球孢白僵菌(Beauveria bassiana)NO6的分离、鉴定与除草活性
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摘要
旨在研究土壤真菌NO6发酵液中对杂草野燕麦具有抑制作用的次生代谢产物。从坎布拉土壤中分离纯化微生物菌株,利用培养皿分析法测定其次级代谢产物除草活性,利用菌落形态和ITS序列对除草活性最高的菌株进行鉴定,进而利用正相硅胶柱色谱、TLC和半制备HPLC方法对其代谢产物中的除草活性物质进行分离纯化。结果表明,共分离获得9株微生物菌株,其中菌株NO6对野燕麦的除草活性最高,将其鉴定为球孢白僵菌(Beauveria bassiana)。发酵液经乙酸乙酯萃取、硅胶柱层析和半制备HPLC分离纯化得到保留时间为66.868min的除草活性物质3。
In order to study the herbicidal activity of a soil fungus NO6,our experiment isolated microbial strains from Kanbula soil,using Petri dish analysis to measure the herbicidal activity of strains second metabolite.The ITS sequence and colonial morphology were used to identified the strain having the highest herbicidal activity,and the second metabolites were repeatedly purified by column chromatography on silica gel,TLC and semi-preparative HPLC.The results indicated that 9 microbial strains with herbicidal activity were isolated,and the strain NO6 had the highest activity on Avena fatua,so it was identified as Beauveria bassiana.The substance 3 was isolated from fermented liquid by ethyl acetate extraction,silica gel column chromatography and semi-preparative HPLC purification,with the retention time of 66.868 min.The research concluded that the strain NO6 had the potential to be developed as a micro-herbicide,and the experiment laid a foundation for purifying active substances produced by the second metabolites and developing a new fungal herbicide.
In order to study the herbicidal activity of a soil fungus NO6,our experiment isolated microbial strains from Kanbula soil,using Petri dish analysis to measure the herbicidal activity of strains second metabolite.The ITS sequence and colonial morphology were used to identified the strain having the highest herbicidal activity,and the second metabolites were repeatedly purified by column chromatography on silica gel,TLC and semi-preparative HPLC.The results indicated that 9 microbial strains with herbicidal activity were isolated,and the strain NO6 had the highest activity on Avena fatua,so it was identified as Beauveria bassiana.The substance 3 was isolated from fermented liquid by ethyl acetate extraction,silica gel column chromatography and semi-preparative HPLC purification,with the retention time of 66.868 min.The research concluded that the strain NO6 had the potential to be developed as a micro-herbicide,and the experiment laid a foundation for purifying active substances produced by the second metabolites and developing a new fungal herbicide.
引文
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[16]邵高祥,束长龙,耿丽丽,等.茶卡盐湖中耐盐抗真菌芽孢杆菌的分离和鉴定[J].中国生物防治学报,2016,32(4):440-448.SHAO G X,SHU CH L,GENG L L,et al.Isolation and identification of antifungal Bacillus with salt-tolerant in Caka Salt Lake[J].Chinese Journal of Biological Control,2016,32(4):440-448.
[17]邹辉琴,张小平,彭卫红.3类重要真菌生物活性物质及其研究方法概述[J].菌物研究,2007,5(2):119-124.ZOU H Q,ZHANG X P,PENG W H.Summarization on the advancement and research methods of three main fungal bioactive compounds[J].Journal of Fungal Research,2007,5(2):119-124.
[18]靳丽萍,张蕴,徐晓,等.黄蜻幼虫肠道真菌QTYC-51的鉴定及其除草活性成分[J].微生物学报,2016,56(12):1869-1875.JIN L P,ZHANG Y,XU X,et al.Identification and phytotoxic activity of fungus QTYC-51from the gut of Pantala flavescens larvae[J].Acta Microbiologica Sinica,2016,56(12):1869-1875.
[19]周瑜,王丽云,魏刚,等.放线菌SPRI-709084次级代谢产物的提取分离及结构鉴定[J].化学与生物工程,2009,26(6):66-68.ZHOU Y,WANG L Y,WEI G,et al.The extraction,separation and structural identification of secondary metabolites of Actinomycete SPRI-709084[J].Chemistry&Bioengineering,2009,26(6):66-68.
