炭团菌抑菌活性成分及其对松枯梢病菌的影响
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摘要
本文运用植物病理学、分子生物学,生物化学、有机化学等方法,对炭团菌菌丝体发酵液中抑菌活性物质对樟子松枯梢病菌的影响、抑菌活性成分分离纯化和结构鉴定、抑菌活性物质生态安全性检验、抑菌活性物质稳定性及炭团菌与樟子松枯梢病菌分类的分子生物学验证进行研究。
(1)应用通用引物ITS1与ITS4从试验菌株中分别扩增出目的片段617bp和544bp,GenBank登陆号分别为EU744544和EU796887。通过GenBank中BLAST,确认试验菌株分别为Hypoxylon perforatum和Sphaeropsis sapinea。
(2)炭团菌菌丝体发酵液中活性物质的提取采用水提和酯提2种方式。水提方式采用直接提取法和超声波提取法2种方法,酯提方式选择正丁醇、乙酸乙酯、乙醇3种萃取剂。各提取物对病原菌的抑菌活性研究选用菌丝生长抑制率和孢子萌发抑制率2个指标,对病原菌菌丝生长的抑制测定采用生长速率法,对病原菌孢子萌发的抑制试验采用悬滴法。结果表明炭团菌菌丝体发酵液5种提取物对樟子松枯梢病菌均有一定的抑菌活性,其中乙酸乙酯提取物对病原菌菌丝生长的抑制率最高,达到73.20%;乙酸乙酯提取物、超声波提取物、正丁醇提取物对病原菌孢子萌发抑制率均超过90%。
(3)采用生物活性跟踪技术,运用柱层析、TLC等方法对发酵液提取物中的抑菌活性成分进行了分离、纯化。结果表明该提取物中有3个组分,组分Ⅰ的抑菌率最高(72.94%)。综合气质联用(GC-MS)的检测结果,组分Ⅰ含3种化合物,其中得率最高的为对甲氧基肉桂酸甲酯(57.952%),其他两个化合物分别为邻苯二甲酸二异丁酯(得率13.592%)和邻苯二甲酸二丁酯(得率28.455%)。
(4)炭团菌液体培养菌液乙酸乙酯提取物稳定性的研究选用紫外线、温度、pH值、氧化剂与还原剂以及贮存时间5个因子。乙酸乙酯提取物对自然环境下的紫外线、温度具有很高的稳定性,并且具有一定的抗氧化还原能力和良好的耐热性,长期贮存不影响其抑菌活性。
(5)小鼠毒性试验中未发生小鼠死亡情况。炭团菌发酵液乙酸乙酯提取物对小鼠的半致死剂量(LD_(50))＞13200mg/kg,按照我国化学物质的急性毒性分级标准,该提取物为微毒。
The effects of inhibiting active substance extracted from culture liquid of Hypoxylon perforatum on Sphaeropsis sapinea;isolation,purification and structure identification of inhibiting active substance; ecological safety test and stability of inhibiting active substance;the rDNA ITS sequences of Hypoxylon perforatum and Sphaeropsis sapinea were studied by methods of Phytopathology, Molecular Biology,Biochemical and Organic Chemical.The results showed as follows:
(1) rDNA ITS sequences were amplified from Hypoxylon perforatum and Sphaeropsis sapinea strain with the universal primers ITS1 and ITS4.rDNA ITS sequence lenth of Hypoxylon perforatum is 617bp and GenBank Accession is EU744544;rDNA ITS sequence lenth of Sphaeropsis sapinea is 544 bp and GenBank Accession is EU796887.Through the BLAST In GenBank,the strains were confirmed Hypoxylon perforatum and Sphaeropsis sapinea.
