1株亳菊内生菌的分离鉴定及其生物活性分析
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摘要
【目的】分离筛选具有抑菌、降解纤维素及抗氧化等功能的亳菊内生菌,为其在农业上的开发应用奠定基础。【方法】以株龄9个月的安徽亳菊为材料,分别取根、茎、叶、花4部分,采用平板培养法从中分离内生菌菌株,并在平板上筛选和检测分离菌株对玉米弯孢菌、黄瓜枯萎菌、小麦赤霉菌、串珠镰刀菌、瓜炭疽菌、茶叶轮斑菌等6种植物病原菌的抑菌活性,测定目标菌株BJF10对纤维素的降解活性和DPPH的清除能力,根据菌体形态特征和ITS序列系统发育树对菌株BJF10进行鉴定。【结果】从亳菊的根、茎、叶、花中共获得了56株内生菌菌株,其中菌株BJF10对6种植物供试病原菌均有较强的拮抗作用,抑菌率均在50%以上;菌株BJF10培养第4天的纤维素降解活性达到最强,培养3d时羧甲基纤维素酶(CMCase)和滤纸酶(FPase)的活力达到最高,分别为180.32和81.46U/mL;BJF10的7d培养上清液对DPPH的清除率可达67.7%;根据形态特征及ITS序列的系统发育分析,将菌株BJF10鉴定为柄孢壳菌属(Zopfiellasp.)。【结论】柄孢壳菌属亳菊内生真菌BJF10菌株具有拮抗植物病原菌、降解纤维素、抗氧化等生物活性。
【Objective】The paper aimed to isolate and screen endophytic fungi with ability of antifungus,antioxidation and cellulose degradation from Chrysanthemum morifolium cv.Boju.It may lay the foundation for further development and application in agriculture.【Method】The 9-month-old Chrysanthemum morifoliumcv.Boju was divided into roots,stems,leaves,and flower parts.Antagonism of endophytic fungus BJF10 against Curvularia lunata,Fusarium graminearum,Fusarium moniliforme,Fusarium oxysporum,Colletotrichum lagenarium and Pestalotiopsis theae were evaluated by plate confrontation assay.The CMC enzyme activity,FPA activity and ability of scavenging DPPH were also studied.The strain BJF10 was identified according to the morphological features and ITS sequence analysis.【Result】The endophytic fungus strain BJF10 has strong antagonistic effects on six plant pathogens and the inhibitory rate was above 50%.The strain reached the highest cellulase activity after incubation for four days.The CMC enzyme activity and FPA activity reached 180.32 and 81.46 U/mL,respectively.The DPPH scaveging reached 67.7% by fermentation supernatant of BJF10.The strain BJF10 was identified as Zopfiella sp.based on morphological characteristics and ITS sequence analysis.【Conclusion】The endophytic fungus BJF10 has good ability of resistance to plant pathogens,cellulose degradation,and antioxidation.
【Objective】The paper aimed to isolate and screen endophytic fungi with ability of antifungus,antioxidation and cellulose degradation from Chrysanthemum morifolium cv.Boju.It may lay the foundation for further development and application in agriculture.【Method】The 9-month-old Chrysanthemum morifoliumcv.Boju was divided into roots,stems,leaves,and flower parts.Antagonism of endophytic fungus BJF10 against Curvularia lunata,Fusarium graminearum,Fusarium moniliforme,Fusarium oxysporum,Colletotrichum lagenarium and Pestalotiopsis theae were evaluated by plate confrontation assay.The CMC enzyme activity,FPA activity and ability of scavenging DPPH were also studied.The strain BJF10 was identified according to the morphological features and ITS sequence analysis.【Result】The endophytic fungus strain BJF10 has strong antagonistic effects on six plant pathogens and the inhibitory rate was above 50%.The strain reached the highest cellulase activity after incubation for four days.The CMC enzyme activity and FPA activity reached 180.32 and 81.46 U/mL,respectively.The DPPH scaveging reached 67.7% by fermentation supernatant of BJF10.The strain BJF10 was identified as Zopfiella sp.based on morphological characteristics and ITS sequence analysis.【Conclusion】The endophytic fungus BJF10 has good ability of resistance to plant pathogens,cellulose degradation,and antioxidation.