[20]薛章荣,徐文平,陈杰,等.浅灰链霉菌CGMCC1370及其代谢产物除草活性研究[J].农药学学报,2011,13(2):155-161.XUE ZH R,XU W P,CHEN J,et al.Study of Streptomyces griseolus CGMCC 1370and its herbicidal metabolite[J].Chinese Journal of Pesticide Science,2011,13(2):155-161.
[21]邵明伟,靳丽萍,张应烙.黄蜻幼虫肠道菌QTYC01除草活性代谢产物的分离和鉴定[J].微生物学报,2016,56(9):1513-1520.SHAO M W,JIN L P,ZHANG Y L.Phytotoxic metabolite from QTYC01,a fungus residing in the gut of Pantala flavescens larvae[J].Acta Microbiologica Sinica,2016,56(9):1513-1520.
[22]程亮,罗明明,吴继纲,等.海洋放线菌Y12-26中抗真菌活性代谢产物的分离纯化与结构鉴定[J].中国抗生素杂志,2016,42(8):631-638.CHENG L,LUO M M,WU J G,et al.Purification and structure elucidation of antifungal bioactive metabolites frommarine actinomycete Y12-26[J].Chinese Journal of Antibiotics,2016,42(8):631-638.
[23]李晓彬.5株内生真菌的化学成分及其生物活性研究[D].济南:山东大学药学院,2014.LI X B.Chemical investigations of five strains of endophytic fungi and their biological activities[D].Jinan:School of Pharmaceutical Science,Shandong University,2014.
[2]赵威,王艳杰,李琳,等.野燕麦繁殖和抗逆特性及其对小麦的他感效应研究[J].中国农业生态学报,2017,25(11):1684-1692.ZHAO W,WANG Y J,LI L,et al.Reproductive and stress resistance characteristics of wild oat and its allelopathic effects on common wheat[J].Chinese Journal of Eco-Agriculture,2017,25(11):1684-1692.
[3]CZAJA K,GORALCZYK K,STRUCINSKIP,et al.Biopesticides-towards increased consumer safety in the European Union[J].Pesticide Management Science,2015,71(1):3-6.
[4]陈世国,强胜.生物除草剂研究与开发的现状及未来的发展趋势[J].中国生物防治学报,2015,31(5):770-779.CHENG SH G,QIANG SH.The status and future directions of bioherbicide study and development[J].Chinese Journal of Biological Control,2015,31(5):770-779.
[5]孔涛,张德胜,徐慧,等.盐碱地及其改良过程中土壤微生物生态特征研究进展[J].土壤,2014,46(4):581-588.KONG T,ZHANG D SH,XU H,et al.Microbial ecological characteristics of Alkaline-saline lands and its amelioration process:A review[J].Soils,2014,46(4):581-588.
[6]唐永红,曹庸,卢成瑛,等.特殊生境微生物及其活性代谢产物研究进展[J].微生物学通报,2006,33(4):163-166.TANG Y H,CAO Y,LU CH Y,et al.Research progress of the active metabolites of special eco-environment microorganisms[J].Microbiology China,2006,33(4):163-166.
[7]方中达.植病研究方法[M].第3版.北京:中国农业出版社,1998:12-164.FANG ZH D.Plant Pathology Research Methods[M].3rd version.Beijing:China Agriculture Press,1998:12-164.
[8]刘学,顾宝根.农药生物活性测试标准操作规范·除草剂卷[M].北京:化学工业出版社,2016:120-122.LIU X,GU B G.Pesticide Bioassay Testing Standard Operating Procedure(Herbicide Volume)[M].Beijing:Chemical Industry Press,2016:120-122.
[9]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979.WEI J CH.Manual of Fungi Identification[M].Shanghai:Shanghai Science&Technology Press,1979.
[10]REHNER S A,BUCKLEY E P.Isolation and characterization of microsatellite loci from the entomopathogenic fungus Beauveria bassiana(Ascomycota:Hypocreales)[J].Molecular Ecology Notes,2003,3(3):409-411.
[11]BROWNBRIDGE M,REAY S D,NELSON T L,et al.Persistence of Beauveria bassiana(Ascomycota:Hypocreales)as an endophyte following inoculation of radiata pine seed and seedlings[J].Biological Control,2012,61(3):1195-2000.