(2) Methods of water-extract and ester-extract were used for extracting active substances from culture liquid of Hypoxylon perforatum.Method of water-extract included two ways,one is extracting directly from cultural liquid,and the others is extracting by ultrasonic.N-butyl alcohol, ethyl acetate and alcohol were used as extractant in ester-extract test.Growth-inhibiting rate and germination-inhibiting rate were used as the index of antifungal activity.Growth rate method was used for determining growth-inhibiting rate and pendent drop method for determining germination-inhibiting rate.The results is that five extracts from culture liquid of Hypoxylon perforatum all have definite antifungal activities to Sphaeropsis sapinea growth and germination.Ethyl acetate-extract is the best one on inhibiting Sphaeropsis sapinea growth,the growth-inhibiting rate is 73.20%;the germination-inhibiting rates of ethyl acetate-extract,ultrasonic-extract and n-butyl alcohol-extract are all over 90%.
(3) The result of isolation,purification of inhibiting active substance shows that there are three components in ethyl acetate-extract.Component I has the highest inhibiting effect on Sphaeropsis sapinea growth,the growth-inhibiting rate is 72.94%.Component I has three compounds,one is Methoxy-cinnamic acid methyl ester whose yield is highest(57.952%),the other two are iso-butyl phthalate(yield is 13.592%) and dibutyl phthalate(yield is 28.455%).
(4) Ultraviolet rays,temperature,pH,oxidants and reductant,storage time were selected as actors of the stability of ethyl acetate-extract.In the natural environment,ethyl acetate-extract has a high stability to ultraviolet rays and temperature,definite anti-oxidation and anti-reduction capacity, and good heat resistance.Long-term storage does not affect its antifungal activity.
(5) In the oral acute toxic examination,the mice'death did not happen.The LD_(50) of the extraction is less than13200mg/kg,which was micro-toxic.
(1)应用通用引物ITS1与ITS4从试验菌株中分别扩增出目的片段617bp和544bp,GenBank登陆号分别为EU744544和EU796887。通过GenBank中BLAST,确认试验菌株分别为Hypoxylon perforatum和Sphaeropsis sapinea。
(2)炭团菌菌丝体发酵液中活性物质的提取采用水提和酯提2种方式。水提方式采用直接提取法和超声波提取法2种方法,酯提方式选择正丁醇、乙酸乙酯、乙醇3种萃取剂。各提取物对病原菌的抑菌活性研究选用菌丝生长抑制率和孢子萌发抑制率2个指标,对病原菌菌丝生长的抑制测定采用生长速率法,对病原菌孢子萌发的抑制试验采用悬滴法。结果表明炭团菌菌丝体发酵液5种提取物对樟子松枯梢病菌均有一定的抑菌活性,其中乙酸乙酯提取物对病原菌菌丝生长的抑制率最高,达到73.20%;乙酸乙酯提取物、超声波提取物、正丁醇提取物对病原菌孢子萌发抑制率均超过90%。
(3)采用生物活性跟踪技术,运用柱层析、TLC等方法对发酵液提取物中的抑菌活性成分进行了分离、纯化。结果表明该提取物中有3个组分,组分Ⅰ的抑菌率最高(72.94%)。综合气质联用(GC-MS)的检测结果,组分Ⅰ含3种化合物,其中得率最高的为对甲氧基肉桂酸甲酯(57.952%),其他两个化合物分别为邻苯二甲酸二异丁酯(得率13.592%)和邻苯二甲酸二丁酯(得率28.455%)。
(4)炭团菌液体培养菌液乙酸乙酯提取物稳定性的研究选用紫外线、温度、pH值、氧化剂与还原剂以及贮存时间5个因子。乙酸乙酯提取物对自然环境下的紫外线、温度具有很高的稳定性,并且具有一定的抗氧化还原能力和良好的耐热性,长期贮存不影响其抑菌活性。
(5)小鼠毒性试验中未发生小鼠死亡情况。炭团菌发酵液乙酸乙酯提取物对小鼠的半致死剂量(LD_(50))＞13200mg/kg,按照我国化学物质的急性毒性分级标准,该提取物为微毒。
The effects of inhibiting active substance extracted from culture liquid of Hypoxylon perforatum on Sphaeropsis sapinea;isolation,purification and structure identification of inhibiting active substance; ecological safety test and stability of inhibiting active substance;the rDNA ITS sequences of Hypoxylon perforatum and Sphaeropsis sapinea were studied by methods of Phytopathology, Molecular Biology,Biochemical and Organic Chemical.The results showed as follows:
(1) rDNA ITS sequences were amplified from Hypoxylon perforatum and Sphaeropsis sapinea strain with the universal primers ITS1 and ITS4.rDNA ITS sequence lenth of Hypoxylon perforatum is 617bp and GenBank Accession is EU744544;rDNA ITS sequence lenth of Sphaeropsis sapinea is 544 bp and GenBank Accession is EU796887.Through the BLAST In GenBank,the strains were confirmed Hypoxylon perforatum and Sphaeropsis sapinea.