引文
[1]王志伟,纪燕玲,陈永敢.植物内生菌研究及其科学意义[J].微生物学通报,2015,42(2):349-363.Wang Z W,Ji Y L,Chen Y G.Studies and biological significances of plant endophytes[J].Microbiology China,2015,42(2):349-363.
[2]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.Chen X D.Endophytic microorganisms are valuable resources worthy to be explored deeply[J].Microbiology China,2012,39(2):282.
[3]赫荣乔.植物内生菌成为我国当前微生物研究领域的热点[J].微生物学通报,2009,36(1):1.He R Q.Plant endophyte becomes one of hot topics in the current microbiological study in China[J].Microbiology China,2009,36(1):1.
[4]陈传文,孙前光,朱军,等.三种药用植物内生菌的分离及其抗肿瘤活性菌株的筛选[J].微生物学通报,2010,37(10):1462-1466.Chen C W,Sun Q G,Zhu J,et al.Isolation and screening for endophyte with antitumor activities from three medicinal plantss[J].Microbiology,2010,37(10):1462-1466.
[5]许进娇,宋萍,封磊,等.雷公藤内生细菌的促生作用及其对雷公藤甲素生成的影响[J].应用生态学报,2014,25(6):1681-1687.Xu J J,Song P,Feng L,et al.Growth-promoting effect and triptolide production regulation of endophytic bacteria from Tripterygium wilfordii[J].Chinese Journal of Applied Ecology,2014,25(6):1681-1687.
[6]Rudgers J A,Koslow J M,Clay K.Endophytic fungi alter relationships between diversity and ecosystem properties[J].Ecol Lett,2004,7(1):42-51.
[7]Saikkonen K.Review:forest structure and fungal endophytes[J].Fungal Biol Rev,2007,21(3):67-74.
[8]国家药典委员会.中华人民共和国药典:2010年版[M].北京:中国医药科技出版社,2010.National Pharmacopoeia Committee.People’s Republic of China Pharmacopoeia:2010[M].Beijing:China Medical Science and Technology Press,2010.
[9]颜晓航.亳菊的遗传学和药理学研究进展[J].亚太传统医药,2014,10(13):38-39.Yan X H.Advances in genetic and pharmacological studies of Dendranthema morifolium[J].Asia-Pacific Traditional Medicine,2014,10(13):38-39.
[10]李友连,王珊,朱晶晶,等.硫磺熏蒸对亳菊化学成分的影响[J].中国中药杂志,2015,40(13):2624-2628.Li Y L,Wang S,Zhu J J,et al.Study on effects of sulfur fumigationon chemical constituents of Chrysanthemum morifoliumcv.Boju[J].China Journal of Chinese Materia Medica,2015,40(13):2624-2628.
[11]纪薇,王瑞,狄娟,等.亳菊试管嫁接苗褐变生理机制研究[J].山西农业大学学报(自然科学版),2016,36(7):489-494.Ji W,Wang R,Di J,et al.Study on physiological mechanism of browning in vitro grafting of Chrysanthemum morifoliumcv.Boju[J].Journal of Shanxi Agricultural University(Natural Science Edition),2016,36(7):489-494.
[12]张伟,左亚锋,金传山,等.不同加工方法对亳菊中2种苯丙素类和6种黄酮类成分的影响[J].中国医院药学杂志,2017,37(2):103-107.ZhangW,Zuo Y F,Jin C S,et al.Effects of different processing methods on contents of 2phenylpropanoids and 6flavonoids in Boju[J].Chinese Journal of Hospital Pharmacy,2017,37(2):103-107.
[13]顾文杰,徐有权,徐培智,等.酸性土壤中高效半纤维素降解菌的筛选与鉴定[J].微生物学报,2012,52(10):1251-1259.Gu W J,Xu Y Q,Xu P Z,et al.Screening and identification of hemicellulose degrading microorganisms in acid soil[J].Acta Microbiologica Sinica,2012,52(10):1251-1259.
[14]孙盈,田永强,赵丽坤.纤维素酶的CMC酶活测定条件的研究[J].食品工业科技,2013,34(2):68-71.Sun Y,Tian Y Q,Zhao L K.Study on the optimum conditions of determination of cellulose activity[J].Science and Technology of Food Industry,2013,34(2):68-71.