[12]OWNLEY B H,PEREIRA R M,KLINGEMAN W E,et al.Beauveria bassiana,a dual purpose biocontrol organism,with activity against insect pests and plant pathogens[C]//LARTEY R T,CAESAR A J.Emerging concepts in plant health management 2004.Trivandrum,2004:255-269.
[13]GURULINGAPPA P,MCGEE P A,SWORD G.Endophytic Lecanicillium lecanii and Beauveria bassiana reduce the survival and fecundity of Aphis gossypii following contact with conidia and secondary metabolites[J].Crop Protection,2011,30(3):349-353.
[14]丁壮,李德海,顾谦群,等.极地微生物次级代谢产物及其活性研究进展[J].中国抗生素杂志,2014,39(1):6-13.DING ZH,LI D H,GU Q Q,et al.Research progress of polar microorganisms secondary metabolites and their bioactivities[J].Chinese Journal of Antibiotics,2014,39(1):6-13.
[15]李江,李光友.极端微生物-生物活性物质的新源泉[J].自然杂志,2011,33(5):275-279.LI J,LI G Y.Extremophilic microorganism:new resources of bioactive subatances[J].Chinese Journal of Nature,2011,33(5):275-279.
[16]邵高祥,束长龙,耿丽丽,等.茶卡盐湖中耐盐抗真菌芽孢杆菌的分离和鉴定[J].中国生物防治学报,2016,32(4):440-448.SHAO G X,SHU CH L,GENG L L,et al.Isolation and identification of antifungal Bacillus with salt-tolerant in Caka Salt Lake[J].Chinese Journal of Biological Control,2016,32(4):440-448.
[17]邹辉琴,张小平,彭卫红.3类重要真菌生物活性物质及其研究方法概述[J].菌物研究,2007,5(2):119-124.ZOU H Q,ZHANG X P,PENG W H.Summarization on the advancement and research methods of three main fungal bioactive compounds[J].Journal of Fungal Research,2007,5(2):119-124.
[18]靳丽萍,张蕴,徐晓,等.黄蜻幼虫肠道真菌QTYC-51的鉴定及其除草活性成分[J].微生物学报,2016,56(12):1869-1875.JIN L P,ZHANG Y,XU X,et al.Identification and phytotoxic activity of fungus QTYC-51from the gut of Pantala flavescens larvae[J].Acta Microbiologica Sinica,2016,56(12):1869-1875.
[19]周瑜,王丽云,魏刚,等.放线菌SPRI-709084次级代谢产物的提取分离及结构鉴定[J].化学与生物工程,2009,26(6):66-68.ZHOU Y,WANG L Y,WEI G,et al.The extraction,separation and structural identification of secondary metabolites of Actinomycete SPRI-709084[J].Chemistry&Bioengineering,2009,26(6):66-68.
[20]薛章荣,徐文平,陈杰,等.浅灰链霉菌CGMCC1370及其代谢产物除草活性研究[J].农药学学报,2011,13(2):155-161.XUE ZH R,XU W P,CHEN J,et al.Study of Streptomyces griseolus CGMCC 1370and its herbicidal metabolite[J].Chinese Journal of Pesticide Science,2011,13(2):155-161.
[21]邵明伟,靳丽萍,张应烙.黄蜻幼虫肠道菌QTYC01除草活性代谢产物的分离和鉴定[J].微生物学报,2016,56(9):1513-1520.SHAO M W,JIN L P,ZHANG Y L.Phytotoxic metabolite from QTYC01,a fungus residing in the gut of Pantala flavescens larvae[J].Acta Microbiologica Sinica,2016,56(9):1513-1520.
[22]程亮,罗明明,吴继纲,等.海洋放线菌Y12-26中抗真菌活性代谢产物的分离纯化与结构鉴定[J].中国抗生素杂志,2016,42(8):631-638.CHENG L,LUO M M,WU J G,et al.Purification and structure elucidation of antifungal bioactive metabolites frommarine actinomycete Y12-26[J].Chinese Journal of Antibiotics,2016,42(8):631-638.
[23]李晓彬.5株内生真菌的化学成分及其生物活性研究[D].济南:山东大学药学院,2014.LI X B.Chemical investigations of five strains of endophytic fungi and their biological activities[D].Jinan:School of Pharmaceutical Science,Shandong University,2014.
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