(2) Methods of water-extract and ester-extract were used for extracting active substances from culture liquid of Hypoxylon perforatum.Method of water-extract included two ways,one is extracting directly from cultural liquid,and the others is extracting by ultrasonic.N-butyl alcohol, ethyl acetate and alcohol were used as extractant in ester-extract test.Growth-inhibiting rate and germination-inhibiting rate were used as the index of antifungal activity.Growth rate method was used for determining growth-inhibiting rate and pendent drop method for determining germination-inhibiting rate.The results is that five extracts from culture liquid of Hypoxylon perforatum all have definite antifungal activities to Sphaeropsis sapinea growth and germination.Ethyl acetate-extract is the best one on inhibiting Sphaeropsis sapinea growth,the growth-inhibiting rate is 73.20%;the germination-inhibiting rates of ethyl acetate-extract,ultrasonic-extract and n-butyl alcohol-extract are all over 90%.
(3) The result of isolation,purification of inhibiting active substance shows that there are three components in ethyl acetate-extract.Component I has the highest inhibiting effect on Sphaeropsis sapinea growth,the growth-inhibiting rate is 72.94%.Component I has three compounds,one is Methoxy-cinnamic acid methyl ester whose yield is highest(57.952%),the other two are iso-butyl phthalate(yield is 13.592%) and dibutyl phthalate(yield is 28.455%).
(4) Ultraviolet rays,temperature,pH,oxidants and reductant,storage time were selected as actors of the stability of ethyl acetate-extract.In the natural environment,ethyl acetate-extract has a high stability to ultraviolet rays and temperature,definite anti-oxidation and anti-reduction capacity, and good heat resistance.Long-term storage does not affect its antifungal activity.
(5) In the oral acute toxic examination,the mice'death did not happen.The LD_(50) of the extraction is less than13200mg/kg,which was micro-toxic.
引文
[1]任玉环,潘丽梅,阎明真.美洲山杨炭团菌溃疡病研究现状.吉林林学院学报,1995,11(4):251-256
[2]J.E.Bier,M.H.Rowat,徐梅卿.树皮腐生菌对炭团菌腐烂病影响的进一步研究.林业实用技术,1964,14:6-7
[3]李厚金,蓝文健,林永成.海洋真菌Hypoxylon oceanicum对1,2,3,4-四氢萘的转化及其产物测定.分析测试学报,2005,24(4):45-47
[4]乐长高,林永成,姜广策等。海洋炭团菌Hypoxylon sp.低沸点代谢物的研究.中国海洋药物,CHINESE JOURNAL OF MARINE DRUGS,1999,(2):12
[5]李厚金,林永成,王立等.南海海洋真菌Hypoxylon oceanicum的代谢产物.中山大学学报(自然科学版),2001,(4):70-72
L6]James M.Onsando,王国华 杀菌剂对引起茶树木腐病的麻饼炭团菌的试管效应.贵州茶叶,1989,(4):51
[7]任玉环,潘丽梅,阎明真.美洲山杨炭团菌溃疡病研究现状.吉林林学院学报,1995,11(4):251-253
[8]Miller,J.H.,A monograph of the world species of Nypaxylott.University of Georgia Press,Athens.1961:158
[9]Hubbes,M.The invasion site of Hypoxylon Pruinatum on Populus trcmuloides.Phytopathology,1964:54:896
[10]S.Bagga.D.K,and smallcy E.B.Foctors affecting inoculated with Hypoyylon pruinatum.Can.J.Bot.1969,47:907-914
[11]Griffin,D.H,Ehrenshaft.M,Monion P.DHostselective toxins of Hypoxylon mammatum for Populus tremuloides,1980:209-214
[12]Schipper,A.L.Jr.A Hypoxylon mammatum pothotoxin responsible for camker formation in quaking aspen.Phy-topatthology,1978,68:866-872
[13]Stermer,B.A,Scheffer,R.P.and Hare.J.H.Isolation of toxins of Hypoxylon mammatum and demonstration of some toxin effects on sdected dones of Populus tremuloides.Phytopathology,1984,74:654-658
[141刘治根.香菇、黑木耳段木上的害菌调查与其生态学初步研究 华中农学院学报,1984,3(4):10-19
[15]叶海亚,南仁燕,施立钦等.新型萃取技术在天然有机物提取及纯化中的应用.宁波教育学院学报,2006,8(4).27-30
[16]张鑫,张峻松.超临界CO_2流体萃取辛夷挥发油化学组分的研究[J].香科香精化妆品,1999,(2):9-12
[17]原永芳,周践.超临界CO_2流体萃取辛夷挥发油化学组分的研究[J].中国药学杂志,2000,35(2):84-87.