[15]王志勇,刘秀娟.植物内生菌分离方法的研究现状[J].贵州农业科学,2014,42(1):152-155.Wang Z Y,Liu X J.Research status on separation methods of plant endophytes[J].Guizhou Agricultural Sciences,2014,42(1):152-155.
[16]刘欣.木霉菌对大豆几种病原真菌的拮抗作用及其耐药性的研究[D].黑龙江大庆:黑龙江八一农垦大学,2007.Liu X.Study on the antagonistic mechanism to pathogens of soybean root rot and preparation of Trichoderma spp[D].Daqing,Heilongjiang:Heilongjiang Bayi Agricultural University,2007.
[17]Brandwilliams W,Cuvelier M E,Berset C.Use of a free radical method to evaluate antioxidant activity[J].LWT-Food Sci Technol,1995,28(1):25-30.
[18]Li X,Li X M,Xu G M,et al.Antioxidant metabolites from marine alga-derived fungus Aspergilluswentii,EN-48[J].Phytochem Lett,2014,7(1):120-123.
[19]赵斌.微生物学实验教程[M].北京:高等教育出版社,2013.Zhao B.Microbiology experiment course[M].Beijing:Higher Education Press,2013.
[20]Hamilton C E,Gundel P E,Helander M,et al.Endophytic mediation of reactive oxygen species and antioxidant activity in plants:a review[J].Fungal Divers,2012,54(1):1-10.
[21]Reissinger A,Vilich V,Sikora R A.Detection of fungi in planta:effectiveness of surface sterilization methods[J].Mycological Research,2001,105(5):563-566.
[22]赵玉萍,杨娟.四种纤维素酶酶活测定方法的比较[J].食品研究与开发,2006,27(3):116-118.Zhao Y P,Yang J.Comparion of four cellulase activity measurement methods[J].Food Research and Development,2006,27(3):116-118.
[23]Yang J K,Zhang J J,Yu H Y,et al.Community composition and cellulase activity of cellulolytic bacteria from forest soils planted with broad-leaved deciduous and evergreen trees[J].Appl Microbiol Biotechnol,2014,98(3):1449-1458.
[24]AndréS,Monika S.Biology and biotechnology of Trichoderma[J].Appl Microbiol Biotechnol,2010,87(3):787-99.
[25]Miller J D,Adams G W,Sumarah M.Antifungal metabolites from fungal endophytes of Pinus strobus[J].Phytochemistry,2011,72(14/15):1833-1837.
[26]Qiu M,Zhang R,Xue C,et al.Application of bio-organic fertilizer can control Fusarium,wilt of cucumber plants by regulating microbial community of rhizosphere soil[J].Biol Fert Soils,2012,48(7):807-816.
[27]Huang X,Zhang N,Yong X,et al.Biocontrol of Rhizoctonia solani,damping-off disease in cucumber with Bacillus pumilus SQR-N43[J].Microbiol Res,2012,167(3):135-143.
[28]刘雅莉,黄一泓,武文斌,等.蒺藜内生真菌抗氧化活性菌株的筛选[J].天然产物研究与开发,2013,25(4):435-439.Liu Y L,Huang Y H,Wu W B,et al.Screening of antioxidant endophytic fungi isolated from Tribulus terrestris L.[J].Natural Product Research and Development,2013,25(4):435-439.
[29]陈金阳,陆儒涵,王玲,等.药用植物内生菌抗氧化活性研究进展[J].中草药,2016,47(20):3720-3727.Chen J Y,Lu R H,Wang L,et al.Recent progress instudy on anti-oxidant activity of endophytes in medicinal plants[J].Chinese Traditional and Herbal Drugs,2016,47(20):3720-3727.
[30]Harper J K,Arif A M,Ford E J,et al.Pestacin:a 1,3-dihydro isobenzofuran from Pestalotiopsis microspora,possessing antioxidant and antimycotic activities[J].Tetrahedron,2003,59(14):2471-2476.
