现伟,等.濒危植物明党参种子散布和种子库动态研究[J].生物多样性,2002,10(3):269-273.
[12] Zhang Y,Chen JW,Xiang LI.Accumulation and Distribution of Furanocoumarins in Rare Medicinal Plants Changium smyrnioides Wolff[J].Natural Product Research & Development,2012,24(4):510-513.
[13] Qiu Y,Fu C.Studies on comparative anatomy of endangered plant Changium smyrnioides Wolff[J].Journal of Zhejiang Agricultural University,2000,26(4):427-432.
[14] Fang-Fang HU.Study on Tissue Culture of Changium smyrnioides Wolff Leaf[J].Journal of Anhui Agricultural Sciences,2007,35(8):2232-2233.
[15] Da BU,Duan ZF,En-Bin LI,et al.Study on γ-aminicAcid CumμLation from Roots of CμLtivated Changium smyrnioides Wolff in Jiangsu[J].Research & Practice on Chinese Medicines,2012,26(6):26-28.
[16] Wang M.Study on Anti-tumor Activity of Isoimperatorin in the Root Bark of Changium Smyrnioides Wolff in Vivo[J].Value Engineering,2016.
[17] 郑峰,周婷婷,孙倩男,等.金黄色葡萄球菌α-溶血素的研究进展[J].中华微生物学和免疫学杂志,2017,37(10):795-801.
[18] 徐振波,刘晓晨,李琳,等.金黄色葡萄球菌肠毒素在食源性微生物中的研究进展[J].现代食品科技,2013,(9):2317-2324.
[19] 冷冰峰.大蒜素对金黄色葡萄球菌外毒素表达的抑制作用[D].长春:吉林大学,2012.
[20] 赵智灵,刘学周,魏晓雨,等.人参可利用内生菌株的筛选和鉴定[J].中草药,2015,46(14):2143-2148.
[21] 刘学周,赵智灵,李绍宾,等.西洋参内生菌群落结构与多样性[J].微生物学报,2015,55(3):330-340.
[22] 刘学周,李绍宾,赵智灵,等.西洋参内生菌株的分离及拮抗活性菌株的筛选和鉴定[J].中草药,2014,45(22):3332-3336.
[23] 周磊,云宝仪,汪业菊,等.大黄素对金黄色葡萄球菌的抑菌作用机制[J].中国生物化学与分子生物学报,2011,V27(12):1156-1160.
[24] 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.
[25] An J,Zhu W,Liu Y,et al.Purification and characterization of a novel bacteriocin CAMT2 produced by Bacillus amyloliquefaciens,isolated from marine fish Epinephelus areolatus[J].Food Control,2015,51:278-282.
[26] 朱大恒,刘丽,刘雨松,等.芽胞杆菌LL17的分离鉴定及其对仓储烟叶霉变菌的抑制效果[J].烟草科技,2017,50(8):23-29.