[18]Polesello S,Lovati F,Rizzolo A,et al.Supercritical fluid extraction as a preparative tool for strawberry aroma analysis[J].Journal of High Resolution Chromatography,1993,16(9):555-559.
[19]郭振德,张相年.超临界CO_2萃取姜油的组成研究[J].色谱,1995,13(3):156-160.
[20]陈开勋,葛红光.超临界CO_2萃取茶籽油[J].中国油脂,1996,21(5):30-33.
[21]尹卓容.超临界CO_2萃取辣根精油[J].中国调味品,1996,(7):10-12.
[22]蔡建国.超临界CO_2提取金银花浸膏[J].香科香精化妆品,1999,(2):1-3.
[23]Berna A,Tarrega A,BlascoM,et al.Supercritical CO_2 extraction of essential oil fromorange peel:effect ofthe height ofthe bed[J].Journal Supercritical Fluids,2000,18(3):227-237.
[24]Papamichail I,Louli V,Magoulas K.Supercritical fluid extraction of celery seed oil[J].Journal of Supercritical Fluid,2000,18(3):213-226.
[25]Modlin RF,Alred PA,Tjemeld EUtilization of temperature-induced phase separation for the purification of ecdysone and 20-.hydroxyecdysone from spinach[J].Chromatogr,1994,668:229-236.
[26]李伟,朱自强,梅乐和等.黄芩甙在伴有温度诱导效应双水相系统中的分配行为[J].化工学报,1998,49(1):92-95.
[27]Carneiro-da-Cunha,M.G,Aires-Barros,M.R.,et al.Continuous Extraction of a Recombinant Cutinase from Eseheriehia coli Disrupte Cells with Reversed Micelles Using a Perforated Rotating Disc Contactor[J].Bioproc.Eng.,1996,15:253-256.
[28]Regalado,C.,Asenjo,JAand Pyle,DL.Studieson the purification of peroxidase frornhorseradish rootsusingreversemicelles[J].EnzymeMicrobTechnol,1996,18:332-339.
[29]Krieger N,Taipa MA,Aires-BarrosM R.J-chem.Purification Of The Penicillium Citrinum-Lipase Using Aot-Isooctane Reversed Micelles[J]Tech.Biotechno,1997,69(1):77-85.
[30]Rahaman R S,Chee J Y,Cabral J MS,Hatton TA.Recovery of an Extracellular Alkaline Protease From Whole Fermentation Broth Using ReverseMicelles[J].Biotechnol Prog,1998,(4):218-224.
[31]蔡宝玉,许林妹,彭远宝.反胶束萃取大豆蛋白的研究[J].中国油脂,2004,29(6):37-39.
[32]冯青然,陈燕军.中药提取工艺研究进展[J].中国实验方剂学杂志,2003,9(10):61-64.
[33]张代佳,刘传斌,修志龙等.微波技术在植物细胞内有效成分提取中的应用[J].中草药,2000,31(9):86-87
[34]Pare JRJ,Bel anger JMR,St afford SS.Mcrowave-Assisted Process(MAPTMa):a new tool for the analytical laboratory[J].Trends in Analtical,1994,13(4):176.