[31]Zhao J T,Ma D H,Luo M,et al.In vitro,antioxidant activities and antioxidant enzyme activities in HepG2cells and main active compounds of endophytic fungus from pigeon pea[Cajanuscajan(L.)Millsp.][J].Food Res Int,2014,56(2):243-251.
[2]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.Chen X D.Endophytic microorganisms are valuable resources worthy to be explored deeply[J].Microbiology China,2012,39(2):282.
[3]赫荣乔.植物内生菌成为我国当前微生物研究领域的热点[J].微生物学通报,2009,36(1):1.He R Q.Plant endophyte becomes one of hot topics in the current microbiological study in China[J].Microbiology China,2009,36(1):1.
[4]陈传文,孙前光,朱军,等.三种药用植物内生菌的分离及其抗肿瘤活性菌株的筛选[J].微生物学通报,2010,37(10):1462-1466.Chen C W,Sun Q G,Zhu J,et al.Isolation and screening for endophyte with antitumor activities from three medicinal plantss[J].Microbiology,2010,37(10):1462-1466.
[5]许进娇,宋萍,封磊,等.雷公藤内生细菌的促生作用及其对雷公藤甲素生成的影响[J].应用生态学报,2014,25(6):1681-1687.Xu J J,Song P,Feng L,et al.Growth-promoting effect and triptolide production regulation of endophytic bacteria from Tripterygium wilfordii[J].Chinese Journal of Applied Ecology,2014,25(6):1681-1687.
[6]Rudgers J A,Koslow J M,Clay K.Endophytic fungi alter relationships between diversity and ecosystem properties[J].Ecol Lett,2004,7(1):42-51.
[7]Saikkonen K.Review:forest structure and fungal endophytes[J].Fungal Biol Rev,2007,21(3):67-74.
[8]国家药典委员会.中华人民共和国药典:2010年版[M].北京:中国医药科技出版社,2010.National Pharmacopoeia Committee.People’s Republic of China Pharmacopoeia:2010[M].Beijing:China Medical Science and Technology Press,2010.
[9]颜晓航.亳菊的遗传学和药理学研究进展[J].亚太传统医药,2014,10(13):38-39.Yan X H.Advances in genetic and pharmacological studies of Dendranthema morifolium[J].Asia-Pacific Traditional Medicine,2014,10(13):38-39.
[10]李友连,王珊,朱晶晶,等.硫磺熏蒸对亳菊化学成分的影响[J].中国中药杂志,2015,40(13):2624-2628.Li Y L,Wang S,Zhu J J,et al.Study on effects of sulfur fumigationon chemical constituents of Chrysanthemum morifoliumcv.Boju[J].China Journal of Chinese Materia Medica,2015,40(13):2624-2628.
[11]纪薇,王瑞,狄娟,等.亳菊试管嫁接苗褐变生理机制研究[J].山西农业大学学报(自然科学版),2016,36(7):489-494.Ji W,Wang R,Di J,et al.Study on physiological mechanism of browning in vitro grafting of Chrysanthemum morifoliumcv.Boju[J].Journal of Shanxi Agricultural University(Natural Science Edition),2016,36(7):489-494.
[12]张伟,左亚锋,金传山,等.不同加工方法对亳菊中2种苯丙素类和6种黄酮类成分的影响[J].中国医院药学杂志,2017,37(2):103-107.ZhangW,Zuo Y F,Jin C S,et al.Effects of different processing methods on contents of 2phenylpropanoids and 6flavonoids in Boju[J].Chinese Journal of Hospital Pharmacy,2017,37(2):103-107.
[13]顾文杰,徐有权,徐培智,等.酸性土壤中高效半纤维素降解菌的筛选与鉴定[J].微生物学报,2012,52(10):1251-1259.Gu W J,Xu Y Q,Xu P Z,et al.Screening and identification of hemicellulose degrading microorganisms in acid soil[J].Acta Microbiologica Sinica,2012,52(10):1251-1259.
[14]孙盈,田永强,赵丽坤.纤维素酶的CMC酶活测定条件的研究[J].食品工业科技,2013,34(2):68-71.Sun Y,Tian Y Q,Zhao L K.Study on the optimum conditions of determination of cellulose activity[J].Science and Technology of Food Industry,2013,34(2):68-71.