[35]张兆旺,孙秀梅.“半仿生提取法”的特点与应用[J].世界科学技术:中药现代化,2000, 2(1):35-38.
[36]许松林,徐世民,于爱华等.天然产物分离的新技术-分子蒸馏[J].中草药,2001,32(6):562-563.
[37]陈来同.生化工艺学[M].北京:科学出版社,2004:8-229
[38)汪茂田,谢培山等.天然有机化合物提取分离与结构鉴定.北京:化学工业出版社,2004:69-156
[39]袁黎明.制备色谱技术及应用.北京:化学工业出版社,2005:12-54
(40]刘湘,汪秋安.天然产物化学。北京:化学工业出版社,2005:32-34
[41]邹良,孟庆超.东北内蒙地区樟子松病害及防治[J].防护林科技,1995.2:42-44
[42]黄敬林,张力.樟子松枯梢病研究进展[J].东北林业大学学报,2005,33(2):83-85
L43]吴小芹.全球松树枯梢病发生状况及防治策略[J].世界林业研究,1999,12(1):16-20.
[44]SwartW J,Knox-DaviesP S,W ingfieldM J.Sphaeropsis sapinea,with special reference to its occurrence onPinusspp,in South Africa[J].SouthAfrican Forestry Jouma,1985,135:1-8.
[45]W ilson L F.Pine spittlebug:its ecology andmanagement[M].United States of Agriculture-Forest service,AgricultureHandbook,1991.30-35
[46]沈伯葵.松枯梢病及其病原的研究[J].林业科学研究,1992,5(6):659-664.
[47]苏开君,谭松山,邓群.国外松枯梢病病状和病原的研究[J].森林病虫通讯,1991(1):2-5.
[48]SwartW J,W ingfieldM J,Knox-DaviesP S.Relative susceptibilities to Sphaeropsis sapineaof sixPinusspp,cultivated in South Africa[J].Eur J ForPath,1998,18:184-189.
[49]Waterman AM.Diplodia pinea,the cause of disease ofhard pines[J].Phytopathology,1993,33:1018-1031.
[50]W ingfield M J,Knox-Davies P S.Associations ofDiplodia pinea with root disease of pines in South Africa[J].PlantDisease,1980,64:221-223.
[51]魏初奖,刘利玲.松枯梢病的研究[J].福建林学院学报,1997,17(1):25-29
[52]项存悌,原树忠.樟子松枯梢病的研究[J].东北林学院学报,1981,2:1-10
[53]杨旺.森林病理学[M]北京:中国林业出版社,1996:154-156
[54]吴景云.樟子松幼苗枯梢病危害与防[J].防护林科技,2001,48(3):70-71
[55]王德宏,徐志和.樟子松枯梢病及其防治措施[J].林业科技,2000,25(2):27-28
[56]Weindling R.,Studies on a Lethal Principle Effective in the Parasitic Action of Trichoderma lignorum on Rhizoctoctonia solani and other Soilfungi Phytopathology,1932,22:837-845
[57]宋瑞清,周秀华.Trichodermaspp.对樟子松枯梢病病原菌的影响[J].东北林业大学学报,2004,32(4):29-30
[58]祁金玉,宋瑞清.毒蘑菇菌株及毒素粗提液对樟子松枯梢病菌生长的影响.林业科技,2006,31(3)20-23
[59]杨建雄.生物化学与分子生物学实验技术教程[M].北京科学出版社,2002:125-200
[60]祁金玉.毒蘑菇菌株及其提取物对樟子松枯梢病病原菌的影响.东北林业大学硕士学位论文.2006:10-11
[61]宋瑞清,冀瑞卿.四种毒蘑菇菌株及其毒素对杨树烂皮病菌生长的抑制作用[J].北京林业大学学报,2005,27(2):88
[62]Song Ruiqing,Ji Ruiqing.Inhibiting effect of 4 toxic mushroom strains and their toxins on Cytospora chrysosperma(Pers.)Fr.KMITL SCIENCE JOURNAL[J].2004,1(4):125-132
[63]Wieland T.Schmidt G.Werth L.Uber die Giflstoffe deskrollen blarrerpilzes.Liebigs Ann Chem,1952,577:215-233
[64]张桥.卫生毒理学基础.北京:人民卫生出版社,2000:213-218
[65]张蕴晖.邻苯二甲酸二丁酯研究进展.北京:卫生研究,2003,32(4)407-408
[66]青岛医院卫生学教研组.增塑剂邻苯二甲酸二异丁酯毒性试验.青岛大学医学院学报,1977,(1):9-11
[67]WHO/IPCS.Di-n-butyl phthalate.Environmental Health Criteria 189Geneva,World Health Organization,1997
[68]张红,区国勇.防晒剂对甲氧基肉桂酸酯类的合成进展.香料香精化妆品,2001,(3):18-22
[69]曾庆友,曾明荣,许瑞安.对甲氧基肉桂酸甲酯的一锅式绿色合成.成都:合成化学,2007,15(5):663-664
[70)曹祺风,宋文生,朱长春等.邻苯二甲酸二丁酯研究概述.中国胶粘剂,2007,16(10):47
[71]柯昌英.邻苯二甲酸二异丁酯的酯化合成.上海:化学世界,1986:440-442
[2]J.E.Bier,M.H.Rowat,徐梅卿.树皮腐生菌对炭团菌腐烂病影响的进一步研究.林业实用技术,1964,14:6-7
[3]李厚金,蓝文健,林永成.海洋真菌Hypoxylon oceanicum对1,2,3,4-四氢萘的转化及其产物测定.分析测试学报,2005,24(4):45-47
[4]乐长高,林永成,姜广策等。海洋炭团菌Hypoxylon sp.低沸点代谢物的研究.中国海洋药物,CHINESE JOURNAL OF MARINE DRUGS,1999,(2):12
[5]李厚金,林永成,王立等.南海海洋真菌Hypoxylon oceanicum的代谢产物.中山大学学报(自然科学版),2001,(4):70-72
L6]James M.Onsando,王国华 杀菌剂对引起茶树木腐病的麻饼炭团菌的试管效应.贵州茶叶,1989,(4):51
[7]任玉环,潘丽梅,阎明真.美洲山杨炭团菌溃疡病研究现状.吉林林学院学报,1995,11(4):251-253
[8]Miller,J.H.,A monograph of the world species of Nypaxylott.University of Georgia Press,Athens.1961:158
[9]Hubbes,M.The invasion site of Hypoxylon Pruinatum on Populus trcmuloides.Phytopathology,1964:54:896
[10]S.Bagga.D.K,and smallcy E.B.Foctors affecting inoculated with Hypoyylon pruinatum.Can.J.Bot.1969,47:907-914
[11]Griffin,D.H,Ehrenshaft.M,Monion P.DHostselective toxins of Hypoxylon mammatum for Populus tremuloides,1980:209-214
[12]Schipper,A.L.Jr.A Hypoxylon mammatum pothotoxin responsible for camker formation in quaking aspen.Phy-topatthology,1978,68:866-872
[13]Stermer,B.A,Scheffer,R.P.and Hare.J.H.Isolation of toxins of Hypoxylon mammatum and demonstration of some toxin effects on sdected dones of Populus tremuloides.Phytopathology,1984,74:654-658
[141刘治根.香菇、黑木耳段木上的害菌调查与其生态学初步研究 华中农学院学报,1984,3(4):10-19
[15]叶海亚,南仁燕,施立钦等.新型萃取技术在天然有机物提取及纯化中的应用.宁波教育学院学报,2006,8(4).27-30
[16]张鑫,张峻松.超临界CO_2流体萃取辛夷挥发油化学组分的研究[J].香科香精化妆品,1999,(2):9-12
[17]原永芳,周践.超临界CO_2流体萃取辛夷挥发油化学组分的研究[J].中国药学杂志,2000,35(2):84-87.
[18]Polesello S,Lovati F,Rizzolo A,et al.Supercritical fluid extraction as a preparative tool for strawberry aroma analysis[J].Journal of High Resolution Chromatography,1993,16(9):555-559.