[15]王志勇,刘秀娟.植物内生菌分离方法的研究现状[J].贵州农业科学,2014,42(1):152-155.Wang Z Y,Liu X J.Research status on separation methods of plant endophytes[J].Guizhou Agricultural Sciences,2014,42(1):152-155.
[16]刘欣.木霉菌对大豆几种病原真菌的拮抗作用及其耐药性的研究[D].黑龙江大庆:黑龙江八一农垦大学,2007.Liu X.Study on the antagonistic mechanism to pathogens of soybean root rot and preparation of Trichoderma spp[D].Daqing,Heilongjiang:Heilongjiang Bayi Agricultural University,2007.
[17]Brandwilliams W,Cuvelier M E,Berset C.Use of a free radical method to evaluate antioxidant activity[J].LWT-Food Sci Technol,1995,28(1):25-30.
[18]Li X,Li X M,Xu G M,et al.Antioxidant metabolites from marine alga-derived fungus Aspergilluswentii,EN-48[J].Phytochem Lett,2014,7(1):120-123.
[19]赵斌.微生物学实验教程[M].北京:高等教育出版社,2013.Zhao B.Microbiology experiment course[M].Beijing:Higher Education Press,2013.
[20]Hamilton C E,Gundel P E,Helander M,et al.Endophytic mediation of reactive oxygen species and antioxidant activity in plants:a review[J].Fungal Divers,2012,54(1):1-10.
[21]Reissinger A,Vilich V,Sikora R A.Detection of fungi in planta:effectiveness of surface sterilization methods[J].Mycological Research,2001,105(5):563-566.
[22]赵玉萍,杨娟.四种纤维素酶酶活测定方法的比较[J].食品研究与开发,2006,27(3):116-118.Zhao Y P,Yang J.Comparion of four cellulase activity measurement methods[J].Food Research and Development,2006,27(3):116-118.
[23]Yang J K,Zhang J J,Yu H Y,et al.Community composition and cellulase activity of cellulolytic bacteria from forest soils planted with broad-leaved deciduous and evergreen trees[J].Appl Microbiol Biotechnol,2014,98(3):1449-1458.
[24]AndréS,Monika S.Biology and biotechnology of Trichoderma[J].Appl Microbiol Biotechnol,2010,87(3):787-99.
[25]Miller J D,Adams G W,Sumarah M.Antifungal metabolites from fungal endophytes of Pinus strobus[J].Phytochemistry,2011,72(14/15):1833-1837.
[26]Qiu M,Zhang R,Xue C,et al.Application of bio-organic fertilizer can control Fusarium,wilt of cucumber plants by regulating microbial community of rhizosphere soil[J].Biol Fert Soils,2012,48(7):807-816.
[27]Huang X,Zhang N,Yong X,et al.Biocontrol of Rhizoctonia solani,damping-off disease in cucumber with Bacillus pumilus SQR-N43[J].Microbiol Res,2012,167(3):135-143.
[28]刘雅莉,黄一泓,武文斌,等.蒺藜内生真菌抗氧化活性菌株的筛选[J].天然产物研究与开发,2013,25(4):435-439.Liu Y L,Huang Y H,Wu W B,et al.Screening of antioxidant endophytic fungi isolated from Tribulus terrestris L.[J].Natural Product Research and Development,2013,25(4):435-439.
[29]陈金阳,陆儒涵,王玲,等.药用植物内生菌抗氧化活性研究进展[J].中草药,2016,47(20):3720-3727.Chen J Y,Lu R H,Wang L,et al.Recent progress instudy on anti-oxidant activity of endophytes in medicinal plants[J].Chinese Traditional and Herbal Drugs,2016,47(20):3720-3727.
[30]Harper J K,Arif A M,Ford E J,et al.Pestacin:a 1,3-dihydro isobenzofuran from Pestalotiopsis microspora,possessing antioxidant and antimycotic activities[J].Tetrahedron,2003,59(14):2471-2476.
[31]Zhao J T,Ma D H,Luo M,et al.In vitro,antioxidant activities and antioxidant enzyme activities in HepG2cells and main active compounds of endophytic fungus from pigeon pea[Cajanuscajan(L.)Millsp.][J].Food Res Int,2014,56(2):243-251.