[19]郭振德,张相年.超临界CO_2萃取姜油的组成研究[J].色谱,1995,13(3):156-160.
[20]陈开勋,葛红光.超临界CO_2萃取茶籽油[J].中国油脂,1996,21(5):30-33.
[21]尹卓容.超临界CO_2萃取辣根精油[J].中国调味品,1996,(7):10-12.
[22]蔡建国.超临界CO_2提取金银花浸膏[J].香科香精化妆品,1999,(2):1-3.
[23]Berna A,Tarrega A,BlascoM,et al.Supercritical CO_2 extraction of essential oil fromorange peel:effect ofthe height ofthe bed[J].Journal Supercritical Fluids,2000,18(3):227-237.
[24]Papamichail I,Louli V,Magoulas K.Supercritical fluid extraction of celery seed oil[J].Journal of Supercritical Fluid,2000,18(3):213-226.
[25]Modlin RF,Alred PA,Tjemeld EUtilization of temperature-induced phase separation for the purification of ecdysone and 20-.hydroxyecdysone from spinach[J].Chromatogr,1994,668:229-236.
[26]李伟,朱自强,梅乐和等.黄芩甙在伴有温度诱导效应双水相系统中的分配行为[J].化工学报,1998,49(1):92-95.
[27]Carneiro-da-Cunha,M.G,Aires-Barros,M.R.,et al.Continuous Extraction of a Recombinant Cutinase from Eseheriehia coli Disrupte Cells with Reversed Micelles Using a Perforated Rotating Disc Contactor[J].Bioproc.Eng.,1996,15:253-256.
[28]Regalado,C.,Asenjo,JAand Pyle,DL.Studieson the purification of peroxidase frornhorseradish rootsusingreversemicelles[J].EnzymeMicrobTechnol,1996,18:332-339.
[29]Krieger N,Taipa MA,Aires-BarrosM R.J-chem.Purification Of The Penicillium Citrinum-Lipase Using Aot-Isooctane Reversed Micelles[J]Tech.Biotechno,1997,69(1):77-85.
[30]Rahaman R S,Chee J Y,Cabral J MS,Hatton TA.Recovery of an Extracellular Alkaline Protease From Whole Fermentation Broth Using ReverseMicelles[J].Biotechnol Prog,1998,(4):218-224.
[31]蔡宝玉,许林妹,彭远宝.反胶束萃取大豆蛋白的研究[J].中国油脂,2004,29(6):37-39.
[32]冯青然,陈燕军.中药提取工艺研究进展[J].中国实验方剂学杂志,2003,9(10):61-64.
[33]张代佳,刘传斌,修志龙等.微波技术在植物细胞内有效成分提取中的应用[J].中草药,2000,31(9):86-87
[34]Pare JRJ,Bel anger JMR,St afford SS.Mcrowave-Assisted Process(MAPTMa):a new tool for the analytical laboratory[J].Trends in Analtical,1994,13(4):176.
[35]张兆旺,孙秀梅.“半仿生提取法”的特点与应用[J].世界科学技术:中药现代化,2000, 2(1):35-38.
[36]许松林,徐世民,于爱华等.天然产物分离的新技术-分子蒸馏[J].中草药,2001,32(6):562-563.
[37]陈来同.生化工艺学[M].北京:科学出版社,2004:8-229
[38)汪茂田,谢培山等.天然有机化合物提取分离与结构鉴定.北京:化学工业出版社,2004:69-156
[39]袁黎明.制备色谱技术及应用.北京:化学工业出版社,2005:12-54
(40]刘湘,汪秋安.天然产物化学。北京:化学工业出版社,2005:32-34
[41]邹良,孟庆超.东北内蒙地区樟子松病害及防治[J].防护林科技,1995.2:42-44
[42]黄敬林,张力.樟子松枯梢病研究进展[J].东北林业大学学报,2005,33(2):83-85
L43]吴小芹.全球松树枯梢病发生状况及防治策略[J].世界林业研究,1999,12(1):16-20.
[44]SwartW J,Knox-DaviesP S,W ingfieldM J.Sphaeropsis sapinea,with special reference to its occurrence onPinusspp,in South Africa[J].SouthAfrican Forestry Jouma,1985,135:1-8.
[45]W ilson L F.Pine spittlebug:its ecology andmanagement[M].United States of Agriculture-Forest service,AgricultureHandbook,1991.30-35
[46]沈伯葵.松枯梢病及其病原的研究[J].林业科学研究,1992,5(6):659-664.
[47]苏开君,谭松山,邓群.国外松枯梢病病状和病原的研究[J].森林病虫通讯,1991(1):2-5.
[48]SwartW J,W ingfieldM J,Knox-DaviesP S.Relative susceptibilities to Sphaeropsis sapineaof sixPinusspp,cultivated in South Africa[J].Eur J ForPath,1998,18:184-189.
[49]Waterman AM.Diplodia pinea,the cause of disease ofhard pines[J].Phytopathology,1993,33:1018-1031.
[50]W ingfield M J,Knox-Davies P S.Associations ofDiplodia pinea with root disease of pines in South Africa[J].PlantDisease,1980,64:221-223.
[51]魏初奖,刘利玲.松枯梢病的研究[J].福建林学院学报,1997,17(1):25-29
[52]项存悌,原树忠.樟子松枯梢病的研究[J].东北林学院学报,1981,2:1-10
[53]杨旺.森林病理学[M]北京:中国林业出版社,1996:154-156
[54]吴景云.樟子松幼苗枯梢病危害与防[J].防护林科技,2001,48(3):70-71
[55]王德宏,徐志和.樟子松枯梢病及其防治措施[J].林业科技,2000,25(2):27-28
[56]Weindling R.,Studies on a Lethal Principle Effective in the Parasitic Action of Trichoderma lignorum on Rhizoctoctonia solani and other Soilfungi Phytopathology,1932,22:837-845
[57]宋瑞清,周秀华.Trichodermaspp.对樟子松枯梢病病原菌的影响[J].东北林业大学学报,2004,32(4):29-30
[58]祁金玉,宋瑞清.毒蘑菇菌株及毒素粗提液对樟子松枯梢病菌生长的影响.林业科技,2006,31(3)20-23
[59]杨建雄.生物化学与分子生物学实验技术教程[M].北京科学出版社,2002:125-200
[60]祁金玉.毒蘑菇菌株及其提取物对樟子松枯梢病病原菌的影响.东北林业大学硕士学位论文.2006:10-11
[61]宋瑞清,冀瑞卿.四种毒蘑菇菌株及其毒素对杨树烂皮病菌生长的抑制作用[J].北京林业大学学报,2005,27(2):88
[62]Song Ruiqing,Ji Ruiqing.Inhibiting effect of 4 toxic mushroom strains and their toxins on Cytospora chrysosperma(Pers.)Fr.KMITL SCIENCE JOURNAL[J].2004,1(4):125-132
[63]Wieland T.Schmidt G.Werth L.Uber die Giflstoffe deskrollen blarrerpilzes.Liebigs Ann Chem,1952,577:215-233
[64]张桥.卫生毒理学基础.北京:人民卫生出版社,2000:213-218
[65]张蕴晖.邻苯二甲酸二丁酯研究进展.北京:卫生研究,2003,32(4)407-408
[66]青岛医院卫生学教研组.增塑剂邻苯二甲酸二异丁酯毒性试验.青岛大学医学院学报,1977,(1):9-11
[67]WHO/IPCS.Di-n-butyl phthalate.Environmental Health Criteria 189Geneva,World Health Organization,1997
[68]张红,区国勇.防晒剂对甲氧基肉桂酸酯类的合成进展.香料香精化妆品,2001,(3):18-22
[69]曾庆友,曾明荣,许瑞安.对甲氧基肉桂酸甲酯的一锅式绿色合成.成都:合成化学,2007,15(5):663-664
[70)曹祺风,宋文生,朱长春等.邻苯二甲酸二丁酯研究概述.中国胶粘剂,2007,16(10):47
[71]柯昌英.邻苯二甲酸二异丁酯的酯化合成.上海:化学世界,1986:440-442